european light microscopy initiative

Transcription

european
light microscopy
initiative
elmi
LEICA TCS SP8 DLS
› Enjoy easy sample handling and multi-position experiments
› Discover new fields of applications
› Upgrade from Leica TCS SP8 to Leica TCS SP8 DLS at any time
THE VERTICAL TURN
Expand your options
with combined light sheet
and confocal
From Eye to Insight
A Shift of PerSPective –
LeicA DMi8 inverteD MicroScoPe PLAtforM
Match the future of life sciences
•Frombasicmicroscopytohigh-endimaging–LeicaDMi8growswithyourever-changingneeds.
The solution for live cell experiments
•Fromfastdynamicstolongtermimaging–LeicaDMi8wascreatedwithyourlivingsampleinmind.
Delve in deeper
• Fromimagestoanswers–LeicaLASXsoftwareenablesyourscientificdiscoveries.
Meet the future: Leica DMi8 and Leica LAS X software workshop
in seminar room 102 (WS2, WS5)
www.leica-microsystems.com/dmi8
Copyright©byLeicaMicrosystemsCMSGmbH,Wetzlar,Germany,2016
16th international
ELMI meeting
24-27 May 2016, Debrecen, Hungary
Kölcsey Center - Hotel Lycium****
The organizing committee and ELMI are
grateful to all industrial sponsors for their
financial contribution and support and for
their participation at the meeting.
ELMI meeting
european
light microscopy
initiative
elmi
16th international
ELMI meeting
Committees
MAIN ORGANIZERS:
György Vámosi, Dept. of Biophysics and Cell Biology, University of Debrecen, Coordinator of the
Hungarian BioImaging Network
Gábor Csúcs, Scientific Center for Optical and Electron Microscopy (ScopeM), ETH Zürich
ORGANIZING COMMITTEE:
• János Szöllősi, University of Debrecen, Hungary
• László Mátyus, University of Debrecen, Hungary
• György Vereb, University of Debrecen, Hungary
• Péter Nagy, University of Debrecen, Hungary
• Győző Garab, Biological Research Center, Szeged, Hungary
• Miklós Kellermayer, Semmelweis University, Budapest, Hungary
• Katalin Tóth, DKFZ, Heidelberg, Germany
• Jörg Langowski, DKFZ, Heidelberg, Germany
• Gábor Steinbach, Centre ALGATECH, Trebon, Czech Republic
General Information
VENUE
Kölcsey Center
Hunyadi u. 1–3.
4026 Debrecen,
Hungary
www.kolcseykozpont.hu
TECHNICAL ORGANISER
Remedicon Kft.
Ganz u. 16.
1027 Budapest,
Hungary
www.remedicon.hu
REGISTRATION DESK
EXHIBITION
Opening hours
24th MAY 08-20
25th MAY 08-20
26th MAY 08-17
27th MAY 08-13
Opening hours
24th MAY 17-20
25th MAY 09-20
26th MAY 09-17
27th MAY 09-13
6
16th international
ELMI meeting
TRANSPORTATION
Free shuttle bus service:
travelling time will be about 3 hours
These coaches will have the following departure times:
european
light microscopy
initiative
elmi
16th international
ELMI meeting
• from Budapest Airport to Debrecen Congress Venue
23rd MAY 17.00
23rd MAY 19.00
24th MAY 12.00
• from Debrecen Congress Venue to Budapest Airport
27th MAY 12.00
27th MAY 14.00
Agora transfer company:
In case your arrival time does not match the bus departures then there is an opportunity to use
minibus transfer for HUF 8 700 (one way) or HUF 14 490 (2 ways).
E-mail: [email protected],
Phone:+36 20 7762163 (Monday-Friday, 8 am-5 pm)
LIABILITY
The ELMI meeting 2016 Secretariat and Organizers cannot accept liability for personal accidents or
loss of or damage to private property of participants and accompanying persons.
Only your personal badge allows you to access all scientific sessions, exhibition and
social events.
SOCIAL EVENTS
24th MAY 20.00 Concert by Debrecen Dixieland Jazz Band and dinner, Kölcsey Center
25th MAY 20.00 Hungarian Folk Dance Show by Hajdú Group and dinner, Kölcsey Center
26th MAY 16.50 Meeting point: Hotel Lycium reception (outdoor program)
Buses leave at 17.00, the trip is ca. 45 minutes
18.00 „Máta Ménes” Equestrian and Carriage Driving Show
19.00 Gala dinner, Hortobágy Csárda
th
27 MAY 13.50 Meeting point: Hotel Lycium reception (outdoor program)
Buses leave at 14.00, the trip is ca. 2 hours
Sightseeing and Wine Tasting Tour, Tokaj (facultative)
7
ELMI meeting
european
light microscopy
initiative
elmi
16th international
ELMI meeting
Platina Sponsor
Seite 1 von 1
http://zeissnet.zeiss.org/41256774004FB1CC/EmbedTitelIntern/LogoGIF/$File/CZ-L... 16.04.2007
8
AL TURN
Gold Sponsors
16th international
ELMI meeting
General Sponsors
european
light microscopy
initiative
elmi
16th international
ELMI meeting
Company: Scientific Volume Imagi
Title: Huygens gpu-accelerated image restoration; now also for light
Workshops at Booth MB05 (all workshop time slots)
We very much welcome you at our Huygens booth (also during the workshops), wher
latest developments, including GPU acceleration, floating licenses, and Light Sheet de
Nowadays, deconvolution is widely accepted as a fundamental technique for restoring
from widefield, confocal, spinning disk, multiphoton, and STED image data.9We have
module to Huygens for the deconvolution of images from a variety of Light Sheet /Se
Ilumination Microscopy (SPIM) imaging setups.
KÖLCSEY
CENTER
Ground floor
MB1
MB2
MB3
MB5
MB4
MB6
MB8
MB9
Entrance
MB7
ć
MB15
MB16
Co
Registration
ffe
e
Stairs
br
ea
k
Restaurant
MB14
MB13
MB21
MB12
MB11
MB17
GRAND HALL
MB18
MB10
MB19
MB20
SB8
SB0
102
103
104
105
LEICA
OLYMPUS
BRUKER
CARL ZEISS
SB7
SB1
SB2
SB3
SB4
SB5
SB6
16th international
ELMI meeting
MEDIUM BOOTHS:
MB 01 CONFOCAL.NL
MB 02 IBIDI
MB 03 ARIVIS
MB 04 NIKON
MB 05 SCIENTIFIC VOLUME IMAGING
MB 06 PHASICS
MB 07 FEI MUNICH
MB 08 ARGOLIGHT
MB 09 TOPTICA
MB 10 RAPP OPTOELECTRONIC,
AHF ANALYSENTECHNIK
MB 11 OMICRON
MB 12 INTELLIGENT IMAGING
INNOVATIONS
MB 13 ANDOR
MB 14 LASOS
MB 15 LEICA
MB 16 VISITRON
MB 17 BRUKER
MB 18 ACQUIFER
MB 19 LUXENDO
MB 20 MAD CITY LABS
MB 21 HAMAMATSU PHOTONICS
european
light microscopy
initiative
elmi
GLASSROOM16th
I. international
meeting
NIKON, 25-26ELMI
May
24-27
May 2016, Debrecen, Hungary
GLASSROOM III.
BITPLANE, 25-26 May
GLASSROOM IV.
ARIVIS 25th May
GE HEALTCARE 26th May
SEMINAR ROOMS:
GROUND FLOOR
102 LEICA
103 OLYMPUS
104 BRUKER
105 CARL ZEISS
2ND FLOOR
402 ANDOR
403 PICOQUANT
404 ACQUIFER, LUXENDO
405 THERMO FISHER
SMALL BOOTHS:
SB 00 MICROTRADE
SB 01 LUMENERA
SB 02 GE HEALTHCARE
SB 03 PHOTOMETRICS
SB 04 SUPERTECH
SB 05 TOKAI
SB 06 COBOLT AB
SB 07 EXCELITAS TECHNOLOGIES
SB 08 PICOQUANT
11
KÖLCSEY
CENTER
1st floor
POSTERS
KÖLCSEY
CENTER
2nd floor
402
403
404
405
ANDOR
PICOQUANT
ACQUIFER, LUXENDO
THERMO FISHER
Hotel
LYCIUM
Ground floor
25th May
26 May
th
GLASSROOM I. NIKON, 25-26 May
GLASSROOM III. BITPLANE, 25-26 May
GLASSROOM IV. ARIVIS 25th May
GE HEALTCARE 26th May
ELMI meeting
european
light microscopy
initiative
16th international
ELMI meeting
16
elmi
16th international
ELMI meeting
Program
light m
16th internation
ELMI meeting
24-27 May 2016, Debrecen
Kölcsey Center - Hotel Lyc
16th international
ELMI meeting
ELMI 2016 at a glance
Tuesday, 24th May 2016
european
light microscopy
initiative
elmi
16th international
ELMI meeting
09:00 – 17:30
Core Facility meeting (Grand Hall and Ballroom)
18:00 – 18:15
18:15 – 19:00
19:00 – 20:00
20:00 – 20:30
20:30 – 22:00
Opening ceremony (Grand Hall)
Opening lecture
Short presentation of selected posters
Concert of the Debrecen Dixieland Jazz Band (Grand Hall)
Welcome reception (restaurant)
Wednesday, 25th May 2016
08:30 – 09:45
10:15 – 12:35
12:35 – 14:00
14:00 – 15:00
15:00 – 16:00
16:30 – 17:30
18:00 – 20:00
20:00 – 21:00
21:00 – 22:30
Session I: Probes
Session II: Functional and live cell imaging
Lunch
Workshop 1
Workshop 2
Workshop 3
Poster session
Dinner
Folk dance show and taster
Thursday, 26th May 2016
Session III: Image analysis and intelligent imaging
08:00 – 10:00
Session IV: Label free imaging
10:30 – 12:00
Lunch
12:00 – 13:00
Workshop 4
13:00 – 14:00
Workshop 5
14:00 – 15:00
Workshop 6
15:30 – 16:30
Buses leave for Hortobágy conference dinner
17:00
Friday, 27th May 2016
Session V: Imaging for neurobiology and developmental biology
08:30 – 10:15
Session VI: Superresolution imaging
10:35 – 13:00
Take-away sandwich lunch
13:00 – 13:45
Buses leave for Tokaj wine trip
14:00
19
ELMI meeting
european
light microscopy
initiative
elmi
16th international
ELMI meeting
ELMI 2016 Meeting Program
09:00 – 17:30
Core Facility meeting (Grand Hall and Ballroom) – see page 26.
18:00 – 18:15
Opening ceremony (Grand Hall)
18:15 – 19:00
Opening lecture
James Pawley, University of Wisconsin-Madison (Madison, WI USA)
Don’t forget the photons!
19:00 – 20:00
Short presentation of selected posters
Michal Kozubek , Masaryk University (Brno, Czech Republic)
Benchmarking and selection of algorithms and software in bioimage
analysis (P015)
Szabolcs Osváth, Semmelweis University (Budapest, Hungary)
Multi-scale transport image of the living cell (P025)
Aliaksandr Halavatyi, Cell Biology and Biophysics Unit, EMBL
(Heidelberg, Germany)
High-throughput measurements of COPII coat turnover with
automated FRAP (P012)
Edina Szabó- Meleg, University of Pécs (Pécs, Hungary)
In the footsteps of “intercellular highways” – formation and function
of membrane nanotubes (P037)
Olga Oleksiuk, University of Heidelberg (Heidelberg, Germany)
Challenges in the labeling and detection of viral RNA by confocal and
super-resolution fluorescent microscopy (P023)
Nikoletta Szalóki, University of Debrecen (Debrecen, Hungary)
Evidence for homodimerization of the c-Fos transcription factor in live
cells revealed by FRET, SPIM-FCCS and MD-modeling (P038)
József Sinkó, University of Szeged (Szeged, Hungary)
Localization analysis with rainSTORM (P041)
20
16th international
ELMI meeting
european
light microscopy
initiative
elmi
Christopher J. Guérin, VIB Bio Imaging Core (Ghent,
16thBelgium)
international
Correlative light and electron microscopy inELMI
3D: meeting
new developments and
applications (P010)
20:00 – 20:30
Concert of the Debrecen Dixieland Jazz Band (Grand Hall)
20:30 – 22:00
Welcome reception (restaurant)
Sponsored by
Wednesday, 25th May 2016
Session I: Probes
Donna Arndt-Jovin, Max Planck Institute for Biophysical Chemistry
(Göttingen, Germany)
An overview of probes for microscopic imaging with consideration of
the biological questions being addressed
09:00 – 09:30
Theodorus Gadella, Swammerdam Institute for Life Sciences, University of
Amsterdam (Amsterdam, The Netherlands)
mScarlet, a novel high quantum yield monomeric red fluorescent
protein with strongly enhanced properties for functional imaging
microscopy
09:30 - 09:45
Christian A. Combs, NHLBI Light Microscopy Facility (Bethesda, MD USA)
Intravital imaging of pHi using two-photon excitation of the red
fluorescent protein mKeima
09:45 – 10:15
Coffee break
our options
Sponsored by
E VERTICAL TURN
08:30 – 09:00
21
ELMI meeting
european
light microscopy
initiative
elmi
16th international
ELMI meeting
Session II: Functional and live cell imaging
10:15 – 10:45
Jörg Langowski, German Cancer Research Center DKFZ (Heidelberg, Germany)
What determines random motion in cells? Chromatin dynamics studied
by fluorescence correlation light sheet microscopy
10:45 – 11:15
Maïté Coppey-Moisan, Institut Jacques Monod (Paris, France)
Chromatin breathing evidenced by fast time lapse FLIM regulates
accessibility of TAFII250-double bromodomain to acetylated histone H4
11:15 – 11:45
Na Ji, Howard Hughes Medical Institute (Ashburn, VA USA)
Probing neural circuits with shaped light
11:45 – 12:00
Peter Hemmerich, Leibniz Institute on Aging – Fritz-Lipman-Institute (Jena, Germany)
Multimodal and multidimensional live-cell imaging of the assembly of
PML nuclear bodies
12:00 – 12:15
Tytus Bernas , Nencki Institute of Experimental Biology (Warsaw, Poland)
DNA replication factories form a dynamic compartment in cell nuclei
12:15 – 12:35
Antje Keppler (EMBL, Heidelberg, Germany)
Euro-BioImaging
12:35 – 14:00
Lunch
14:00 – 15:30
Euro-Bioimaging national coordinator meeting (Glass room II.)
14.00 – 15:00
Workshop 1
15:00 – 16:00
Workshop 2
16:00 – 16:30
Coffee break
16:30 – 17:30
Workshop 3
18:00 – 20:00
Poster session with drinks(1st floor)
20:00 – 21:00
Dinner (Grand Hall)
21:00 – 22:30
Folk dance show and taster
22
16th international
ELMI meeting
Thursday, 26th May 2016
Session III: Image analysis and intelligent imaging
european
light microscopy
initiative
elmi
16th international
ELMI meeting
08:00 – 08:30
Jyoti K. Jaiswal, Children’s National Health System (Washington, DC USA)
Imaging mitochondrial and sub-mitochondrial localization of proteins
08:30 – 09:00
Jason Swedlow, Centre for Gene Regulation & Expression, University of Dundee
(Dundee, Scotland, United Kingdom)
The open microscopy environment: open source image informatics for
the biological sciences
09:00 – 09:30
Daniel Sage, Ecole Polytechnique Fédérale de Lausanne, EPFL (Lausanne,
Switzerland)
Open software tools for microscopy image processing
09:30 – 09:45
Jeremy Pike, Cancer Research UK Cambridge Institute (University of Cambridge,
Cambridge, UK)
Event driven automated microscopy
09:45 – 10:00
Péter Horváth, Biological Research Center of HAS (Szeged, Hungary)
Analysis of large scale imaging data using machine learning and image
analysis methods
10:00 – 10:30
Coffee break
Session IV: Label free imaging
Chiara Stringari, CNRS, INSERM, Université Paris-Saclay, Palaiseau cedex (Paris,
10:30 – 11:00
France)
Efficient multicolor two-photon imaging of endogenous fluorophores
in live tissues by wavelength mixing
11:00 – 11:30
Michael Schmitt, Friedrich-Schiller University (Jena, Germany)
Raman Microspectroscopy a powerful tool for spectral histopathology
11:30 – 12:00
Abhishek Kumar, Medical University of Vienna (Vienna, Austria)
Digital adaptive optics for achieving space invarient resolution in
optical coherence tomography
12:00 – 13:00
Lunch
23
ELMI meeting
european
light microscopy
initiative
elmi
16th international
ELMI meeting
13:00 – 14:00
Workshop 4
14:00 – 15:00
Workshop 5
15:00 – 16:00
ELMI steering committee meeting (Glass room II.)
15:00 – 15:30
Coffee break
15:30-16:30
Workshop 6
17:00
Buses leave for Hortobágy conference dinner
Friday, 27th May 2016
Session V: Imaging for neurobiology and developmental biology
08:30 – 09:00
Periklis Pantazis, Department of Biosystems Science and Engineering (D-BSSE),
(Basel, Switzerland)
Mapping single neurons in vivo with confined primed conversion
09:00 – 09:30
Balázs Rózsa, Institute of Experimental Medicine, Hungarian Academy of
Sciences, (Budapest, Hungary)
Fast 3D functional imaging of neuronal networks and dendritic spine
assemblies in behaving animals
09:30 – 10:00
Anthony de Vries, Max Planck Institute for Biophysical Chemistry
Generation 3 Programmable Array Microscope (PAM) for Adaptive, high
speed, large format optical sectioning
10:00 – 10:15
Christopher Schmied, Max Planck Institute of Molecular Cell Biology and Genetics
(Dresden, Germany)
Light sheet imaging for studying development and evolution of
development
10:15 – 10:35
Coffee break
24
16th international
ELMI meeting
Session VI: Superresolution imaging
european
light microscopy
initiative
elmi
16th international
ELMI meeting
10:35 – 11:05
Ingo Gregor, 3rd Institute of Physics, Georg-August-University
Multi-target microscopy by spectrally resolved fluorescence lifetime
imaging
11:05 – 11:35
István Katona, Institute of Experimental Medicine,
Hungarian Academy of Sciences, (Budapest, Hungary)
Correlated confocal and super-resolution imaging in brain circuits by
VividSTORM
11:35 – 11:50
Alexander Demchenko, Palladin Institute of Biochemistry,
National Academy of Sciences of Ukraine (Kiev, Ukraine)
New and prospective nanocomposites for super-resolution and singlemolecular studies
11:50 – 12:05
Sven C. Sidenstein, Max Planck Institute for Biophysical Chemistry
Coordinate-targeted fluorescence nanoscopy with multiple off states
12:05 – 12:20
Erik M. M. Manders, Swammerdam Institute for Life Sciences,
University of Amsterdam (Amsterdam, The Netherlands)
Re-scan Confocal Microscopy (RCM): scanning twice for better
resolution and high sensitivity; characterisation and applications.
12:20 – 12:35
Jan Schmoranzer, Charité-Universitätsmedizin (Berlin, Germany)
Registration free multicolor ‘caged’ dSTORM with novel dyes resolves
ultra-structure of synaptic vesicles
12:35 – 13:00
Poster prizes, closing remarks
13:00 – 13:45
Take-away sandwich lunch
14:00
Buses leave for Tokaj wine trip (facultative)
25
ELMI meeting
european
light microscopy
initiative
elmi
16th international
ELMI meeting
Core Facility Satellite Meeting Program
09:00 – 09:15
Welcome and introduction (Grand Hall)
09:15 – 09:30
Arne Seitz, Ecole Polytechnique (Lausanne, Switzerland)
Teaching users
09:30 – 09:45
Martin Spitaler, Max Planck Institute (Martinsried, Germany)
Teaching CF staff
09: 45 – 10: 00
Urs Ziegler, University of Zürich (Zürich, Switzerland)
Big Data processing
10:00 – 10:15
Timo Zimmermann, Center for Genomic Regulation (Barcelona, Spain)
Big Data processing
10:15 – 11:00
Discussion: Big Data Processing
10:15 – 11:00
Discussion: teaching users and staff (Ball room)
11:00 – 11:10
Break
11:10 – 11:25
Summary of discussions by moderators
11:25 – 11:40
Peter O’Toole, University of York (York, UK)
Long-term staff perspective / career path
11:40 – 11:55
(speaker TBA)
Cooperation of facilities with companies
11:55 – 12:25
Discussion: Long-term staff perspective / career path
11:55 – 12:25
Discussion: Cooperation of facilities with companies (Ball room)
12:25 – 12:35
Break
26
16th international
ELMI meeting
12:35 – 12:50
Summary of discussions by moderators
european
light microscopy
initiative
elmi
16th international
ELMI meeting
12:50 – 13:30
Sandwich lunch
13:30
“Companies vs. Academics” football match
Sponsored by
15:30 – 15:45
Julien Colombelli, Institute for Research in Biomedicine (Barcelona, Spain)
NEUBIAS
15:45 – 16:00
Ann Wheeler, University of Edinburgh (Edinburg, UK)
Super-resolution in Core Facilities and Standards
16:00- 16:15
Jan Schmoranzer, Charité-Universitätsmedizin (Berlin, Germany)
Superresolution in Core Facilities and Standards
16:15- 17:15
Discussion: Superresolution in Core Facilities and Standards
17:15
Wrap up of CF day
27
16th international
ELMI meeting
Oral abstracts
light m
16th internation
ELMI meeting
ELMI meeting
european
light microscopy
initiative
elmi
16th international
ELMI meeting
opening lecture
Don’t forget the photons!
James Pawley
Zoology Department, University of Wisconsin-Madison, Madison, WI, USA
[email protected]
Although the laser was invented in 1960, it made virtually no contribution to microscopy until the
late 1980s. Starting then, first the confocal and 2photon microscopes added the third dimension, then
light-sheet microscopy permitted us to follow embryological development at the subcellular level
and now we have an ever-growing stable of super-resolution fluorescence microscopies that allow
us to break the Abbe-resolution limit, often even in living cells. Supported by related developments
in optical and laser engineering, in computer data display and in the ability to use genetic tags to
attach fluorescent markers to specific proteins, these new microscopies have returned LM to the central
location in biological research that it held prior to the introduction of the electron microscope in the
1950s. Progress continues with the development of up-converter labels, nano-particle tags and many
of the other innovations that will be described later at this meeting.
Most of the discussion about the operation of this new instrumentation seems to focus on how the
wave nature of light can be manipulated to our advantage. At least in part, this is because a vast array
of mathematical formulae allow the accurate prediction of optical results. However, although light may
travel as a wave, it is created and detected as a particle, and sadly both of these processes are limited
by Poisson Statistics, a factor not easily turned into elegant mathematics. From Rose’s early work on
how statistics limited the visibility of structure in an image(1) to the recognition by Glaeser that these
considerations also limited what could be seen in the electron microscope(2), the importance of the
detection channel became ever more evident (3).
In response, the optical improvements have been paralleled by major improvements in
photodetectors. The sequence from the photodiode, to the photomultiplier tube and the avalanche
photodiode and now the hybrid-PMT and the multi-pixel photon counter, has seen a massive increase
in the quantum efficiency (QE) and a reduction of read-noise in single-channel photodetectors.
Starting with the vidicon, and moving through the age of CCD and CMOS sensors and on to that of
the electron-multiplier CCD and the scientific-CMOS, we see a similar improvement in image sensors.
With the QE now often reaching 80-90% over a wide range of wavelengths and noise levels that hover
at or below one count/pixel, it is now hard to imagine that much additional improvement is possible.
32
opening lecture
16th international
ELMI meeting
european
light microscopy
initiative
elmi
The remaining statistical limitations are now set by photosensitivity
even simply by the small
16thorinternational
ELMI meeting
number of fluorescent molecules present in the voxel interrogated by the various imaging techniques.
This presentation will review the important stages in our efforts to reduce the limitations placed
by Poisson Noise on our ability to probe structure and function with light. It will highlight both the
importance of counting as large a fraction as possible of any photons that are excited and remind
us of the remaining limitations imposed by the lumpiness of light and of the discrete nature of the
molecules and particles that emit it.
1. Rose, A. (1948).Television pickup tubes and the problem of noise. Adv. Electron. 1, 131.
2. Glaeser, R. M. (1971). Limitations to significant information in biological electron microscopy as a result of radiation
damage. J. Ultrastruct. Res. 36, 466.
3. Pawley, J.B. (1994) The sources of noise in three-dimensional microscopical data sets, in Three Dimensional Confocal
Microscopy: Volume Investigation of Biological Specimens ed. J. Stevens, (U. Toronto), Academic Press. NY. 47-94
33
ELMI meeting
european
light microscopy
initiative
elmi
invited speaker
16th international
ELMI meeting
Wednesday, 25th May
An overview of probes for microscopic imaging with
consideration of the biological questions being addressed
Donna Arndt-Jovin
Laboratory of Cellular Dynamics, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany
[email protected]
I will discuss the following topics with examples from our own work and those of other scientific
colleagues.
Probe considerations
Size of probe, length of
conjugation sequences
Wavelength of the probe or
probes
Fluorescent vs. fluorogenic
Quencher replacing one
fluorophore
Photostability, physical and
chemical properties
34
Biological questions
Oligomerization or complex
formation
Sensors for pH, ion, small
molecule concentrations
Transport kinetics
location, multiplexing
Imaging techniques
FRET, BRET, FiC, Fluorescence
induced complementation
Ratiometric or lifetime
Live cell imaging
4-D imaging
Structural analysis
STED, PALM, STORM, RESOLFT
invited speaker
Wednesday, 25th May
16th international
ELMI meeting
european
light microscopy
initiative
elmi
16th international
mScarlet, a novel high quantum yield monomeric
red
ELMI meeting
fluorescent protein with strongly enhanced properties for
functional imaging microscopy
Lindsay Haarbosch1, Daphne Bindels1, Laura van Weeren1, Marieke Mastop1, Marten Postma1, Antoine Royant2,
Dorus Gadella1
1) Section of Molecular Cytology & van Leeuwenhoek Centre for Advanced Microscopy, Swammerdam Institute for
Life Sciences, University of Amsterdam, Science Park 904, NL- 1098 XH, The Netherlands
2) Structural Biology Group, European Synchrotron Radiation Facility, 38043 Grenoble, France
[email protected]
mScarlet, a novel red fluorescent protein was generated from a synthetic template based on a
consensus amino acid sequence derived from naturally occurring red fluorescent proteins and purple
chromoproteins and on consensus monomerization mutations. The encoded synthetic red fluorescent
protein was optimized by molecular evolution through site directed and random mutagenesis.
Improved variants were selected by quantitative multimode screening for increased fluorescence
lifetime, increased photo stability, increased quantum yield and for increased chromophore maturation.
Very bright variants were obtained with high fluorescence lifetimes up to 3.8 ns, quantum yields >70
% and complete maturation. The monomeric status of the variants was confirmed by OSER analysis
and with α-tubulin fusions. The brightness of mScarlet is >2- fold increased as compared to bright red
fluorescent proteins such as mCherry, mRuby2 and tagRFP-T as was analyzed with quantitative (single
plasmid with viral 2A sequence) coexpression with mTurquoise2 in mammalian cells. During evolution
mScarlet variants with substantially altered spectroscopic properties were generated including
fluorescence lifetime variants, photo labile variants and strongly spectrally shifted variants.
mScarlet can be used as a bright red fluorescent fusion tag for staining various subcellular structures
in live cells. Because of their efficient maturation and high quantum yield, mScarlet vastly outperforms
existing monomeric red fluorescent proteins in ratiometric FRET-microscopy applications due to
seriously enhanced sensitized emission.
35
ELMI meeting
european
light microscopy
initiative
elmi
16th international
ELMI meeting
Wednesday, 25th May
Intravital imaging of pHi using two-photon excitation of the red
fluorescent protein mKeima
Christian A. Combs1, Jeanho Yun2, Nuo Sun2, Eric A. Alba3, Aleksandr Smirnov4, George Patterson3, Jay
Knutson4, Toren Finkel2
1) NHLBI Light Microscopy Facility
2) NHLBI Laboratory of Molecular Biology
3) NIBIB Section on Biophotonics
4) NHLBI Optical Spectroscopy Section, National Institutes of Health, Bethesda, Maryland 20892-1061
[email protected]
Intravital imaging of pHi using two-photon excitation of the red fluorescent
Intracellular pH (pHi) isprotein
a critical
modulator of many cellular processes including mitochondrial
mKeima
metabolism, protein function,
regulation
of the cell cycle, lysosomal function, and apoptosis. Targeted
Christian A. Combs1, Jeanho Yun2, Nuo Sun2, Eric A. Alba3, Aleksandr Smirnov4, George
3
2
, Jay Knutson4,can
and be
Toren
Finkel
Patterson
pHi imaging in specific cellular
compartments
done
with
genetically encoded pH sensors. Most
NHLBI
Facility;
Laboratory
of Molecular
Biology,
NIBIBshown
Section onthe
Biophotonics,
of these are GFP based or rely
onLight
FRETMicroscopy
between
twoNHLBI
proteins.
Tantama
et al.
(1) have
utility
NHLBI Optical Spectroscopy Section, National Institutes of Health, Bethesda, Maryland 20892-1061
of using RFP based ratio-metric imaging of pHi. Their work using pHRed showed an apparent pKa of
[email protected]
6.6 with a ratiometric dynamic range >10 when excited at 440 and 610nm . Here we show that the
Intracellular pH (pHi) is a critical modulator of many cellular processes including mitochondrial
RFP variant mKeima (targeted
to mitochondria) can be ratiometrically imaged using (two) two-photon
metabolism, protein function, regulation of the cell cycle, lysosomal function, and apoptosis.
pHi imaging
in specific cellular
compartments
canOptical
be done Parametric
with genetically
encoded
excitation lines (860nm andTargeted
1120nm)
from a standard
Ti:Sapph
laser and an
Oscillator
pH sensors. Most of these are GFP based or rely on FRET between two proteins. Tantama et al.
or by Fluorescent Lifetime Imaging
(FLIM)
at based
980nm.
These modalities
allow
forwork
intravital
(1) have shown
thewith
utilityexcitation
of using RFP
ratio-metric
imaging of pHi.
Their
using
pHRed showed an apparent pKa of 6.6 with a ratiometric dynamic range >10 when excited at
imaging with all of the benefits
of610nm
standard
two-photon
microscopy
depthcanofbe
440 and
. Here
we show thatexcitation
the RFP variant
mKeima (TPEM)
(targetedincluding
to mitochondria)
ratiometrically
using (two)
two-photon
excitationThis
linestechnique
(860nm andis1120nm)
from a
penetration in tissues, speed
of imaging,imaged
and inherent
optical
sectioning.
demonstrated
standard Ti:Sapph laser and an Optical Parametric Oscillator or by Fluorescent Lifetime Imaging
using the isolated protein (FLIM)
in solution,
culturedatcells,
andThese
freshly
harvested
In our
workwith
mKeima
with excitation
980nm.
modalities
allow tissues.
for intravital
imaging
all of the
of standard two-photon excitation microscopy (TPEM) including depth of penetration in
shows at least a > 10 foldbenefits
dynamic
range
between
pH
8
and
4.0
in
the
samples
we
have
imaged.
tissues, speed of imaging, and inherent optical sectioning. This technique is demonstratedWe
using
isolated
protein
in solution,
cultured
cells, and freshly
harvested
tissues.different
In our work
mKeima
show a mitochondrial (highthe
pH)
and
lysosomal
(low
pH)
distribution
of
mKeima
in
many
mouse
shows at least a > 10 fold dynamic range between pH 8 and 4.0 in the samples we have imaged.
We show
a mitochondrial
(high pH)method
and lysosomal
(low pH) distribution
of mKeima
in many
tissues. Representative figure
showing
the ratiometric
in harvested
liver is shown
in Figure
1.
different mouse tissues. Representative figure showing the ratiometric method in harvested liver
Utility of this technique is isdiscussed
with
an
eye
towards
examining
levels
of
autophagy
and
deep
twoshown in Figure 1. Utility of this technique is discussed with an eye towards examining levels
autophagy
and deep two-photon imaging using red shifted TPEM.
photon imaging using red ofshifted
TPEM.
1
2
3
4
A
B
Figure 1: (A) Overlay of 1120 (red) and 860 (green) excited mKeima in mouse liver. (B) pH map
Figure 1: (A) Overlay of 1120
and
860
(green) excited
mKeima
in mouse
(B)image
pH map
based
on A.
based(red)
on the
ratio
of 1120/860
excited mKeima
in mouse
liverliver.
from the
shown
in panel
the ratio of 1120/860 excited mKeima
in
mouse
liver
from
the
image
shown
in
panel
A.
1. Tantama, M., Hung, Y.P, and Gary Yellen. (2011). JACS. 133: 10034-10037.
1. Tantama, M., Hung, Y.P, and Gary Yellen. (2011). JACS. 133: 10034-10037.
36
invited speaker
16th international
ELMI meeting
Wednesday, 25th May
european
light microscopy
initiative
elmi
16th international
What determines random motion in cells? Chromatin
dynamics
ELMI meeting
studied by fluorescence correlation light sheet microscopy
Giulia Marcarini1,2, Dominik Zeller1, Buse Isbilir1, Jan W. Krieger1,3, Giuseppe Chirico2, Jörg Langowski1,3*
1) Division Biophysics of Macromolecules, DKFZ Heidelberg
2) Department of Physics, University Milano-Bicocca
3) Interdisciplinary Center for Scientific Computation (IWR), University of Heidelberg
[email protected]
A fundamental paradigm in biology is that molecular interactions are driven by random motion.
However, in contrast to an equilibrium system where random motion is purely thermal, biological
systems are in a steady state, and active processes such as molecular motors and active transport will also
contribute to the random motion of cellular constituents. In particular, the viscoelastic properties of the
cell nucleus and their connection with gene function have become a focus of interest recently [1]. We are
trying to understand the interplay of thermal and active contributions by analyzing the random motion
using advanced single molecule imaging methods.
Fluorescence correlation spectroscopy (FCS) is a typical microscopic technique for characterizing
intracellular mobility in the focus of a laser beam. It offers fast time resolution but so far has been limited
to single-point measurements. Although we have collected protein mobility maps by point-to-point FCS
[2], this is extremely time-consuming and impractical for live cell measurements.
SPIM-FCS is a new method that combines the speed of FCS with the possibility of acquiring mobility data
on an entire two-dimensional cross-sections of cells [3], providing diffusion coefficients, flow velocities
and concentrations in an imaging mode. Two-color fluorescence crosscorrelation spectroscopy (SPIMFCCS) also allows imaging of molecular interactions [4,5].
Here we present new data on the dynamics of interphase chromatin, measured by FCS analysis of fast
image series from light sheet microscopy of fluorescently labeled histones in interphase cell nuclei. We
show that the random motion of the chromatin network is subdiffusive, that is, the effective diffusion
coefficient decreases for slow time scales. In a lamin A knockout cell line, on the other hand, the diffusion
changes to normal. While a similar effect has been observed by single particle tracking of telomers [1],
here we have established that this is a property of the entire chromatin network. The correlated motion
of lamin A and histones measured by imaging-FCCS shows unambiguously the cross-linking of the
chromatin network by lamin A. Spatial correlation analysis of chromatin motion further suggests that in
lamin A-containing cells the random motion of chromatin is correlated over distances of several µm. Our
conclusion is that lamin A plays a central role for determining the elasticity of the chromatin network and
to help maintaining local ordering of interphase chromosomes.
37
ELMI meeting
european
light microscopy
initiative
16th international
ELMI meeting
elmi
invited speaker
Wednesday, 25th May
To separate the effects of random motion and active processes, we compared mobility measurements of
cells in the presence and absence of agents that influence active motion, e.g. sodium azide that causes ATP
depletion, or blebbistatin that inhibits myosin motors. Our first data show a significant influence of these
agents on the characteristics of diffusional motion, indicating that active processes determine random
intracellular motion beyond the effect of thermal motion alone.
References
[1] I. Bronstein, Y. Israel, E. Kepten, S. Mai, Y. Shav-Tal, E. Barkai, Y. Garini, Phys Rev Lett 2009, 103, 018102.
[2] N. Dross, C. Spriet, M. Zwerger, G. Muller, W. Waldeck, J. Langowski, PLoS One 2009, 4, e5041.
[3] T. Wohland, X. Shi, J. Sankaran, E. H. Stelzer, Opt Express 2010, 18, 10627-10641.
[4] J. W. Krieger, A. P. Singh, C. S. Garbe, T. Wohland, J. Langowski, Opt Express 2014, 22, 2358-2375.
[5] J. W. Krieger, A. P. Singh, N. Bag, C. S. Garbe, T. E. Saunders, J. Langowski and T. Wohland, Nat Protoc 2015, 10, 1948-1974.
38
invited speaker
16th international
ELMI meeting
Wednesday, 25th May
european
light microscopy
initiative
elmi
international
Chromatin breathing evidenced by fast time16th
lapse
FLIM
ELMI meeting
regulates accessibility of TAFII250-double bromodomain to
acetylated histone H4
Chromatin breathing evidenced by fast time lapse FLIM regulates
1
2
accessibility
of TAF
250-double
bromodomain
to 3acetylated
histone1 H4
Nicolas Audugé
, SergiIIPadilla-Parra
, Nicolas
Borghi1, Marc Tramier
, Maïté Coppey-Moisan
1
2
1
3
Nicolas
Audugé
, Sergi
Padilla-Parra
1) Institut
Jacques
Monod,
Paris, France , Nicolas Borghi , Marc Tramier and Maïté CoppeyMoisan1
2) Wellcome Trust Centre for Human Genetics, University of Oxford, UK
3) Institut
Génétique
et Développement,
Rennes,Trust
France
1) Institut
JacquesdeMonod,
Paris,
France 2) Wellcome
Centre for Human Genetics, University of Oxford, UK
3) Institut
de Génétique et Développement, Rennes, France
[email protected]
[email protected]
Genome accessibility
to proteic factors is an important issue for the regulation of genomic and
epigenetic activities. Due to nucleosomal DNA compaction and higher order chromatin structures,
Genome accessibility to proteic factors is an important issue for the regulation of genomic and
specific sites are made accessible for the recruitment of regulatory factors and transcriptional machinery
epigenetic activities. Due to nucleosomal DNA compaction and higher order chromatin
through
dynamical
in chromatin
structure.
However,
how chromatin
dynamical
properties
structures,
specific
siteschanges
are made
accessible
for the
recruitment
of regulatory
factors
and
transcriptional
machinery
throughindynamical
in chromatin
structure.
how
regulate genome
accessibility
living cells ischanges
poorly understood.
Here,
we take However,
advantage of
the
chromatin
dynamical sensitivity
propertiesof EGFP
regulate
genomelifetime
accessibility
living
is poorly
microenvironment
fluorescence
measuredinby fast
timecells
lapse Fluorescence
understood. Here, we take advantage of the microenvironment sensitivity of EGFP fluorescence
Lifetime Imaging Microscopy (FLIM) to map dynamical chromatin changes and accessibility of the
lifetime measured by fast time lapse Fluorescence Lifetime Imaging Microscopy (FLIM) to map
doublechromatin
bromodomain
(dBD)and
module
of humanof
TAFII250
to acetylated
lysines of(dBD)
EGFP-Histone
dynamical
changes
accessibility
the double
bromodomain
moduleH4ofin
acetylated
EGFP-Histone
H4“breathing
in the nucleus
cells. We
humantheTAF
nucleus
cells. We lysines
reveal theofexistence
of distinct
modes”ofof live
sub-micron
sized
II250 oftolive
revealchromatin
the existence
of unrestrained
distinct “breathing
modes”
of sub-micron
sizedbychromatin
domains:
and restrained
chromatin,
characterized
high or low domains:
fluctuation
unrestrained and restrained chromatin, characterized by high or low fluctuation amplitudes of
amplitudes of EGFP-H4 fluorescence lifetime, respectively. Moreover, we show that accessibility of
EGFP-H4 fluorescence lifetime, respectively. Moreover, we show that accessibility of dBDdBD-mCherry
to acetylated
EGFP-H4
and transient
their transient
interaction
revealed
FörtserResonant
Resonant
mCherry
to acetylated
EGFP-H4
and their
interaction
revealed
by by
Förtser
Energy
Transfer
(FRET)
amplitudeofofthese
thesefluctuations.
fluctuations.
results
support
a
Energy
Transfer
(FRET)depends
depends on
on the amplitude
OurOur
results
support
a model
modelbybywhich
which
restrained
chromatin
dynamics,
rather
than
a
stable
condensed
state,
impedes
restrained chromatin dynamics, rather than a stable condensed state, impedes mCherry-dBD
mCherry-dBD access to its chromatin recognition site, the access requiring unrestrained
access
to
its
chromatin
requiringbackground
unrestrainedstate.
chromatin dynamics, which
chromatin dynamics,
whichrecognition
can occur site,
eventhe
in access
a condensed
can occur even in a condensed background state.
spatio-temporal
map ofmap
interactions
between
spatio-temporal
of interactions
betweenmCherry-dBD
mCherry-dBD
and hyperacetylated
H4H4
ininenergetized
depleted
cells (Snapshot
ofFRET)
time
and hyperacetylated
energetized ororATPATP
depleted
cells (Snapshot
of time lapse
lapse FRET)
39
ELMI meeting
european
light microscopy
initiative
elmi
16th international
ELMI meeting
invited speaker
Wednesday, 25th May
Probing neural circuits with shaped light
Na Ji1
1) Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia, United States
[email protected]
To understand computation in the brain, one needs to understand the input-output relationships for
neural circuits and the anatomical and functional relationships between individual neurons therein.
Optical microscopy has emerged as an ideal tool in this quest, as it is capable of recording the activity of
neurons distributed over millimeter dimensions with sub-micron spatial resolution. I will describe how
we use concepts in astronomy and optics to develop next-generation microscopy methods for imaging
neural circuits at higher resolution, greater depth, and faster speed. By shaping the wavefront of the
light, we have achieved synapse-level spatial resolution through the entire depth of primary visual
cortex, optimized microendoscopes for imaging deeply buried nuclei, and developed a video-rate (30
Hz) volumetric imaging method. We apply these methods to understanding neural circuits, using the
mouse primary visual cortex as our model system.
40
16th international
ELMI meeting
elmi
Multimodal and multidimensional live-cell imaging of the assem
Wednesday, 25th May
nuclear bodies
16th international
Multimodal and multidimensional
live-cell imaging
of the
Christian Hoischen1, Shamci Monajembashi1ELMI
& Peter
Hemmerich1
meeting
assembly of PML nuclear bodies
european
light microscopy
initiative
1) Leibniz Institute for Aging Research - Fritz-Lipman-Institute, Jena, Germany, 2)
Christian Hoischen1, Shamci Monajembashi1, Peter Hemmerich1
[email protected]
1) Leibniz Institute for Aging Research - Fritz-Lipman-Institute, Jena, Germany
[email protected]
The tumor suppressor PML gene product is the basic protein building unit
structurePML
in gene
mammalian
named
PMLunit
nuclear
body (PML-NB).
The tumor suppressor
product is cells
the basic
proteinthe
building
of a subnuclear
structure in PMLdynamic
multi-protein
complexes
which
function
as
hubs
for
nuclear signa
mammalian cells named the PML nuclear body (PML-NB). PML-NBs are focal yet dynamic multi-protein
pathways involved in genome maintenance. The biochemical role(s) of PML b
complexes which function as hubs for nuclear signaling transduction pathways involved in genome
this function however is not known so far.
maintenance. The biochemical role(s) of PML bodies underlying this function however is not known so far.
We have employed a range of fluoresecnce fluctuation microscopy (FFM) tech
We have employed
a range of fluoresecnce
fluctuation
microscopy
(FFM)cells,
techniques
to unravel
the mechanisms
of PML-NB
assembly
in living
including
UV the
microbeam
mechanisms ofFRAP,
PML-NB assembly
in
living
cells,
including
UV
microbeam,
optical
tweezer,
FRAP,
FCS,
FCCS,
FCS, FCCS, RICS, and FRET. In addition, STED superresolution
micr
the architecture
ofwas
PML-NBs.
Weunravel
will report
on the results w
RICS, and FRET.toIn further
addition, unravel
STED superresolution
microscopy
used to further
the architecture
theweassembly
mechanism(s)
biochemical
function(s) of
of PML-NBs. Wethe
willpast
reportyears
on theon
results
collected over
the past years onand
the assembly
mechanism(s)
state-of-the-art
FFM
methods.
and biochemical function(s) of PML-NBs using state-of-the-art FFM methods.
This project highlights the needfulness of sophistated light microscopy ap
This project highlights the needfulness of sophistated light microscopy applications in the elucidation of
elucidation of multi-protein complexes in human cells.
multi-protein complexes in human cells.
References:
References: (1) Münch S. et al. (2014) MOL. CELL. BIOL. 34. 1733-1746
(1) Münch S.(2)
et al.Ulbricht
(2014) MOL.
34. 1733-1746
T. CELL.
et al.BIOL.
(2012)
J. CELL BIOL. 199. 49-63
Hemmerich
(2011)
CHROMOSOME RES. 19. 131-151
(2) Ulbricht T.(3)
et al.
(2012) J. CELLP.
BIOL.
199. 49-63
(3) Hemmerich P. (2011) CHROMOSOME RES. 19. 131-151
41
ELMI meeting
european
light microscopy
initiative
elmi
16th international
ELMI meeting
Wednesday, 25th May
DNA replication factories form a dynamic compartment in cell
nuclei.
Hannsa Sas-Nowosielska, Maciej Krupa, Tytus Bernas
Nencki Institute of Experimental Biology, Polish Academy of Sciences, Warsaw, Poland
[email protected]
DNA replication is a fundamental process in cell proliferation and DNA damage control, regulated by
sequential assembly and reorganization of diverse protein complexes. PCNA (proliferating cell nuclear
antigen), a DNA sliding clamp, recruits these complexes to replication forks. It postulated that active
forks form clusters which forks correspond to nuclear regions of high PCNA concentration, observable
with optical microscopy. Biochemical models predict that PCNA is stably bound in the factories during
replication and released upon completion of this process. We verify these predictions with imaging of
replicating nuclei in living cells. We use FCS as internal calibration standard to construct nuclear map of
absolute PCNA concentration, in replication factories and nucleoplasm. Our data indicate that the number
of PCNA molecules in the former nuclear compartment exceeds, by an order of magnitude, the number
of potential DNA replication forks. We apply FRAP to demonstrate that a fraction of PCNA is rapidly
exchanged between the factories and nucleoplasm. Moreover, this exchange is coupled to translocation
of factories within nucleus. On the other hand, FLIP experiments indicate that the release of PCNA occurs
at the same time scale as the movement of the protein through nucleoplasm. In addition, the rate of
PCNA release from factories undergoes periodic changes. These data suggest that PCNA dynamics in the
replication factories is not directly determined by involvement of this protein in replication forks. We apply
3D time-lapse imaging to demonstrate replication factories move in the nuclear space in constrained
manner. This notion corresponds to the structure of factories visualized with SIM microscopy. Furthermore,
we apply N&B to demonstrate that the PCNA dynamics in the factories comprises a range of components.
Their characteristic times fall between the PCNA exchange/release times and mobility times of the
factories. Information from the range of microscopy techniques is combined to propose a dynamic model
of replication factories.
42
invited speaker
16th international
ELMI meeting
Wednesday, 25th May
Euro-BioImaging
Antje Keppler
european
light microscopy
initiative
elmi
16th international
ELMI meeting
Euro-BioImaging Interim Phase Secretariat, EMBL, Heidelberg, Germany
[email protected]
The European Research Infrastructure for Imaging Technologies in Biological and Biomedical Sciences
(Euro-BioImaging, EuBI or EuBI ERIC) will provide open physical user access to a broad range of stateof-the-art technologies in biological and biomedical imaging for life scientists. It will offer image data
support and training for infrastructure users and providers, as well as continuously evaluating and
acquiring new imaging technologies to ensure the sustained delivery of cutting-edge services. The
EuBI ERIC will consist of a set of complementary and strongly interlinked, geographically distributed
Nodes (specialised imaging facilities) to grant access to scientists from all EU Member States and
beyond. A strong supporting and coordinating entity, the EuBI Hub, will empower the infrastructure.
The Hub will provide the virtual access entry point from which users will be directed to their desired
imaging technology as served by the respective EuBI Nodes. The Hub will coordinate dedicated data
management and training activities tailored to the needs of users of the imaging infrastructure. In
the EuBI Interim Board, 15 member countries and EMBL are currently finalizing the statutes for the
EuBI ERIC, and together they have identified the EuBI Hub and 1st generation of 29 Node Candidates.
Together with Finland as statutory seat of the ERIC, and Italy as site for medical imaging coordination,
EMBL will host the EuBI Hub for coordinating open user access to and training in biological imaging
technologies, as well as image data repositories and tools. EMBL together with the two Hub partners
and the Node Candidates now prepare for Interim Operation to start in May 2016 and to welcome the
first EuBI pilot users for physical access to their imaging platforms.
On behalf of EuBI, EMBL is coordinating the EC H2020 funded Global BioImaging Project, which
allows EuBI to extent its international collaborations and establish common services for core facility
staff together with imaging infrastructure partners in Australia, Argentina, India, Japan, South Africa
and USA.
43
ELMI meeting
european
light microscopy
initiative
16th international
ELMI meeting
elmi
invited speaker
Thursday, 26th May
Imaging mitochondrial and sub-mitochondrial localization of
proteins
Kyle Salka1, Shivaprasad Bhuvanendran1, Kassandra Wilson1, Kristin Rainey2, George H. Patterson2,
Jonathan Boyd3, and Anamaris M. Colberg-Poley1,4,5, Jyoti K. Jaiswal1,4
1) Center for Genetic Medicine Research, Children’s National Health System, 111 Michigan Ave, NW Washington, DC
20010 USA
2) Section on Biophotonics, National Institute of Biomedical Imaging and Bioengineering, National Institutes of
Health, Bethesda, MD 20892 USA
3) Life Science Division, Leica Microsystems, Inc., 1700 Leider Lane, Buffalo Grove, IL 60089, USA.
4) Department of Integrative Systems Biology, and of Pediatrics, George Washington University School of Medicine
and Health Sciences, Washington, DC 20037 USA
5) Departments of Biochemistry & Molecular Medicine, and of Microbiology, Immunology & Tropical Medicine,
George Washington University School of Medicine and Health Sciences, Washington, DC 20037 USA
[email protected]
Proteins regulate cellular function by the virtue of their subcellular localization, nanoscale
organization, and interactions with other proteins. Precise monitoring of these events in individual cells
and subcellular compartments requires the use of high resolution microscopic imaging. Mitochondria
function by organizing their membrane proteins at the inner, outer mitochondrial membrane (OMM),
and at sites of contact between mitochondria and other organelle membranes. Using a combination
of confocal, superresolution imaging, and fluorescence lifetime imaging microscopy (FLIM), we have
imaged protein localization at the OMM and at the sites where mitochondria forms contact with the
Endoplasmic Reticulum. Superresolution imaging approach allowed visualizing protein trafficking to
and organization at the OMM, detecting nanoscale clusters at the OMM. While use of FLIM offered
insights into protein interactions that facilitate trafficking and organization of the proteins at the
OMM. Additionally, we successfully employed FLIM to monitor homo-FRET interactions in live cells.
Monitoring homo-FRET by FLIM has enabled studies that require imaging diffraction limited transient
events in live cells. We will discuss this use of FLIM, and the biological and technical insights offered by
these studies regarding the hierarchical subcellular organization of mitochondrial membrane proteins
in situ.
44
invited speaker
16th international
ELMI meeting
Thursday, 26th May
european
light microscopy
initiative
elmi
16th international
The Open Microscopy Environment: Open Source
Image
ELMI meeting
Informatics for the Biological Sciences
Jason R. Swedlow1,2 and the OME Consortium3
1) Centre for Gene Regulation & Expression, University of Dundee, Dundee, Scotland, UK.
2) Glencoe Software, Inc. Seattle, WA, USA
3) http://www.openmicroscopy.org/site/about/who-ome
[email protected]
Despite significant advances in cell and tissue imaging instrumentation and analysis algorithms,
major informatics challenges remain unsolved: file formats are proprietary, facilities to store, analyze
and query numerical data or analysis results are not routinely available, integration of new algorithms
into proprietary packages is difficult at best, and standards for sharing image data and results are
lacking. We have developed an open-source software framework to address these limitations called
the Open Microscopy Environment (http://openmicroscopy.org). OME has three components—an
open data model for biological imaging, standardised file formats and software libraries for data file
conversion and software tools for image data management and analysis.
The OME Data Model (http://openmicroscopy.org/site/support/ome-model/) provides a common
specification for scientific image data and has recently been updated to more fully support fluorescence
filter sets, the requirement for unique identifiers, screening experiments using multi-well plates.
The OME-TIFF file format (http://openmicroscopy.org/site/support/ome-model/ome-tiff) and the
Bio-Formats file format library (http://openmicroscopy.org/site/products/bio-formats) are easy-touse tools for converting data from proprietary file formats. These resources enable access to data by
different processing and visualization applications, sharing of data between scientific collaborators
and interoperability with third-party tools like ImageJ, Fiji, CellProfiler, Matlab, and several others.
The Java-based OMERO platform (http://openmicroscopy.org/site/products/omero) includes server
and client applications that combine an image metadata database, a binary image data repository
and visualization and analysis by remote access. The current stable release of OMERO (OMERO 5.2;
http://downloads.openmicroscopy.org) includes a single mechanism for accessing image data of all
types-- regardless of original file format-- via Java, C/C++ and Python and a variety of applications
and environments (e.g., ImageJ, Matlab and CellProfiler). OMERO includes SSL-based secure access,
distributed compute facility, filesystem access for OMERO clients, and a scripting facility for image
processing. An open script repository allows users to share scripts with one another. A permissions
system controls access to data within OMERO and enables sharing of data with users in a specific
group or even publishing of image data to the worldwide community. OMERO 5 includes updates
and resources that specifically support large datasets that appear in digital pathology, high content
45
ELMI meeting
european
light microscopy
initiative
elmi
16th international
ELMI meeting
invited speaker
Thursday, 26th May
screening and long term timelapse imaging. Importing these large datasets is fast, and data are stored
in their original file format and can be accessed by 3rd party software. Several tools that use OMERO
are now released by the OME Consortium: FLIMfit, a fluorescence lifetime analysis module; u-track,
an object tracking module; WND-CHARM, for image-based search; an automatic image tagging
application and a biobanking application (http://www.openmicroscopy.org/site/products/partner).
OMERO and Bio-Formats run the JCB DataViewer (http://jcb-dataviewer.rupress.org/), the world’s
first on-line scientific image publishing system and are used to publish multidimensional images
in the EMDataBank (http://emdatabank.org/), the IMPC (http://mousephenotype.org), the EuroBioImaging IDR (http://idr-demo.openmicroscopy.org). They also power several large institutional
image data repositories (e.g., http://odr.stowers.org and http://lincs.hms.harvard.edu/).
All OME software is available at http://openmicroscopy.org.
46
invited speaker
Thursday, 26th May
16th international
ELMI meeting
european
light microscopy
initiative
elmi
16th international
Open software tools for microscopy image processing
ELMI meeting
Daniel Sage
Biomedical Imaging Group (BIG), Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland
[email protected]
Recent advances in microscope technology combined with new digital tools now provide outstanding
images (3D, time-lapse, multichannel, fluorescence), allowing us to address fundamental questions in
developmental biology, molecular biology and neuroscience. The analysis of this unprecedented flow of
imaging data requires the development of sophisticated software packages to numerically reconstruct
images and to automatically perform segmentation, quantification and tracking of structures of interest.
This has led to the emergence of a new field of research, “bioimage informatics,” which aims to develop
computational procedures to process, analyze, and visualize images coming from various light microscopy
techniques.
Here, we report our experience in the development of open-source software tools. These tools are
written as Java plugins for the popular software suites: ImageJ, Fiji or Icy. In particular, we are focusing
on the reconstruction of images from incomplete data measurements. This is often a challenging imageprocessing task in terms of algorithmic tuning and computational runtime. In this context, we show the
importance of carefully identifying the image formation model in properly designing algorithms. We
describe bioimaging applications such as restoration of details with deconvolution methods, recovery
of shape from phase images, segmentation of cellular compartments from the photobleaching decay,
reconstruction of nanoscale images by applying super-resolution localization microscopy, and generation
of theoretical point-spread functions.
Often overlooked in software development, the validation and usability are finally what counts for the
end-users. In this respect, we report our effort to propose reference datasets and quantitative benchmarks
of software through the organization of Grand Challenges.
47
ELMI meeting
european
light microscopy
initiative
elmi
16th international
ELMI meeting
Event
Driven Automated Microscopy
Thursday, 26th May
1
EventPike
Driven
Jeremy
, JoanaAutomated
Grah1,2, PatriceMicroscopy
Mascalchi1, Stefanie Reichelt1
1
1) Light
Research
UK1, Cambridge
Institute,
University of Cambridge, CB20RE
Jeremy
Pike1,microscopy,
Joana Grah1,2Cancer
, Patrice
Mascalchi
Stefanie Reichelt
2) Department of Applied Mathematics and Theoretical Physics, University of Cambridge, CB30WA
1) Light microscopy, Cancer Research UK Cambridge Institute, University of Cambridge, CB20RE
[email protected]
2) Department of Applied Mathematics and Theoretical Physics, University of Cambridge, CB30WA
[email protected]
Automation
of the image acquisition process can greatly enhance the scope and scale of a
microscopy
an event
driven
approach
imageenhance
analysis
incorporated
into the
Automationstudy.
of the In
image
acquisition
process
can greatly
theprotocols
scope andare
scale
of a microscopy
acquisition
and
run
on
the
data
as
it
is
acquired.
The
results
of
the
analysis
are
used
as
a
feedback
study. In an event driven approach image analysis protocols are incorporated into the acquisition and run
mechanism to control the subsequent stages of the acquisition.
on the data as it is acquired. The results of the analysis are used as a feedback mechanism to control the
subsequent
the acquisition.
Using
the stages
Zeiss ofOpen
Application Development (OAD) environment within Matlab we are
Using the Zeiss
Openapplications
Application with
Development
(OAD) environment
Matlab work
we arefrom
developing
developing
several
this approach.
This buildswithin
on previous
our group
where
automated
confocal
microscopy
was
used
to
detect
and
image
intestinal
crypt
bottoms.
several applications with this approach. This builds on previous work from our group where automated
confocal microscopy was used to detect and image intestinal crypt bottoms.
In the first application a (low frame-rate) time-lapse phase contrast experiment is used to detect
In the cells
first application
a (low
phasemitotic
contrastevent
experiment
is used
to detectin 3D
mitotic
in real time.
The frame-rate)
duration oftime-lapse
each detected
can then
be imaged
mitotic
cells
in
real
time.
The
duration
of
each
detected
mitotic
event
can
then
be
imaged
in
3D
with
with high frame-rate and multiple fluorescent channels. In live cell microscopy thehigh
sample
frame-rate
and multiple
channels.ofInlight.
live cellThis
microscopy
thetargets
samplethe
should
bebudget
exposedtotoevents
a
should
be exposed
to afluorescent
minimal amount
approach
light
minimal
amount
light. This
approach
thesample
light budget
to events
of interestexperiments
thus greatly reducing
of
interest
thus of
greatly
reducing
thetargets
overall
exposure.
Therefore
can be run
the longer
overall sample
exposure.
Therefore
be run for
longer and events of interest can be
for
and events
of interest
canexperiments
be imaged can
in greater
detail.
imaged in greater detail.
In the second application whole slides can be imaged at low resolution and analyzed to detect the
In the second application whole slides can be imaged at low resolution and analyzed to detect
location of metaphase spreads. Each spread can then be imaged at high resolution and in 3D. An
the
locationapproach
of metaphase
spreads. Each as
spread
then beand
imaged
at high
resolution
and in 3D.
An and
automated
is advantageous
it is acan
lengthy
tedious
process
to manually
locate
automated
approach
is advantageous
as it is abias
lengthy
and tedious
process
to manually
image
spreads.
Moreover
user selection
is removed
from
the process.
locate and image
spreads. Moreover user selection bias is removed from the process.
2
Figure:
fromaa1cm2
1cmtile
tile
Scan
using
a 20X,
air objective
(left). spreads
Detected
Figure:AAsingle
single tile
tile from
Scan
using
a 20X,
0.4NA0.4NA
air objective
(left). Detected
arespreads
are
imaged
in
high
resolution
with
a
100X,
1.4NA
oil
objective
(right).
imaged in high resolution with a 100X, 1.4NA oil objective (right).
48
16th international
ELMI meeting
Thursday, 26th May
european
light microscopy
initiative
elmi
international
Reginam occidere nolite timere bonum est si16th
omnes
ELMI meeting
consentiunt ego non contradico
Peter Horvath1, 2
1) Institute for Molecular Medicine Finland, University of Helsinki, Finland
2) Synthetic and Systems Biology Unit, BRC HAS, Szeged, Hungary
[email protected]
In this talk I will give an overview of the computational steps in the analysis of a single cell-based
high-content screen. First, I will present a novel microscopic image correction method designed to
eliminate vignetting and uneven background effects which, left uncorrected, corrupt intensity-based
measurements. A novel image analysis method will be presented to reconstruct label-free microscopic
images and make DIC microscopy quantitative. I will discuss the Advanced Cell Classifier (ACC) (www.
cellclassifier.org), a software tool capable of identifying cellular phenotypes based on features extracted
from the image. It provides an interface for a user to efficiently train machine learning methods to predict
various phenotypes. We developed the Suggest a Learner (SALT) toolbox, which selects the optimal
machine learning algorithm and parameters for a particular classification problem. For cases where
discrete cell-based decisions are not suitable, we propose a method to use multi-parametric regression
to analyze continuous biological phenomena. To improve the learning speed and accuracy, we recently
developed an active learning scheme which automatically selects the most informative cell samples.
Lastly a new initiative the EUCAI (Eurpoean Cell-based Assays Interest Group) will be shortly presented.
49
ELMI meeting
european
light microscopy
initiative
16th international
ELMI meeting
elmi
invited speaker
Thursday, 26th May
Efficient multicolor two-photon imaging of endogenous
fluorophores in live tissues by wavelength mixing
Chiara Stringari1, Lamiae Abdeladim1, Willy Supatto1, Ana-Maria Pena2, Sébastien Brizion2, Jean-Baptiste
Galey2, Renaud Legouis3, Emmanuel Beaurepaire1
1) Laboratory for Optics and Biosciences, Ecole polytechnique, CNRS, INSERM, Université Paris-Saclay, 91128
Palaiseau cedex, France.
2) L'Oréal Research and Innovation, 93600 Aulnay sous Bois, France
3) Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Univ. Paris-Sud, Université Paris-Saclay, 91198
Gif-sur-Yvette, France.
[email protected]
Two-photon imaging of endogenous fluorescence can provide important physiological and metabolic
information from intact tissues in a label-free and non-invasive way. However imaging of multiple intrinsic
fluorophores, such as NADH, FAD, retinoids and porphyrins in living systems is generally hampered by
sequential multi-wavelength excitation resulting in long acquisition times and motion artifacts.
We report on efficient and simultaneous multicolor two-photon excitation of endogenous fluorophores
with absorption spectra spanning the 700-1040nm range using wavelength mixing. By using two
synchronized pulse trains at two different wavelengths, we generate an additional “virtual” two-photon
excitation wavelength, and achieve simultaneous excitation of blue, green and red fluorophores.
Our method permits to implement fast and reliable simultaneous imaging of the metabolic coenzymes
NADH and FAD, overcoming the difficulties associated to their difference in absorption spectra and disparity
in concentration. We achieve efficient ratiometric redox imaging and simultaneous efficient two-photon
fluorescence lifetime imaging (FLIM) of NADH and FAD in living tissues. We measure lifetime gradients of
NADH and FAD associated to different cellular metabolic and differentiation states in reconstructed human
skin and in live C. elegans worms.
50
invited speaker
Thursday, 26th May
16th international
ELMI meeting
european
light microscopy
initiative
elmi
16thspectral
international
Raman Microspectroscopy a powerful tool for
ELMI meeting
histopathology
Michael Schmitt1, Jürgen Popp1,2
1) Institute of Physical Chemistry & Abbe-Center of Photonics, Friedrich-Schiller University Jena, Helmholtzweg 4,
D-07743 Jena, Germany
2) Leibniz Institute of Photonic Technology Albert-Einstein-Str. 9, D-07745 Jena, Germany
[email protected]
During the last years Raman microspectroscopy has been recognized as an extremely powerful tool
for bioanalytical and biomedical applications because the method provides molecular fingerprint
information of the molecular structure and biochemical composition of cells and tissues without
external markers, which is crucial for a non-invasive histopathologic examination of tissue. In this
contribution, we will focus on highlighting the potential of linear and non-linear Raman microscopic
approaches as a sensitive and selective tool to supplement routine pathological diagnostics.
First, it will be shown that the processing of chemically specific Raman-maps of biopsy specimen via
mathematical approaches enables an objective evaluation of the tissue samples for an early disease
diagnosis. The potential to couple the Raman system via optical fibers to the point of measurements
has enabled also in-vivo Raman studies, i.e. Raman endospectroscopy. We will introduce novel Raman
fiber probes for in-vivo tissue screening.
Finally, we present the combination of Raman approaches with other imaging techniques to a
multimodal imaging approach in order to further improve the diagnostic result of Raman spectroscopy.
In this context, we introduce among others the combination of non-linear Raman techniques as CARS
(coherent anti-Stokes Raman spectroscopy) with imaging approaches of similar image acquisition
times like two-photon excited autofluorescence (TPEF) and second harmonic generation (SHG). We
will present a compact CARS/SHG/TPEF multimodal nonlinear microscope for use in clinics. It will be
shown that CARS/TPEF/SHG imaging in combination with advanced image processing algorithms
offers great potential to augment standard intraoperative clinical assessment with multimodal images
to highlight functional activity and tumor boundaries.
Overall, the presented examples show the potential of Raman approaches to provide a pathologist
with adequate support in the form of clinically-relevant information under both ex vivo and in vivo
conditions.
Acknowledgements
Financial support of the EU, the ”Thüringer Kultusministerium”, the ”Thüringer Aufbaubank”, the
Federal Ministryof Education and Research, Germany (BMBF), the German Science Foundation, the
Fonds der Chemischen Industrie and the Carl-Zeiss Foundation are greatly acknowledged.
51
ELMI meeting
european
light microscopy
initiative
elmi
16th international
ELMI meeting
invited speaker
Thursday, 26th May
Digital adaptive optics for achieving space invarient resolution
in optical coherence tomography
Abhishek Kumar*1, Laurin Ginner1, Wolfgang Drexler1, Rainer A. Leitgeb1
1) Center of Medical Physics and Biomedical Engineering, Medical University of Vienna, Währinger Gürtel 18-20
A-1090 Vienna, Austria
[email protected]
Optical coherence tomography (OCT) has been successfully combined with adaptive optics (AO) in
order to achieve high lateral resolution in combination with the high axial resolution. Visualization
of cone photoreceptors in 3-D has been successfully demonstrated using AO-OCT 1. OCT, being an
interferometric imaging technique, can provide access to phase information which can be exploited
by digital adaptive optics (DAO) techniques to correct optical aberration in the post-processing step
to obtain diffraction-limited space invariant lateral resolution throughout the image volume and
provide the possibility of obtaining depth resolved wavefront aberration measurement 2; 3. Thus
the need for hardware based AO, which requires Shack Hartmann wavefront sensor (SH WFS) and
deformable mirror, can be eliminated which in turn can reduce the system complexity and economical
cost. Kumar et al. have demonstrated a novel sub-aperture based DAO technique, which is the digital
equivalent of SH WFS 4. In this DAO technique the aperture at the pupil plane is digitally segmented
into sub-apertures and the images of the sub-apertures are cross-correlated with the image of central
reference sub-aperture to detect relative shifts in the presence of wavefront aberration. Using the
shift information, local slopes of the wavefront error can be calculated from which phase error can be
estimated analytically in a single step 4. The advantage of this method is that it is non-iterative and it
does not require a priori knowledge of any system parameters such wavelength, focal length, numerical
aperture (NA) or detector pixel size which may be required in other optimization or forward model
based techniques 4. This method has been extended using region of interest (ROI) based processing to
correct anisotropic aberration across the field of view (FOV) in case of imaging with high NA close to
unity 5.The proof of principle is shown using an iron (III) oxide nano-particle phantom sample imaged
with a fiber-based point scanning spectral domain (SD) OCT setup at a high NA of 0.6 and a limited
DOF of 7 . Sub-micron lateral resolution is obtained over a depth range of 218 , thus achieving DOF
improvement by ~30x 5. The sub-aperture based DAO is applied in the images of living human retina
obtained using a high speed (0.5 MHz A-scan rate) line field (LF) OCT system to drastically enhance the
resolution of the cone photoreceptors without the use of any extra AO hardware, as shown in Fig. 1.
52
invited speaker
Thursday, 26th May
16th international
ELMI meeting
european
light microscopy
initiative
elmi
3. References
Liu, Y.-Z., Shemonski, N.D., Adie, S.G., Ahmad, A., Bower,
A.J., Carney, P.S., and
16th international
meeting
S.A.
(2014).
Computed
interferometric
for25,high-speed
1. Miller,Boppart,
D.T., Kocaoglu,
O.P., Wang,
Q., and
Lee, S. (2011).optical
Adaptive optics
and the eye ELMI
(supertomography
resolution OCT). Eye
321-330.
Center - Hotel Lycium****
Opt
5, Kölcsey
2988-3000.
2. Adie,volumetric
S.G., Graf, B.W.,cellular
Ahmad, A.,imaging.
Carney, P.S.,Biomed
and Boppart,
S.A.Express
(2012). Computational
adaptive optics for broadband
optical interferometric
tomography
of biological
tissue.
Proceedings
of the
National Academy
of Sciences. based digital
4. Kumar,
A., Drexler,
W., and
Leitgeb,
R.A.
(2013).
Subaperture
correlation
3. Liu, Y.-Z.,
Shemonski,
N.D., for
Adie,full
S.G., field
Ahmad,optical
A., Bower,coherence
A.J., Carney, P.S.,
and Boppart, S.A.
(2014).
Computed
adaptive
optics
tomography.
Opt
Express
21,optical
10850interferometric
10866. tomography for high-speed volumetric cellular imaging. Biomed Opt Express 5, 2988-3000.
Kumar, A.,A.,
Drexler,
W., and Leitgeb,
R.A. (2013).
correlation
digitalW.,
adaptive
for full field
optical
5. 4.Kumar,
Kamali,
T., Platzer,
R.,Subaperture
Unterhuber,
A., based
Drexler,
andoptics
Leitgeb,
R.A.
(2015).
coherence
tomography.aberration
Opt Express 21,correction
10850-10866. using region of interest based digital adaptive optics
Anisotropic
5. Kumar,
Kamali, T.,domain
Platzer, R.,OCT.
Unterhuber,
A., Drexler,
and Leitgeb,
(2015). Anisotropic aberration correction
inA.,Fourier
Biomed
OptW.,Express
6, R.A.
1124-1134.
using region of interest based digital adaptive optics in Fourier domain OCT. Biomed Opt Express 6, 1124-1134.
Figure
1: 1:(a)(a)Aberrated
imageofof
photoreceptors
of living
retina by
obtained
by LF OCT
Figure
Aberrated image
photoreceptors
of living
human human
retina obtained
LF OCT system,
system, (b) phase corrected image obtained after applying sub-aperture based DAO
(b) phase corrected
imagephase
obtained
afterinapplying
DAO technique,
(c) estimated
technique,
(c) estimated
error
radians,sub-aperture
(d) plot ofbased
coefficients
of Zernike
polynomials
phase
error
in
radians,
(d)
plot
of
coefficients
of
Zernike
polynomials
show
presence
of
strong
defocus,
show presence of strong defocus, astigmatism and coma in the estimated
wavefront
astigmatism and coma in the estimated wavefront aberration.
aberration.
53
ELMI meeting
european
light microscopy
initiative
16th international
ELMI meeting
elmi
invited speaker
Friday, 27th May
Mapping single neurons in vivo with confined primed
conversion
Periklis (Laki) Pantazis
Laboratory of Nano Bio Imaging, Department of Biosystems Science and Engineering (D-BSSE), Basel, Switzerland
[email protected]
Unraveling the structural organization of neurons can provide fundamental insights into brain
function. However, visualizing neurite morphology in vivo remains difficult due to the high density and
complexity of neural packing in the nervous system. Detailed analysis of neural morphology requires
distinction of closely neighboring, highly intricate cellular structures such as neurites with high
contrast. Green-to-red photoconvertible fluorescent proteins have become powerful tools to optically
highlight molecular and cellular structures for developmental and cell biological studies. Yet, selective
labeling of single cells of interest in vivo has been precluded due to inefficient photoconversion when
using high intensity, pulsed, near infrared laser sources that are commonly applied for achieving
axially confined two photon (2P) fluorescence excitation. Here we describe a novel optical mechanism,
“confined primed conversion”, which employs continuous dual-wave illumination to achieve confined
green-to-red photoconversion of single cells in live zebrafish embryos. Confined primed conversion
exhibits wide applicability and here we specifically elaborate on employing it to analyze neural
morphology of optically targeted single neurons in the developing zebrafish brain.
54
invited speaker
Friday, 27th May
16th international
ELMI meeting
european
light microscopy
initiative
elmi
16th international
Fast 3D functional imaging of neuronal networks
and dendritic
ELMI meeting
spine assemblies in behaving animals
Gergely Szalay1*, Linda Judák1*, Zoltán Szadai1,2, Katalin Ócsai3, Máté Veress4, Tamás Tompa2, Balázs Chiovini1,2,
Pál Maák4, Gergely Katona1,3*, Balázs Rózsa1,2
1) Two-Photon Imaging Center, Institute of Experimental Medicine, Hungarian Academy of Sciences, Budapest,
Hungary
2) The Faculty of Information Technology, Pázmány Péter Catholic University, Budapest, Hungary
3) MTA-PPKE ITK-NAP B – 2p measurement technology group, The Faculty of Information Technology, Pázmány
Péter Catholic University, Budapest, Hungary 4) Department of Atomic Physics, Budapest University of
Technology and Economics, Budapest, Hungary.
* These authors contributed equally to this work.
[email protected]
Understanding brain function requires novel imaging methods such as 3D random-access point
scanning that can simultaneously read out neural activity on both the dendritic and somatic scales.
Our 3D AO scanning method can increase measurement speed and signal-to-noise ratio by up to
6-9 orders of magnitude, but suffers from one main disadvantage: fluorescence information is lost
during brain movement in awake, behaving animals as the amplitude of brain motion is much larger
than the diameter of a single excitation spot. In this work we present a novel fluorescent imaging
technology, 3D drift AO scanning microscopy, which can extend each scanning point to small 3D lines
or surface or volume elements, preserving fluorescence information for fast off-line motion correction.
Our method effectively eliminates in vivo motion artifacts, allowing fast 3D measurement of over 150
dendritic spines with 3D lines, over 100 somata with squares and cubes, or multiple spiny dendritic
segments with surface and volume elements in moving animals. Finally, a four-fold improvement in
total excitation efficiency resulted in a large, about 500µm × 500µm × 650µm, scanning volume with
genetically encoded sensors.
We used the new technology to record activity of inhibitory neurons in the moving brain of behaving
animals. We revealed a new, broadcasted signaling pathway which activates learning mechanism
through the entire neocortex during reward and punishment.
55
ELMI meeting
european
light microscopy
initiative
elmi
16th international
ELMI meeting
invited speaker
Friday, 27th May
Generation 3 Programmable Array Microscope (PAM) for
Adaptive, high speed, large format optical sectioning
Generation 3 Programmable Array Microscope (PAM) for Adaptive, high
Anthony H. B. de Vries, Nathan Cook, Donna J. Arndt-Jovin, Thomas M. Jovin
speed, large format optical sectioning
LaboratoryH.
of Cellular
Dynamics,
Max Planck
Chemistry,
Göttingen,
Germany
Anthony
B. de Vries,
Nathan
Cook,Institute
DonnaforJ.Biophysical
Arndt-Jovin,
Thomas
M. Jovin
[email protected]
Laboratory of Cellular Dynamics, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany
Keywords: programmable array microscope, digital micro-mirror device, fluorescence imaging,
[email protected]
confocal, living cells
Keywords: programmable array microscope, digital micro-mirror device, fluorescence
We report
on the current
version of the optical sectioning programmable array microscope (PAM)
imaging,
confocal,
living cells
implemented with a digital micro-mirror device (DMD) as a spatial light modulator utilized for both
We
report on
the current
version of
the optical
sectioning
array microscope
fluorescence
excitation
and emission
detection.
The PAM
is basedprogrammable
on structured illumination
[1]. A
(PAM) implemented with a digital micro-mirror device (DMD) as a spatial light modulator
sequence
of
HD
(1920×1080)
binary
patterns
of
excitation
light
is
projected
into
the
focal
plane
of the
utilized for both fluorescence excitation and emission detection. The PAM is based
on
microscope
at
the
18
kHz
binary
frame
rate
of
the
TI
1080p
DMD.
The
resulting
sequence
of
patterned
structured illumination [1]. A sequence of HD (19201080) binary patterns of excitation light
is
projected
the focal
the microscope
the 18images:
kHz binary
frame(ca.
rate“on-focus”)
of the TI
emissions
is into
captured
in a plane
single ofacquisition
as two atdistinct
conjugate
1080p
DMD.
The
resulting
sequence
of
patterned
emissions
is
captured
in
a
single
acquisition
consisting of signals impinging on and deviated from the “on” elements of the DMD, and the nonas two distinct images: conjugate (ca. “on-focus”) consisting of signals impinging on and
conjugatefrom
(ca. “out-of-focus”)
of those
on andand
deviated
from the “off”(ca.
elements.
The sectioned
deviated
the “on” elements
of falling
the DMD,
the non-conjugate
“out-of-focus”)
of
image
is
gained
from
a
weighted
subtraction
of
the
conjugate
and
non-conjugate
images.
those falling on and deviated from the “off” elements. The sectioned image is gained from a
weighted subtraction of the conjugate and non-conjugate images.
This procedure allows for a high duty cycle (typically 30 to 50%) of on-elements in the
excitation patterns and thus functions well with low light intensities, preventing saturation of
Thisfluorophores.
procedure allows
a high duty cycle
(typically
30 tois50%)
of on-elements
in the
excitation
the
The for
corresponding
acquisition
speed
also very
high, limited
only
by the
bandwidth
thefunctions
camera(s)
withpreventing
the current
sCMOSofcamera)
and the
patterns andofthus
well(100
with fps
low full
lightframe
intensities,
saturation
the fluorophores.
optical
power of the
light source
(lasers,
In contrast
the by
static
typical
of
The corresponding
acquisition
speed
is alsoLEDS).
very high,
limited toonly
the patterns
bandwidth
of the
SIM systems, the programmable array allows optimization of the patterns to the sample (duty
camera(s)
fpssize),
full frame
withas the
currenta sCMOS
camera)
and the optical
power of the
light
cycle
and (100
feature
as well
enabling
wide range
of microscopy
applications,
ranging
sourcepatterned
(lasers, LEDS).
In contrast to(FRAP,
the static
patterns
of SIM systems,
thesuperresolution
programmable
from
photobleaching,
FLIP)
and typical
photoactivation,
spatial
(SIM,
etc.), optimization
automated adaptive
minimized
light
exposure
[2], feature
and photolithography.
array allows
of the patterns
to the
sample
(duty(MLE)
cycle and
size), as well as
This work is supported by BMBF VIP Grant 03V0441 (iPAM: "Intelligentes"
enabling a wide range of microscopy applications, ranging from patterned photobleaching, (FRAP,
Programmierbares Array Mikroskop).
56
[1] P.A.A. De Beule; A.H.B. de Vries, D.J. Arndt-Jovin, T.M. Jovin, “Generation-3
programmable array microscope (PAM) with digital micro-mirror device (DMD)”. In: SPIE
invited speaker
Friday, 27th May
16th international
ELMI meeting
european
light microscopy
initiative
elmi
FLIP) and photoactivation, spatial superresolution (SIM, etc.), automated
adaptive minimized light
16th international
ELMI meeting
exposure (MLE) [2], and photolithography. This work is supported by BMBF VIP Grant 03V0441 (iPAM:
"Intelligentes" Programmierbares Array Mikroskop).
[1] P.A.A. De Beule; A.H.B. de Vries, D.J. Arndt-Jovin, T.M. Jovin, “Generation-3 programmable array microscope (PAM) with
digital micro-mirror device (DMD)”. In: SPIE PROCEEDINGS. Conference on Emerging Digital Micromirror Device Based
Systems and Applications III, San Francisco, Calif., 2011 (2011).
[2] W. Caarls; B. Rieger, A.H.B. de Vries, D.J. Arndt-Jovin, T.M. Jovin, “Minimizing light exposure with the programmable array
microscope”, J. MICROSCOPY, 241, 101-110 (2010).
57
ELMI meeting
european
light microscopy
initiative
16th international
ELMI meeting
elmi
Friday, 27th May
Light sheet imaging for studying development and evolution of
development
Christopher Schmied1, Michaela Rupprecht1, Peter Steinbach1, Jaroslav Icha1, Mihail Sarov1, Frank Schnorrer2,
Volker Hartenstein3, Pavel Tomancak1
1) Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany
2) Max Planck Institute of Biochemistry, Martinsried, Germany
3) University of California, Department of Molecular Cell and Developmental Biology, Los Angeles, United States
[email protected]
Development of metazoans depends on the precise spatio-temporal expression of genes at specific
levels. Variation of this genetic program produces aberrations, however if beneficial leads to the formation
of new morphologies and thus to the diversity of animal form. In order to understand development and
the evolution of morphology, we need to understand gene expression and its variation. In the past the
expression of developmental genes has been studied mostly in fixed tissues, which is unable to visualize
these highly dynamic processes. We now are employing powerful novel methods to study gene expression
and its variation over the entire embryogenesis, live, in toto and at endogenous levels.
We are using fosmid transgenes carrying the endogenously tagged genes of interest and their regulatory
context, which allows studying their expression at endogenous levels (Sarov et al. 2016). For imaging
the entire Drosophila embryogenesis live and in toto at single cell resolution we are using selective plane
illumination microscopy (SPIM). SPIM allows to image developing organisms with unprecedented
temporal resolution over long periods of time with minimal phototoxicity. In order to process the large
datasets generated by SPIM we developed an automated workflow for processing on a high performance
cluster (Schmied 2015).
These technologies allow us to characterize the global expression patterns of various developmentally
important genes in the whole embryo. Further we are interested in how spatiotemporally regulated gene
expression patterns and levels lead to different morphologies across Drosophila species. To this end we
have compared by SPIM the expression of transcription factors encoded by D. melanogaster fosmids to
their orthologous D. pseudoobscura counterparts by expressing both fosmids in D. melanogaster.
Sarov M., Barz C., Jambor H., Hein M. Y., Schmied C., Suchold D., Stender B., Janosch S., Vikas V. K.J., Krishnan R.T., Aishwarya
K., Ferreira I. R. S., Ejsmont R. K., Finkl K., Hasse S., Kämpfer P, Plewka N., Vinis E., Schloissnig S., Knust E., Hartenstein V., Mann
M., Ramaswami M., Raghavan K.V., Tomancak P. and Schnorrer F. A genome-wide resource for the analysis of gene function and
protein localisation in Drosophila. Elife 2016 Feb 20;5. pii: e12068. doi: 10.7554/eLife.12068.
Schmied C., Steinbach P., Pietzsch T., Preibisch S., Tomancak P. An automated workflow for parallel processing of large multiview
SPIM recordings. Bioinformatics 2015 Dec 1 pii: btv706. [Epub ahead of print]
58
invited speaker
Friday, 27th May
16th international
ELMI meeting
european
light microscopy
initiative
elmi
16th international
Multi-target microscopy by spectrally resolved
fluorescence
Multi-target microscopy by spectrallyELMI
resolved
meeting fluorescence lifetim
lifetime imaging
Ingo Gregor1, Thomas Niehörster2, Anna Löschberger2, Benedikt Krämer3, Felix K
1
2
3
3
2
Ingo GregorJörg
, Thomas
Niehörster2,2,Markus
Anna Löschberger
Enderlein
Sauer1, Benedikt Krämer , Felix Koberling , Jörg Enderlein ,
1
Markus Sauer
1) 3rd Institute of Physics, Georg-August-University, Göttingen, Germany 2) Department of
1) 3rd Institute
of Physics, Georg-August-University,
Göttingen, Würzburg,
Germany Germany 3) PicoQuant GmbH, Berlin, Germ
Biophysics,
Julius-Maximilian-University,
2) Department of Biotechnology & Biophysics, Julius-Maximilian-University, Würzburg, Germany
3) PicoQuant [email protected]
GmbH, Berlin, Germany
[email protected]
The development of single-molecule fluorescence techniques has led to an enorm
The development of single-molecule fluorescence techniques has led to an enormous progress in
quantitative cellular biophysics. However, the precise absolute quantification of
quantitativeorcellular
However, the
quantification
of concentrations,
even biophysics.
relative numbers,
of precise
severalabsolute
proteins
at the same
time within a sing
or even relative
numbers,
of
several
proteins
at
the
same
time
within
a
single
cell
remains
difficult.insight in
difficult. For many biomedical studies, this information can
provide
For many biomedical
studies,
this information
can provide
insight into
intracellular
correlations ofe.g. canc
correlations
of protein
levels. Such
information
is the
key to understand
protein levels.
Such
information
is thestrategies
key to understand
e.g. cancer development
and finding possible
and
finding
possible
for therapeutic
intervention.
strategies for therapeutic intervention.
Wea novel
introduce
to efficiently
identify
fluorophore ratio
We introduce
techniqueato novel
efficientlytechnique
identify fluorophore
ratios in complex
multidimensional
multidimensional
fluorescence
signals.
The
technique
is
based
on pattern m
fluorescence signals. The technique is based on pattern matching using reference fluorescence decay
reference fluorescence decay and spectral signature patterns of individual fluoresc
and spectral signature patterns of individual fluorescent probes. We use pulsed interleaved laser
use pulsed interleaved laser excitation at three different wavelengths to e
excitation atexcitation
three different
wavelengths to ensure efficient excitation of fluorophores. Time-resolved
of fluorophores. Time-resolved detection in up to 32 spectrally di
detection inrecords
up to 32aspectrally
distinct
channels records
a maximum
information present
maximum
of information
present
in the offluorescence
signal.in the
fluorescence signal.
Using theUsing
described
fluorescence
lifetime
imaging microscopy
(sFLIM) wemicroscop
the spectrally
describedresolved
spectrally
resolved
fluorescence
lifetime imaging
visualize simultaneously
to molecules
nine different
molecules
within the
visualize simultaneously
up to nine differentuptarget
within thetarget
same sample.
Exploiting
Exploiting
the emission
sensitivity
of and
fluorescence
emission
spectraonand
lifetime of organ
the sensitivity
of fluorescence
spectra
lifetime of organic
fluorophores
environmental
environmental
demonstrate
fluorescence
imaging
of three d
factors, we on
demonstrate
fluorescencefactors,
imaging we
of three
different target
molecules in U2OS
cells with
molecules
in
U2OS
cells
with
the
same
fluorophore.
Our
results
also
the same fluorophore. Our results also demonstrate that sFLIM can be used for multi-target super-demonstr
can be used for multi-target super-resolution imaging by stimulated emission depl
resolution imaging by stimulated emission depletion (STED).
59
ELMI meeting
european
light microscopy
initiative
16th international
ELMI meeting
elmi
invited speaker
Friday, 27th May
Correlated confocal and super-resolution imaging in brain
circuits by VividSTORM
László Barna1, Barna Dudok1, Vivien Miczán1,2, András Horváth2, Zsófia László1, István Katona1*
1) Momentum Laboratory of Molecular Neurobiology, Institute of Experimental Medicine, Hungarian Academy of
Sciences, Budapest, Hungary
2) Faculty of Information Technology and Bionics, Pázmány Péter Catholic University, Budapest, Hungary
[email protected]
A central goal in life sciences is to unravel how protein localization and density determine the
qualitative and quantitative properties of biological processes. For instance, our knowledge on
the molecular parameters underlying synaptic communication between specific neurons still
remains rather limited due to technical obstacles. In our talk, we will introduce an approach,
which combines whole-cell patch-clamp electrophysiological recording, confocal microscopy and
STORM superresolution imaging, thereby enabling cell-specific integrated analysis of physiological
and anatomical properties with nanoscale molecular imaging at identified synapses. The
experimental protocol can be carried out in a few days and uncovers the localization of synaptic
proteins with excellent sensitivity and specificity in intact brain circuits. To support the correlative
visualization and analysis of confocal and STORM microscopy data, we have prepared new
computational analysis tools, including the freely available open-source software called VividSTORM
( http://katonalab.hu/vividstorm2/ ). We will demonstrate how nanoscale molecular investigations
with VividSTORM in association with physiological and anatomical characterization helped to reveal
robust cannabinoid receptor downregulation upon chronic treatment with THC, the psychoactive
substance in marijuana.
60
invited speaker
16th international
ELMI meeting
Friday, 27th May
european
light microscopy
initiative
elmi
16th international
New and prospective nanocomposites for super-resolution
and
ELMI meeting
single-molecular studies
Alexander Demchenko, Maria Dekaliuk, Kyrylo Pyrshev, Mykola Kaniuk, Ihor Panas
Laboratory of Nanobiotechnologies, Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine,
Kiev, Ukraine
[email protected]
The requirements of high brightness and efficient switching or blinking behavior for efficient
applications in super-resolution and single-molecular microscopy can be realized on the level of
fluorescence nanocomposites. Here we report on successful application of carbon nanoparticles
New and prospective nanocomposites for super-resolution and single(C-dots) in Super-resolution
Optical Fluctuation Imaging (SOFI) microscopy and discuss other
molecular studies
possibilities for increase the brightness of different fluorescence emitters (J-aggregates of cyanine
Alexander Demchenko, Mariia Dekaliuk, Kyrylo Pyrshev, Mykola Kaniuk, Ihor Panas
dyes) and for combining fluorescence with high-resolution electron microscopy (complexes of organic
Laboratory of Nanobiotechnologies, Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine,
dyes with the atoms
of noble and heavy atoms).
Kiev, Ukraine
C-dots possess
a unique hybrid combination of fluorescence properties exhibiting characteristics
[email protected]
of both dye molecules
and semiconductor nanocrystals. They demobstrate two-state transitions
The requirements of high brightness and efficient switching or blinking behavior for efficient
applications
in super-resolution
and single-molecular
microscopy
be realized onfor
the their
level ofutilization in
between emissive
and dark
states [1]. These
findings open
up newcanpossibilities
fluorescence nanocomposites. Here we report on successful application of carbon nanoparticles
various super-resolution
microscopy Optical
methods
based Imaging
on stochastic
optical switching
[2]. Depending
(C-dots) in Super-resolution
Fluctuation
(SOFI) microscopy
and discuss other
possibilities for increase the brightness of different fluorescence emitters (J-aggregates of
on surface charge,
unmodified
C-dots
entering
spontaneously
into
the
cells
can
label
cytoplasm or
cyanine dyes) and for combining fluorescence with high-resolution electron microscopy
of organic dyes with the atoms of noble and heavy atoms).
localize also in(complexes
cellular nuclei.
They demonstrate significant differences in localization between living
C-dots[3].
possess a unique hybrid combination of fluorescence properties exhibiting characteristics
and apoptotic cells
of both dye molecules and semiconductor nanocrystals. They demobstrate two-state transitions
between
emissiveofanddramatic
dark states increase
[1]. These in
findings
open up we
new possibilities
for their
Addressing the
problem
brightness,
suggest the
application of
utilization in various super-resolution microscopy methods based on stochastic optical switching
J-aggregates of[2].cyanine
dyes
as
fluorescence
collectors
and
excitation
energy
transfer
Depending on surface charge, unmodified C-dots entering spontaneously into the cells can donors. We
label cytoplasm or localize also in cellular nuclei. They demonstrate significant differences in
demonstrate their
formation
theandsurface
different
types of nanoparticles. For combination of
localization
betweenon
living
apoptoticofcells
[3].
fluorescence microscopy
with x-ray microscopy we suggest the synthesized in our laboratory stable
Addressing the problem of dramatic increase in brightness, we suggest the application of Jaggregates
of cyanine
dyesatoms
as fluorescence
collectors
and excitation
complexes of organic
dyes
with the
of noble
and heavy
metals.energy transfer donors. We
demonstrate their formation on the surface of different types of nanoparticles. For combination
of fluorescence microscopy with x-ray microscopy we suggest the synthesized in our laboratory
stable complexes
of organic
dyes with the atoms of noble and heavy metals.
[1] S Ghosh et al (2014)
NANO LETTERS
14. 5656–5661
[2] A Chizhik et al [1]
(2016)
NANOetLETTERS
16.NANO
237–242
S Ghosh
al (2014)
LETTERS 14. 5656–5661 [2] A Chizhik et al (2016) NANO
16. 237–242 [3] M Dekaliuk et al (2015) JOURNAL NANOBIOTECHNOLOGY 13. 86
[3] M Dekaliuk et alLETTERS
(2015) JOURNAL
NANOBIOTECHNOLOGY 13. 86
61
ELMI meeting
european
light microscopy
initiative
16th international
ELMI meeting
elmi
Friday, 27th May
Coordinate-Targeted Fluorescence Nanoscopy with Multiple Off
States
Sven C. Sidenstein, Johann G. Danzl, Carola Gregor, Nicolai T. Urban, Peter Ilgen, Stefan Jakobs, Stefan W. Hell
Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077
Göttingen, Germany
[email protected]
Far-field superresolution microscopy or nanoscopy techniques “superresolve” features residing
closer than the diffraction-limit by transiently preparing fluorophores in distinguishable (typically onand off-) states and reading them out sequentially. In coordinate-targeted superresolution modalities,
such as stimulated emission depletion (STED) microscopy, this state difference is created by patterns of
light, driving for instance all molecules to the off-state except for those residing at intensity minima.
For high resolution, strong spatial confinement of the on-state is required. However, this also subjects
fluorophores at intensity maxima to excess light intensities and state cycling. In addition, as spatial
confinement of the on-state is increased, state contrast between designated on- and off-regions has
to be improved, too. We show that driving fluorophores to a second off-state that is inert to the excess
intensities enables protection of fluorophores and superior state contrast. In a realization that we
dubbed "protected STED", we used reversibly switchable fluorescent proteins as labels and employed
both STED and reversible photoswitching as off-transitions. This directly translated into reduced
bleaching and enhanced resolution in live-cell nanoscopy. (J. G. Danzl, S. C. Sidenstein et al., Nature
Photonics 10, 122-128 (2016)).
62
Re-scan Confocal Microscopy (RCM): scanning twice for better re
high sensitivity; characterisation and applications.
16th international
1
ELMI meeting
, Emilie C.B. Desclos1, Laura Dolz E
Giulia M.R. De Luca1, Ronald M.P. Breedijk
Friday,
May
Smits2, 27th
Sjoerd
Stallinga3, Ron Hoebe4 and Erik M.M. Manders1
european
light microscopy
initiative
elmi
international
Re-scan
Confocal
Microscopy
(RCM):Institute
scanning
twice
forUniversity of Amsterd
1) Innovative
Microscopy
Group, Swammerdam
for16th
Life
Sciences,
ELMI meeting
better
resolution
and
high Food
sensitivity;
characterisation
2) Molecular
Biology and
Microbial
Safety, Swammerdam
Institute forand
Life Sciences, Unive
3)
Quantitative
Imaging
group,
Department
of
Imaging
Science
&
Technology,
Delft University of
applications.
4) Department of Cell Biology and Histology, Academic Medical Centre, Amsterdam, The Nether
Giulia M.R. De Luca1, Ronald M.P. Breedijk1, Emilie C.B. Desclos1, Laura Dolz Edo2, Gertien J. Smits2, Sjoerd
Stallinga3, Ron Hoebe4, Erik M.M. Manders1
[email protected]
1) Innovative Microscopy Group, Swammerdam Institute for Life Sciences, University of Amsterdam, Amsterdam
2)
Biology
and Microbial Food
Safety,
Swammerdam
Institute for Lifethe
Sciences,
University
of Amsterdam
InMolecular
confocal
microscopy,
the
pinhole
is influencing
axial
resolution,
the lateral
3) Quantitative Imaging group, Department of Imaging Science & Technology, Delft University of Technology, Delft
signal to noise ratio (SNR) of the final image. When the pinhole is almost closed,
4) Department of Cell Biology and Histology, Academic Medical Centre, Amsterdam, The Netherlands
resolution of confocal is √2 times better than wide-field microscopy. Users do not
[email protected]
pinhole
almost zero, because the severe reduction in SNR of the image. When the
In confocal microscopy, the pinhole is influencing the axial resolution, the lateral resolution and the
than
1
A.U.,
gain
infinal
lateral
of confocal
compared
wide-field
signal to noise ratiothe
(SNR)
of the
image.resolution
When the pinhole
is almost closed,
the lateraltoresolution
of micr
confocal is √2 times better than wide-field microscopy. Users do not often set the pinhole almost zero,
We have developed a new “optics-only” super-resolution technique, Re-scan Conf
because the severe reduction in SNR of the image. When the pinhole is larger than 1 A.U., the gain in
(RCM),
based
on standard
extended with an optical unit (relateral
resolution
of confocal
comparedconfocal
to wide-fieldmicroscopy,
microscopy is lost.
projects
the emitted
light
directlysuper-resolution
on a cameratechnique,
with double
amplitude com
We have developed
a new
“optics-only”
Re-scan angular
Confocal Microscopy
(RCM),
basedunit.
on standard
extended
an optical
unit (re-scanner)
that projects
scanning
As aconfocal
result,microscopy,
the width
of thewith
spot
is improved
by a factor
of √2 relat
the
emitted
light
directly
on
a
camera
with
double
angular
amplitude
compared
to
the
first
scanning
unit.
limited resolution. With a 100 nm fluorescent bead sample, the FWHM is reduced
As
a result, the down
width ofto
the170
spot nm
is improved
by a factor
of √2The
relative
to diffraction limitedisresolution.
wide-field
in RCM
(Fig.1).
extra-resolution
obtained by us
With a 100 nm fluorescent bead sample, the FWHM is reduced from 250 nm in wide-field down to 170 nm
state-of-the-art detector (sCMOS or EMCCD) offering extra sensitivity and good o
in RCM (Fig.1). The extra-resolution is obtained by using a very sensitive state-of-the-art detector (sCMOS
live cell imaging.
or EMCCD) offering extra sensitivity and good opportunities for live cell imaging.
Fig. 1: Wide-field (left) vs RCM (right)
beads.
Scale bars
Resolutio
Fig.
1: Wide-field
(left)arevs100
RCMnm.
(right)
improved
tonm
170beads.
by aScale
simple
tric
imaging
of 100
barsoptical
are
100 nm. Resolution of RCM is improved to
170 by a simple optical trick.
We quantify how the pinhole is influencing the performances of RCM and we compare it with
confocal. For RCM, the lateral resolution is independent from the pinhole size: it is not necessary
anymore to close the pinhole down to almost zero to improve the lateral resolution. In RCM, the axial
resolution
is maintained
and pinhole
measurements
show the axialthe
resolution
of RCM is even
improvedand
by we com
We quantify
how the
is influencing
performances
of RCM
10%.
Measurements
on
the
image
quality
of
RCM
show
that
for
the
SNR
is
improved
by
a
factor
confocal. For RCM, the lateral resolution is independent from the pinholeofsize: it i
four for sub-resolution objects, as compared with confocal microscopy with a pinhole of 0.25 AU.
anymore to close the pinhole down to almost zero to improve the lateral resolution
63
resolution is maintained and measurements show the axial resolution of RCM
is ev
10%. Measurements on the image quality of RCM show that for the SNR is impro
ELMI meeting
european
light microscopy
initiative
16th international
ELMI meeting
elmi
Friday, 27th May
We show how the RCM is a flexible system that can be equipped for multi-colour imaging, FRET and
FRAP measurements and measurements of intracellular pH.
• G. M. R. De Luca, R. M. P. Breedijk, E. M. M. Manders et al., “Re-scan confocal microscopy: scanning
twice for better resolution”, Biomedical Optics Express 4 (2013).
• Giulia De Luca, Ronald Breedijk, Ron Hoebe, Sjoerd Stallinga, and Erik Manders. Re-scan Confocal
Microscopy (RCM) improves the resolution of confocal microscopy and increases the sensitivity.
Manuscript submitted.
• Giulia De Luca, Emilie Desclos, Ronald Breedijk, L. Dolz Edo, G.J. Smits, Ron Hoebe, Erik Manders.
Configuration of the Re-scan Confocal Microscope (RCM) for biomedical applications. Manuscript
submitted.
64
16th international
ELMI meeting
Friday, 27th May
european
light microscopy
initiative
elmi
egistration free multicolor dSTORM with novel 16th
dyes resolves ultra‐
international
Registration
free ‘caged’ multicolor
‘caged’
dSTORM
with
novel
dyes
ELMI meeting
tructure of synaptic vesicles resolves ultra-structure of synaptic vesicles
1
1
Martin Lehmann
, Benjamin Gottschalk, Georgi Tadeus, Gregor Lichtner, Haider 1
Martin, Andre Lampe
Lehmann1, Andre Lampe
, Benjamin Gottschalk, Georgi Tadeus, Gregor Lichtner, Haider Klenz, Dmytro
1
1
1
1
1
1 Schwagerus1, Christian 1
Peter Schmieder
, Sergej P. Haucke
R. Hackenberger
, enz, Dmytro Puchkov
Puchkov
, Peter, Schmieder
, Sergej Schwagerus
, Christian P. R. Hackenberger1, Volker
,
1
2
2
Schmoranzer
, and Jan Schmoranzer
olker HauckeJan
1) Leibniz Institute of Molecular Pharmacology, Berlin, Germany
Leibniz Institute of Molecular Pharmacology, Berlin, Germany 2)Charité‐Universitätsmedizin, Berlin, Germany 2) Charité-Universitätsmedizin, Berlin, Germany
Presenter: [email protected]
resenter: [email protected] The precision of multicolor single molecule localization-based super-resolution microscopy (SMLM)
he precision of multicolor single molecule localization‐based super‐resolution microscopy (SMLM) approaches are challenged by several factors including the photo-chemical compatibility of fluorophores,
pproaches are challenged by several factors including the photo‐chemical compatibility of the errors in multi-channel registration and the crosstalk between color channels. We recently introduced
uorophores, the errors in multi‐channel registration and the crosstalk between color channels. We a variant
of direct stochastic
reconstruction
microscopy that ismicroscopy based on spectral
cently introduced a variant of direct optical
stochastic optical reconstruction that demixing
is based (SDon dSTORM)
to
perform
registration
error
free
multicolor
SMLM
with
minimal
channel
crosstalk
(1,
2).minimal To enable
pectral demixing (SD‐dSTORM) to perform registration error free multicolor SMLM with any laboratory to perform rapid SD-based multicolor SMLM we have designed the open-source software
hannel crosstalk (1, 2). To enable any laboratory to perform rapid SD‐based multicolor SMLM we have tool ‘SDmixer’ (3).
By testing
28 commercially
available
dyes for28 their
suitability to super-resolve
a known
esigned the open‐source software tool ‘SDmixer’ (3). By testing commercially available dyes for cellular
nanostructure
we
identified
eight
novel
dyes
with
good
to
excellent
photo-switching
properties
eir suitability to super‐resolve a known cellular nanostructure we identified eight novel dyes with that enable
high quality dSTORM
imaging
differenthigh spectral
regimes
(4). Among
those, in thedifferent spectrally
ood to excellent photo‐switching properties that inenable quality dSTORM imaging close dyes CF647 and CF680 comprise an optimal dye pair for dual-color SD-dSTORM. Combining this dye
pectral regimes (4). Among those, the spectrally close dyes CF647 and CF680 comprise an optimal dye pair with
the separatelyCombining excited CF568this we dye performed
dSTORM
to image excited the relative
nanoscale
air for dual‐color SD‐dSTORM. pair 3-color
with the separately CF568 we distribution
of
components
of
the
endocytic
machinery
and
the
cytoskeleton.
As
the
precision
of
all SMLM
erformed 3‐color dSTORM to image the relative nanoscale distribution of components of the endocytic approaches, including dSTORM, critically depends on the number of detected photons per localization,
achinery and the cytoskeleton. As the precision of all SMLM approaches, including dSTORM, critically we searched for SD-dSTORM suitable dyes that can be reductively quenched (‘caged’) to yield longer ON
epends on the number of detected photons per localization, we searched for SD‐dSTORM suitable dyes states and higher photon counts upon light induced recovery. By screening 39 dyes for their fluorescence
at can be reductively quenched (‘caged’) to yield caging
andhigher recovery
kinetics,counts we identified
nger ON states and photon upon novel dyes By thatscreening yield a multicolor
SDght induced recovery. 39 dyes for dSTORM
localization
precision
below
15
eir fluorescence caging and recovery kinetics, we nm
(5).
Caged
SD-dSTORM
could
resolve
entified novel dyes that yield a multicolor SD‐
the ultrastructure
of below single 4015 nmnm synaptic
STORM localization precision (5). vesicles in brain sections similar to images
aged SD‐dSTORM could resolve the ultrastructure obtained
by immuno-electron
single 40 nm synaptic vesicles in brain microscopy,
sections yet with
much improved
label density.
milar to images obtained by immuno‐electron icroscopy, yet with much improved label density. 1. Lampe A, Haucke V, Sigrist S, Heilemann M, Schmoranzer J, ” Multi-colour direct STORM with red-emitting carbocyanines”,
Lampe A, Haucke V, Sigrist S, Heilemann M, Schmoranzer J, ” Multi‐colour direct STORM with red‐emitting carbocyanines”, Biology of the Biology of the Cell, 2012 Apr;104(4):229-37.
ll, 2012 Apr;104(4):229‐37. 2. Lampe A, Tadeus A, Schmoranzer J, “Spectral demixing avoids registration errors and reduces noise in multicolor localization-
Lampe A, Tadeus A, Schmoranzer J, “Spectral demixing avoids registration errors and reduces noise in multicolor localization‐based super‐
solution microscopy”, Methods Appl. Fluoresc., 2015 Jun, doi:10.1088/2050‐6120/3/3/034006 Tadeus G, Lampe A, Schmoranzer J, “SDmixer – A versatile software tool for spectral demixing of multicolor single molecule localization ta”, Methods Appl. Fluoresc., 2015 Jun, doi:10.1088/2050‐6120/3/3/037001 Lehmann M, Lichtner G, Klenz H, Schmoranzer J, “Novel organic dyes for multicolor localization‐based super‐resolution microscopy”, J 65
ELMI meeting
european
light microscopy
initiative
16th international
ELMI meeting
elmi
Friday, 27th May
based super-resolution microscopy”, Methods Appl. Fluoresc., 2015 Jun, doi:10.1088/2050-6120/3/3/034006
3. Tadeus G, Lampe A, Schmoranzer J, “SDmixer – A versatile software tool for spectral demixing of multicolor single molecule
localization data”, Methods Appl. Fluoresc., 2015 Jun, doi:10.1088/2050-6120/3/3/037001
4. Lehmann M, Lichtner G, Klenz H, Schmoranzer J, “Novel organic dyes for multicolor localization-based super-resolution
microscopy”, J Biophotonics, 2016 Jan;9(1-2):161-70.
5. Lehmann M, Gottschalk B, Haucke V and Schmoranzer J, “Multicolor ‘caged’ STORM resolves ultra-structure of single synaptic
vesicles in brain”, Angew Chem Int Ed Engl. 2015 Nov 2; 54(45):13230-5.
66
16th international
ELMI meeting
Workshop abstracts
light m
16th internation
ELMI meeting
ELMI meeting
european
light microscopy
initiative
elmi
16th international
ELMI meeting
Day 2 Workshops
Wednesday 25th MAY 2016
Workshop 1:
14:00 - 15:00
Acquifer AG
High Content Screening workflows by ACQUIFER
Seminar Room 404
Andor
A New Imaging Platform from Andor
Seminar Room 402
Argolight
Argolight new hardware and software solutions for
the quality control of fluorescence microscopes
Booth MB08
arivis
Solutions for Big Image Data
Glass Room 4
Bitplane
The developmental biologist's best friend - Imaris
Glass Room 3
Bruker
Seminar Room 104
Vutara 352 – Opterra II combo – Correlative high
speed, quantitative super-resolution microscopy and
multipoint live-cell confocal imaging.
Confocal.nl
Re-scan Confocal Microscopy (RCM) for improved
resolution and sensitivity. A new microscope by
researchers for researchers.
Booth MB01
FEI Company
3D cryo-light microscopy enables targeted cryoelectron tomography
Booth MB07
Hamamatsu
A step change in practical usability: Hamamatsu’s
Photonics
simultaneous two-channel imaging system
Deutschland GmbH
Booth MB21
ibidi GmbH
Quantitative, real-time oxygen measurement during Booth MB02
live cell imaging
Intelligent Imging
Innovations GmbH
Lightsheet Microscopy – Lattice Light Sheet System
Booth MB12
Leica Microsystems HyVolution turns super-sensitivity into 140 nm
resolution
Seminar Room 102
Luxendo
A practical approach to light-sheet microscopy –
focus on the essential
Seminar Room 404
Nikon
New N-SIM E, a simple way of doing SIM
Glass Room 1
70
16th international
ELMI meeting
european
light microscopy
initiative
elmi
Olympus
international
FLUOVIEW FV3000 - The new confocal laser16th
scanning
Seminar Room 103
ELMI
meeting
microscope
Omicron-Laserage
Laserprodukte
GmbH
LedHUB® – Flexible multicolor LED light engines for
microscopy
Booth MB11
Phasics
High definition quantitative phase microscopy
combined to fluorescence imaging using a single
camera thanks to a smart plug & play optomechanical module
Booth MB6
PicoQuant GmbH
Rapid2flim: The new and innovative method for
ultra-fast flim imaging of biological processes
Seminar Room 403
PicoQuant GmbH
Time-resolved STED add-on for the MicroTime 200.
Confocal fluorescence lifetime microscope
Seminar Room 403
Rapp
OptoElectronic
GmbH
Solutions for photo-manipulation, deep-UV
microscopy and fluorescence life-time imaging
Booth MB10
Scientific Volume
Imaging
Huygens gpu-accelerated image restoration; now
also for light sheet microscopy
Booth MB5
ThermoFisher
Scientific
Move away from the dark ages- evolve with
Invitrogen™ Evos™ Imaging systems
Seminar Room 405
Visitron
Confocal & FRAP applications: New Visitron
Homogenizer for Confocal Spinning Disk Scan Heads
Booth MB16
Zeiss
Boosting Speed and Sensitivity in Light Microscopy
Seminar Room 105
Workshop 2:
15:00 - 16:00
Acquifer AG
The ACQUIFER HIVE: A Modular Computing Platform Seminar Room 404
for Data Storage, Handling, Visualization and Analysis
AHF
LED light sources and specific LED filter sets – a
analysentechnik AG perfect match
Booth MB10
71
ELMI meeting
european
light microscopy
initiative
elmi
16th international
ELMI meeting
Andor
Mosaic3 - High speed light patterning for microscopy Seminar Room 402
Argolight
Booth MB08
arivis
arivis strategies and application solutions for
visualization and exploration of big images
Booth MB03
Bitplane
Glass Room 3
Bruker
Seminar Room 104
Confocal.nl
FEI Company
Interactive visualization and advanced segmentation Booth MB07
workflows for 3D image data using Amira®
Booth MB01
Hamamatsu
Synchronization and triggering the ORCA-Flash 4.0
Photonics
Scientific CMOS Camera with peripheral equipment
Deutschland GmbH
Booth MB21
Intelligent Imging
Innovations GmbH
Booth MB12
Leica Microsystems LAS X – Guided Image Acquisition and Analysis in 2D Seminar Room 102
and 3D at its best
Luxendo
High-speed imaging of larger objects with the
MuVi-SPIM
Seminar Room 404
Nikon
Seamless SIM-confocal imaging: extend confocal
resolution by C2+ integration with N-SIM E
Glass Room 1
Olympus
FLUOVIEW FV3000 - The new confocal laser scanning Seminar Room 103
microscope
72
16th international
ELMI meeting
european
light microscopy
initiative
elmi
Omicron-Laserage
Laserprodukte
GmbH
16th international
BrixXps – Versatile picosecond/CW diode lasers
formeeting
Booth MB11
ELMI
microscopy
Phasics
Booth MB6
camera thanks to a smart plug & play opto-mechanical module
PicoQuant GmbH
SeminarRoom 403
PicoQuant GmbH
Seminar Room 403
Scientific Volume
Imaging
Booth MB5
ThermoFisher
Scientific
Move away from the dark ages- evolve with Invitrog- Seminar Room 405
en™ Evos™ Imaging systems
Visitron
Optimized for Superresolution: VisiTIRF Condenser
Booth MB16
combines fiber-based with direct-coupled laser lines
Zeiss
Entering new dimensions of enhanced optical resolu- Seminar Room 105
tion in combination with full sample flexibility
Workshop 3:
16:30 - 17:30
Acquifer AG
Seminar Room 404
Andor
Seminar Room 402
Argolight
Booth MB08
arivis
arivis Immersive View – Microscopy goes Virtual
Reality
Booth MB03
Bitplane
Glass Room 3
73
ELMI meeting
european
light microscopy
initiative
elmi
16th international
ELMI meeting
Bruker
Seminar Room 104
Confocal.nl
Booth MB01
ibidi GmbH
New tools to study cell migration with live cell
microscopy
Booth MB02
Intelligent Imging
Innovations GmbH
Booth MB12
Luxendo
Seminar Room 404
Nikon
Glass Room 1
Omicron-Laserage
Laserprodukte
GmbH
microscopy
Booth MB11
Phasics
Booth MB6
PicoQuant GmbH
Seminar Room 403
PicoQuant GmbH
Seminar Room 403
Scientific Volume
Imaging
Booth MB5
ThermoFisher
Scientific
74
16th international
ELMI meeting
european
light microscopy
initiative
elmi
Visitron
16th international
Confocal & FRAP applications: New Visitron HomogBooth MB16
ELMI meeting
enizer for Confocal Spinning Disk Scan Heads
Zeiss
Day 3 Workshops
Thursday 26th MAY 2016
Workshop 4:
13:00 - 14:00
Acquifer AG
Seminar Room 105
AHF
LED light sources and specific LED filter sets – a
analysentechnik AG perfect match
Booth MB10
Andor
Seminar Room 402
Argolight
Booth MB08
arivis
arivis strategies and application solutions for
visualization and exploration of big images
Booth MB03
Bitplane
Glass Room 3
Bruker
Seminar Room 104
Confocal.nl
FEI Company
Interactive visualization and advanced segmentation Booth MB07
workflows for 3D image data using Amira®
GE Healthcare Life
Sciences
Live cell structured illumination imaging: a new
reality
Hamamatsu
A step change in practical usability: Hamamatsu’s
Photonics
simultaneous two-channel imaging system
Deutschland GmbH
Booth MB01
Glass Room 4
Booth MB21
75
ELMI meeting
european
light microscopy
initiative
elmi
16th international
ELMI meeting
ibidi GmbH
Quantitative, real-time oxygen measurement during Booth MB02
live cell imaging
Intelligent Imging
Innovations GmbH
Leica Microsystems HyVolution turns super-sensitivity into 140 nm
resolution
Booth MB12
Seminar Room 102
Luxendo
High-speed imaging of larger objects with the MuVi- Seminar Room 404
SPIM
Nikon
Olympus
microscope
Omicron-Laserage
Laserprodukte
GmbH
BrixXps – Versatile picosecond/CW diode lasers for
microscopy
Booth MB11
Phasics
camera thanks to a smart plug & play optomechanical module
Booth MB6
PicoQuant GmbH
Seminar Room 403
PicoQuant GmbH
Seminar Room 403
Scientific Volume
Imaging
Booth MB5
ThermoFisher
Scientific
Move away from the dark ages- evolve with
Invitrogen™ Evos™ Imaging systems
Seminar Room 405
Visitron
Booth MB16
76
Glass Room 1
16th international
ELMI meeting
european
light microscopy
initiative
elmi
Zeiss
16th international
Entering new dimensions of enhanced optical
Seminar room 105
ELMI meeting
resolution in combination with full sample flexibility
Workshop 5:
14:00 - 15:00
Acquifer AG
Andor
Mosaic3 - High speed light patterning for microscopy Seminar Room 402
Argolight
arivis
arivis strategies and application solutions for visuali- Booth MB03
zation and exploration of big images
Bitplane
Bruker
Seminar Room 104
Confocal.nl
Booth MB01
FEI Company
3D cryo-light microscopy enables targeted
cryo-electron tomography
Booth MB07
Seminar Room 404
Booth MB08
Glass Room 3
Hamamatsu
Synchronization and triggering the ORCA-Flash 4.0
Photonics
Scientific CMOS Camera with peripheral equipment
Deutschland GmbH
Booth MB21
Intelligent Imging
Innovations GmbH
Booth MB12
Leica Microsystems LAS X – Guided Image Acquisition and Analysis in 2D Seminar Room 102
and 3D at its best
Luxendo
Seminar Room 404
77
ELMI meeting
european
light microscopy
initiative
elmi
16th international
ELMI meeting
Nikon
Olympus
microscope
Omicron-Laserage
Laserprodukte
GmbH
microscopy
Phasics
Booth MB6
PicoQuant GmbH
Seminar Room 403
PicoQuant GmbH
Seminar Room 403
Rapp OptoElectronic GmbH
Solutions for photo-manipulation, deep-UV microscopy and fluorescence life-time imaging
Booth MB10
Scientific Volume
Imaging
Booth MB5
ThermoFisher
Scientific
Visitron
Confocal & FRAP applications: New Visitron Homogenizer for Confocal Spinning Disk Scan Heads
Booth MB16
Zeiss
Seminar Room 105
Workshop 6:
15:30 - 16:30
Acquifer AG
Andor
78
Glass Room 1
Booth MB11
Seminar Room 402
16th international
ELMI meeting
european
light microscopy
initiative
elmi
Argolight
16th international
Argolight new hardware and software solutions
Booth MB08
ELMIfor
meeting
arivis
arivis Immersive View – Microscopy goes Virtual
Reality
Booth MB03
Bitplane
Glass Room 3
Bruker
Seminar Room 104
Confocal.nl
Booth MB01
ibidi GmbH
New tools to study cell migration with live cell
microscopy
Booth MB02
Intelligent Imging
Innovations GmbH
Booth MB12
Luxendo
High-speed imaging of larger objects with the
MuVi-SPIM
Seminar Room 404
Nikon
N-SIM E with 40x dry objective, a whole new range of Glass Room 1
applications
Omicron-Laserage
Laserprodukte
GmbH
BrixXps – Versatile picosecond/CW diode lasers for
microscopy
Phasics
Booth MB6
PicoQuant GmbH
Seminar Room 403
PicoQuant GmbH
Seminar Room 403
Booth MB11
79
ELMI meeting
european
light microscopy
initiative
elmi
16th international
ELMI meeting
Scientific Volume
Imaging
Visitron
Booth MB16
Zeiss
Entering new dimensions of enhanced optical resolu- Seminar Room 105
tion in combination with full sample flexibility
80
Booth MB5
16th international
ELMI meeting
ACQUIFER AG
Seminar Room 404AG
(WS1, WS3, WS5)
ACQUIFER
european
light microscopy
initiative
elmi
16th international
ELMI meeting
Seminar room 404 (WS1, WS3, WS5)
In this workshop, we will present the ACQUIFER workflow concept for High Content Screening (HCS).
This
is based
on apresent
tight integration
of the five
principalconcept
requirements
a successful
highIn thisprocess
workshop,
we will
the ACQUIFER
workflow
for HighofContent
Screening
(HCS).
This
process
is
based
on
a
tight
integration
of
the
five
principal
requirements
of a
content or high-throughput imaging experiment:
successful
high-content
or
high-throughput
imaging
experiment:
a) Clever and efficient planning and experimental design;
Development
of optimized
preparation steps
to control understood variables;
a) b)
Clever
and efficient
planningsample
and experimental
design;
c)
Stable,
easy-to-use,
flexible
and
easily
scalable
imaging
devices
whichvariables;
can interface with
b) Development of optimized sample preparation steps to control
understood
automated
sample preparation,
sampleimaging
delivery to
the imaging
and direct
c) Stable,
easy-to-use,
flexible andautomated
easily scalable
devices
whichapparatus,
can interface
with
automated
sample
automated
sample delivery
to the imaging
and direct
integration
intopreparation,
the data handling,
post-processing,
and analysis
hardwareapparatus,
and software;
integration
thePerformance
data handling,
post-processing,
analysis
hardware
and as
software;
d) Reliableinto
High
Computing
and dataand
storage
hardware
as well
high-speed data
d) Reliable High Performance Computing and data storage hardware as well as high-speed data
networks;
networks;
e)
Contextual
integration of the resulting data into local, group, and distributed databases, knowledge
e) Contextual integration of the resulting data into local, group, and distributed databases,
bases
and
publications.
knowledge bases
and publications.
At ACQUIFER we have developed our products around these five principal requirements in the context
At
we projects,
have developed
our products
thesefrom
five fixed
principal
requirements
in the
of ACQUIFER
a number of HCS
with a variety
of modelaround
organisms,
cell, to
live cell including
context of a number of HCS projects, with a variety of model organisms, from fixed cell, to live
yeast,
to modelyeast,
organism
analysis.
Extensive
experience
of our staff
in HCS as
ourindedicated
cell
including
to model
organism
analysis.
Extensive
experience
of well
our as
staff
HCS as
well
as our
dedicated
Imaging
Machines
and datahardware
handlingare
and
are the
Imaging
Machines
and data
handling
and processing
theprocessing
backbone ofhardware
the information
backbone
of
the
information
pipeline
indicated
above.
pipeline indicated above.
thisworkshop
workshopwewepresent
presentand
anddiscuss
discussananexample
exampleproject
projectinvolving
involving an
an extensive
extensive Zebrafish
Zebrafish
In Inthis
screening
protocol.
The
requirements
of
this
screen
on
sample
preparation,
the
imaging
hardware,
screening protocol. The requirements of this screen on sample preparation, the imaging
hardware,
the ACQUIFER
Imaging
andand
thedata
storage
and data
processing
the ACQUIFER
Imaging Machine
IM03,Machine
and theIM03,
storage
processing
pipeline
basedpipeline
on the
based on the ACQUIFER HIVE Data Module are detailed.
ACQUIFER HIVE Data Module are detailed.
Pleaserefer
refertotoourourwebsite
websiteatathttp://www.acquifer.de
http://www.acquifer.deororcall
call us
us atat +49
+49(721)
(721) 83
83 08
08 74-0
74-0 for
for more
Please
more
detailed
information
on
ACQUIFER
products.
detailed information ACQUIFER products.
81
ELMI meeting
european
light microscopy
initiative
elmi
16th international
ELMI meeting
ACQUIFER AG
Seminar Room 404 (WS2, WS4, WS6)
The ACQUIFER HIVE: A Modular Computing Platform for Data
Storage, Handling, Visualization and Analysis
ACQUIFER AG
Biomedical research laboratories are equipped with ever more capable imaging systems. This is
Seminar
roomincreasing
404 (WS1,
WS3, WS5)
dramatically
imaging
data volumes that are provoking a severe challenge with respect to
handling, storing, processing, and ultimately archiving these datasets. Furthermore, an increasingly
In this workshop, we will present the ACQUIFER workflow concept for High Content Screening
collaborative and globally distributed research community calls for reliable tools for remote data
(HCS). This process is based on a tight integration of the five principal requirements of a
access, datahigh-content
sharing and or
communication.
successful
high-throughput imaging experiment:
In this workshop, we present the HIVE, the modular computing platform for distributed handling,
a)storage,
Clever processing,
and efficient
planning and
visualization
andexperimental
analysis of design;
large (multi-Terabyte sized) datasets. The HIVE
b) Development of optimized sample preparation steps to control understood variables;
combines ease of use with a industry standard technology for large data management. The HIVE
c) Stable, easy-to-use, flexible and easily scalable imaging devices which can interface with
provides a sample
digital data
hub withautomated
easy remote
accessdelivery
to server-side
processing,
which can
automated
preparation,
sample
to the imaging
apparatus,
andreplace
direct
or
augment
multiple
acquisition,
processing
and
storage
devices.
The
HIVE
is
designed
integration into the data handling, post-processing, and analysis hardware and software;to enable
simultaneous
connections
of multiple
microscopes
for rapidhardware
acquisition
of data,
or processing
d)
Reliable High
Performance
Computing
and data storage
as well
as high-speed
data
networks;
devices for direct data access. This relieves pressure on institutional networking infrastructure, makes
e)microscopy
Contextual
integration
of theand
resulting
data into from
local,
group, and
and thus
distributed
platforms
more stable
more independent
IT services
brings youdatabases,
one step
knowledge bases and publications.
closer to exploit the full potential of your imaging equipment.
are compatible
commonly
openaround
source and
processing,
visualization
AtHIVE’s
ACQUIFER
we havewith
developed
ourused
products
thesecommercial
five principal
requirements
in the
context
of a number
HCS
projects, with adevice
variety
of model organisms,
fixed to
cell,
live
and analysis
software.ofAny
network-enabled
(OS-independent)
can befrom
connected
thetoHIVE.
cell including yeast, to model organism analysis. Extensive experience of our staff in HCS as
HIVEs can also be connected to the my.acquifer.net portal, a central service allowing secure sharing of
well as our dedicated Imaging Machines and data handling and processing hardware are the
datasets and
project
management
that indicated
enables secure
remote access for project members independent
backbone
of the
information
pipeline
above.
of their geographical location.
In Inthis
and discuss the
an example
an extensive
Zebrafish
thisworkshop
workshop,wewepresent
will demonstrate
flexibilityproject
of the involving
HIVE for handling
and analyzing
screening protocol. The requirements of this screen on sample preparation, the imaging
various image-based
datasets.
Including:
connecting
and streaming
of data to the
HIVE,
hardware,
the ACQUIFER
Imaging
Machine
IM03,microscopes
and the storage
and data processing
pipeline
based
on the ACQUIFER
HIVE Data
detailed.
collaborative
project management
andModule
remote are
access,
setup of a HIVE network, image processing
examples as well as rendering and analysis of Terabyte-sized image datasets (LSM).
Please refer to our website at http://www.acquifer.de or call us at +49 (721) 83 08 74-0 for more
Pleaseinformation
refer to our websites
www.acquifer.de
detailed
on ACQUIFER
products. or call us at +49 (721) 83 08 74-0 for more detailed
information on ACQUIFER products.
82
16th international
ELMI meeting
AHF analysentechnik AG
european
light microscopy
initiative
elmi
16th international
ELMI meeting
LED light sources and specific LED filter sets
–Maya2016,
perfect
match
24-27
Debrecen, Hungary
Booth MB10 (WS2, WS4)
WS2 and WS4 at the AHF / Rapp booth
LED light sources and specific LED filter sets – a perfect match
light
more
and morecommon
substituting
common
Long
LED lightLED
sources
are sources
more andaremore
substituting
arc lamps.
Longarc
termlamps.
stability,
ultraterm
fast stability,
ultra (µs)
fast and
switching
timeintensities
(µs) and adjustable
intensities
LED tool
lightforsources
a perfect
tool
switching time
adjustable
make LED light
sourcesmake
a perfect
any kind
of
for any kind of automated acquisition. We will show measurements comparing different LED
automated acquisition. We will show measurements comparing different LED and arc lamp sources
and arc lamp sources and their output during their lifetimes.
and their output during their lifetimes.
LED are
light
are in
nottheimplemented
in theof the
microscope
software
Some LEDSome
light sources
notsources
implemented
microscope software
manufacturers.
This of the
manufacturers. This problem can be solved in different ways. We will show you how.
problem can be solved in different ways. We will show you how.
hear regarding
questionsfilter
regarding
fit to
andfilter
is an
We oftenWe
hearoften
questions
sets. Dofilter
theysets.
fit toDo
LEDthey
sources
andLED
is ansources
excitation
stillexcitation
filter
still
necessary?
necessary?
This workshop
sets influence
the performance
overall performance
of a fluorescence
This workshop
will showwill
howshow
filterhow
setsfilter
influence
the overall
of a fluorescence
experiment.
Using
the
right
filters
can
increase
signal
intensity
up
to
a
factor
of 5.
experiment. Using the right filters can increase signal intensity up to a factor of 5.
We will show:
We will show:
- equipping
the LED light source with excitation filters
- equipping the LED light source with excitation filters
- using LED light source together with singleband filter sets
- using LED light source together with singleband filter sets
- using LED
light source together with multiband filter sets
- using
LED lightWhich
sourceone
together
withchoice?
multiband filter sets
- singleband
or multiband:
is the right
- singleband or multiband: Which one is the right choice?
83
ELMI meeting
european
light microscopy
initiative
elmi
16th international
ELMI meeting
Andor
Seminar Room 402 (WS1, WS3, WS4, WS6)
Keywords: Confocal, deconvolution, TIRF, live cell, fixed sample, software, 3D capture, 3D visualisation.
Microscopy is now a routine part of many research projects, fundamental to understanding biological
processes or targets like protein localisation, dynamics and function, capturing detail not only at
the sub-cellular scale, but also multi-cellular or whole organism. In order to study a wide variety of
biological mechanisms using multi-scale model systems (e.g. single cell to whole drosophila embryo),
you typically need to capture images using more than one imaging system according to the biological
question you are asking.
Andor has designed and manufactured a new microscopy platform which in a single device includes
high-speed confocal, widefield-deconvolution, and simultaneous multi-colour TIRF imaging. This
novel solution is driven by new and dedicated software to facilitate the multi-modal imaging through
a user friendly interface. The imaging workflow covers capture to real-time multi-dimensional
rendering and deconvolution, designed to then compliment Imaris for analysis. We will present our
new imaging platform and its many benefits including:
1. Improving imaging quality through our patented Borealis illumination technology, delivering better
signal to noise performance, illumination throughput and uniformity, and extended spectral range.
2. Significantly increasing the speed of confocal image capture by at least 10-fold compared to
traditional point scanning technology, and so delivering faster 3D volume data for
a. Studying high-speed multi-dimensional cellular dynamics
b. High-throughput 3D volume capture and rendering.
3. Engaging additional modes of imaging:
a. widefield-deconvolution for highsensitivity photo-sensitive imaging (e.g. yeast &
Dictyosteliida), and
b. TIRF imaging for cell membrane related physiology (e.g. receptor localisation and exocytosis).
4. Reducing phototoxicity and/or photobleaching for prolonged live cell imaging and deeper imaging
before losing signal.
5. Controlling sample illumination and detection parameters as additional creative tools for deeper
investigations such as single molecule and localisation studies.
84
16th international
ELMI meeting
Andor
Seminar Room 402 (WS2, WS5)
european
light microscopy
initiative
elmi
16th international
ELMI meeting
Mosaic3 - High speed light patterning for microscopy
Keywords: Optogenetics, optophysiology, photostimulation, photobleaching, photoactivation,
uncaging, DMD.
Mosaic3 is a patented device designed specifically for active illumination techniques such as
Optogenetics, photobleaching and activation, and uncaging. Using a digital mirror array, Mosaic3 can
deliver illumination patterns across a broad wavelength spectrum and at speeds of up to 5KHz.
This ability to generate complex patterns simultaneously, sequentially or cumulatively, makes
Mosaic3 the ideal tool for optophysiology. The resolution of the system is such that it is possible
to target single cells for photostimulation. The functionality of loading patterns into its on-board
memory and calling them off with external triggers (e.g. from patch-clamp systems), means that
complex physiology such as neuronal network signalling can be investigated with ease.
Mosaic3 has been designed to easily attach to inverted and now upright microscopes for simple
integration into existing or new solutions, and supports a number of different light sources depending
on the intended application. Please join our workshop to learn more about Mosaic3 and how it might
extend your research capabilities.
85
ELMI meeting
elmi
european
light microscopy
initiative
16th international
ELMI meeting
Argolight
Booth MB8 (WS1, WS2, WS3, WS4, WS5, WS6)
Argolight new hardware and software solutions for the quality
Argolight
control
of fluorescence microscopes
Argolight new hardware and software solutions for the quality control of
This workshop
aimsmicroscopes
to present the assessment of the performances a fluorescence (widefield,
fluorescence
confocal, spinning disk) microscope with Argolight new hardware and software solutions.
In particular, different aspects of the microscope will be evaluated:
This workshop
to present
the assessment of the performances a fluorescence (widefield,
- illumination
andaims
collection
homogeneity,
confocal, spinning disk) microscope with Argolight new hardware and software solutions.
- distortion
of the
field ofaspects
view,of the microscope will be evaluated:
In particular,
different
illumination and collection homogeneity,
- spatial-- colocalization,
distortion of the field of view,
spatial colocalization,
- lateral- resolving
power,
- lateral resolving power,
- stages- repositioning
accuracy,
stages repositioning
accuracy,
- intensity
and spectral
responses
the system,
- intensity
and spectral
responses
of theofsystem,
etc.etc.
The demonstration aims to show how the quality control of the instrument is simple and fast with
Argolight solutions.
The demonstration
aims to show how the quality control of the instrument is simple and fast with
Argolight solutions.
86
Argolight
16th international
ELMI meeting
european
light microscopy
initiative
elmi
arivis
arivis
AG AG
ELMI meeting
Glass Room IV. (WS1)
arivis Vision - how to complete your imaging and image processing workflow
16th international
arivis Vision
complete your imaging and image
Solutions
for Big- how
Imageto
Data
processing workflow
Solutions for Big Image Data
Glass room IV. (WS1)
arivis is a specialized biomedical big image data and compliance software company in the life
arivis isindustry.
a specialized biomedical big image data and compliance software company in the life science
science
industry.
ItsIts
revolutionary,
arivisVision4D
Vision4D
allows
to handle
image
revolutionary,award
awardwinning
winning software
software arivis
allows
to handle
image
datadata
withwith
virtually
virtually unlimited file sizes (files of up to 6 TB have been used in the lab to prove
unlimited file sizes (files of up to 6 TB have been used in the lab to prove unprecedented performance)
unprecedented performance) on standard desktop PCs or notebooks, hence allowing for a
on standard desktop PCs or notebooks, hence allowing for a hardware-independent exploration of a
hardware-independent exploration of a wide range of scientific imaging tasks.
wide range of scientific imaging tasks.
Even
handling, visualization
visualizationand
andexploration
explorationkeeping
Evenforforhuge
hugedata
dataarivis
arivisVision4D
Vision4D ensures
ensures robust handling,
keeping
high
performance
in
the
complete
image
processing
workflow.
This
ranges
from
original
high performance in the complete image processing workflow. This ranges from original file
import,
to
file import, to the creation of 3D and 4D rendered volumes, visibility corrections & modification,
the creation of 3D and 4D rendered volumes, visibility corrections & modification, export of screenshots
export of screenshots & movies and quantification by measurements & image analysis.
& movies and quantification by measurements & image analysis.
In In
this,
trulyoutstanding.
outstanding.Among
Among
various
applications,
the benefit
is
this,arivis
arivisVision4D
Vision4D isistruly
various
useruser
applications,
the benefit
is extremely
extremely
impressive
and
substantial
for
customers
applying
modern
tissue
clearing
applications.
impressive and substantial for customers applying modern tissue clearing applications.
arivisproducts
productsare
areused
used by
by small
biotech
companies,
by the
leading
medical
arivis
smalland
andmedium
mediumsized
sized
biotech
companies,
by world’s
the world’s
leading
device
and
pharmaceutical
companies
as
well
as
by
international
renowned
research
associations,
medical device and pharmaceutical companies as well as by international renowned research
organizationsorganizations
and universities.
associations,
and universities.
87
ELMI meeting
european
light microscopy
initiative
elmi
16th international
arivis AG
ELMI meeting
arivis strategies and application solutions for visualization and exploration of
arivis AG
big images
Booth MB03 (WS2, WS4, WS5)
arivis strategies and application solutions for visualization and
New imaging techniques hold many opportunities but also pose various challenges to those
exploration of big images
involved. And as optical engineers, biologist or data analysis specialists learn how to make use
of, or even
of novel
hardware but
systems,
new
scientific
methods
Newextend
imagingfunctions
techniques hold
many opportunities
also posealso
various
challenges
to those
involved.evolve
as optical
biologist
data analysis
learn
how to
make use of, or even
rapidly.And
During
theengineers,
past few
years,orLSFM
has specialists
triggered
novel
developments
andextend
the modificatio
functions
of
novel
hardware
systems,
also
new
scientific
methods
evolve
rapidly.
During
the
past
of existing microscopic devices as well as it demanded for new methods in samplefew
preparation
years, LSFM
triggered novelofdevelopments
and theallows
modification
of existing microscopic
devices
and staining.
Thishascombination
methods today
high-resolution
imaging
of whole
as well
it demanded
in sample
and staining.
This combination
organisms
andasorgans
and for
duenew
to methods
comparable
lowpreparation
photo toxicity,
specimens
may beofimaged in
methods
today
allows
high-resolution
imaging
of
whole
organisms
and
organs
and
due
to
comparable
long-term time lapse experiments. The data sets produced by these new techniques are often
low photo toxicity, specimens may be imaged in long-term time lapse experiments. The data sets
extremely large, and range from several hundred gigabytes to terabytes per data set. How to
produced by these new techniques are often extremely large, and range from several hundred
handle such file sizes in the downstream imaging pipeline, without unnerving long thumbgigabytes to terabytes per data set. How to handle such file sizes in the downstream imaging pipeline,
turning without
time when
trying
open and explore?
and image
processing
unnerving
longtothumb-turning
time when For
tryingvisualization
to open and explore?
For visualization
and &
analysis,
hereprocessing
will show
strategies
in show
arivis
Vision4D
software
how to how
tackle
these
challenges.
image
& analysis,
here will
strategies
in arivis
Vision4D software
to tackle
these
We willchallenges.
present We
solutions
for
selected
imaging
applications
derived
from
state-of-the-art
LSFM,
will present solutions for selected imaging applications derived from state-of-the-art
confocal
microscopy
and slide
LSFM,
confocal microscopy
andscanners.
slide scanners.
88
16th international
ELMI meeting
arivis AG
european
light microscopy
initiative
elmi
16th international
ELMI meeting
arivis
arivisAG
Immersive View – Microscopy goes Virtual Reality
arivis Immersive View – Microscopy goes Virtual Reality
Advances in imaging technology in all fields of microscopy provide more and more highly resolved
Booth
(WS3,data.
WS6)
3D andMB03
4D image
Althoughinthere
are 2D
or 3D tools
to visualize
andprovide
evaluatemore
or work
data, there
Advances
imaging
technology
in available
all fields of
microscopy
and with
morethese
highly
is still a distance
between
scientist and the data.
resolved
3D and 4D
imagethe
data.
Although
there
are 2D ortechnology
3D tools available
to visualizeyour
andmicroscopy
evaluate ordata
work
with these data,
With arivis
Immersive
you can experience
properly.
there is still a distance between the scientist and the data.
This Virtual Reality approach from arivis enables you to travel through your images and interact with
With arivis Immersive technology you can experience your microscopy data properly.
them
more naturally.
You can from
look inarivis
all directions,
behind
or along
structures,
you can
anywhere
This Virtual
Reality approach
enables you
to travel
through
your images
andflyinteract
and
set
yourself
into
the
point
of
view
of
your
biological
structure
of
interest.
We
combine
off-the-shelf
with them more naturally. You can look in all directions, behind or along structures, you can fly
virtual reality
hardware
thethefirst
timeofwith
rendering
approaches
to immerse
anywhere
and set
yourselfforinto
point
viewvolumetric
of your biological
structure
of interest.
We you in
combine
virtual
reality hardware
for graphics
the first time
volumetric
rendering
your 3D off-the-shelf
microscopy data.
A high-end
consumer
card with
and an
Oculus Rift
headset comprise
approaches
to immerse
in your
3D data
microscopy
data.so
A the
high-end
consumer
graphics
card for
andyou
the major hardware.
Theyou
actual
image
is rendered,
real pixel
intensities
are ready
an Oculus Rift headset comprise the major hardware. The actual image data is rendered, so the
to examine, change thresholds, opacity, etc. Because you’re in the actual data, not a surface model,
real pixel intensities are ready for you to examine, change thresholds, opacity, etc. Because
you caninmore
easilydata,
develop
strategies
andeasily
interactively
them with our
you’re
the actual
not acreative
surfaceanalysis
model, you
can more
developimplement
creative analysis
powerful
arivis
Vision4D
analysis
tools.
strategies and interactively implement them with our powerful arivis Vision4D analysis tools.
stillininthetheprocess
processofofevaluating
evaluatingappropriate
appropriateapplications
applicationsand
andworkflows
workflowsfor
forthis
this technology.
WeWe
areare
still
technology.
Therefore
any
suggestions
on
new
application
and
ideas
are
very
much
appreciated.
Therefore any suggestions on new application and ideas are very much appreciated.
Mouse brain
brain tissue,
with
LUMOS
andand
imaged
withwith
Lightsheet
Z.1 Data
and 3D and
rendering
in arivis Vision4D.
Mouse
tissue,optically
opticallycleared
cleared
with
LUMOS
imaged
Lightsheet
Z.1processing
Data processing
3D rendering
in arivis Vision4D.
Sample was kindly prepared and provided by Olga Efimova, National Research Center, Kurchatov Institute, Moscow, Russia Sample vas kindly prepared and provided by Olga Efimova, National Research Center, Kurchatov Institute, Moscow, Russia.
89
ELMI meeting
european
light microscopy
initiative
elmi
Developmental, stem and in‐vivo cell biologists often find themselv
technology for high‐resolution temporal and spatial microscopy. A
16th international
ELMI meeting
the first to be faced with the struggles associated with these techn
degradation, the requirement for quick image acquisition in 3D, te
BITPLANE
Glass Roomthe need for efficient data visualization, storage and analysis. More
III. (WS1, WS2, WS3, WS4, WS5, WS6)
launched its first application for interactive rendering of 3D data se
The developmental
biologist’s best friend – Imaris.
introduced key tools, which cater to the needs of researchers doin
Developmental,
stem and in-vivo cell biologists often find themselves using state-of-the-art
particular, the needs of developmental biologists. 24-27 May 2016, Debrecen, Hungary
technology for high-resolution temporal and spatial microscopy. As a result, this group is often the
first to beThe workshops at ELMI2016 will cover the high‐performance tools
faced with the struggles associated with these techniques, namely, sample degradation, the
requirement for quick image acquisition in 3D, terabyte-sized data sets, and the need for efficient data
namely, visualization of terabyte multidimensional data sets, detec
visualization, storage and analysis. More than 22 years ago Bitplane launched its first application for
interactivecells and organelles, tracking of cell division, lineage analysis, drift rendering of 3D data sets. Over the years we have introduced key tools, which cater to the
needs of researchers
doing live cell imaging and, in particular, the needs of developmental biologists.
measurements, a wide range of plugins (XTensions) and advanced The workshops at ELMI2016 will cover the high-performance tools Imaris has to offer today, namely,
results exploration. In addition, we will introduce as part of Imaris visualization of terabyte multidimensional data sets, detection, tracking and analysis of cells and
organelles,a new algorithm which detects cells that have nothing but a cell m
tracking of cell division, lineage analysis, drift correction, angle measurements, a wide
range of plugins
(XTensions) and advanced interactive plotting tools for results exploration. In addition,
hand independent coordinate system. we will introduce as part of Imaris 8.3 two innovative solutions: a new algorithm which detects cells
that have nothing but a cell membrane label and a free-hand independent coordinate system.
Image (example o
publication using I
CW, Hadjantonaki
imaging of cell dyn
sheet microscopy.
10.1242/dev.1110
http://dev.biologi
Featured here, htt
hour‐time‐lapse‐im
morphogenesis‐de
Image (example of recent developmental biology publication using Imaris) from Udan RS, Piazza
VG, Hsu CW,
Hadjantonakis AK, Dickinson ME. 2014. Quantitative imaging of cell dynamics in mouse
embryos using light-sheet microscopy. Development. 141(22):4406-14. doi: 10.1242/dev.111021
http://dev.biologists.org/content/141/22/4406.long
Featured here, http://www.bitplane.com/learning/24-hour-time-lapse-imaging-of-vertebrate-embryo-morphogenesis-development
90
Bruker
16th international
ELMI meeting
european
light microscopy
initiative
elmi
Vutara 352 – Opterra II combo – Correlative high speed, quantitative superresolution microscopy and multipoint live-cell confocal Imaging.
Bruker
SeminarSeminar
roomRoom
104 104
- WS1
WS3WS4,
WS4
(WS1,WS2
WS2, WS3,
WS5,WS5
WS6)WS6
16th international
ELMI meeting
Bruker’s Vutara 352 is at the leading edge of super-resolution microscopy, providing the fastest
Vutara 352 – Opterra II combo – Correlative high speed,
super-resolution, Single Molecule Localization (SML) system commercially available. The
super-resolution
microscopy
andthrough
multipoint
live-proprietary
Vutara quantitative
352 enables 3D
SML and video-rate
nanoscopy
Bruker’s
cell confocal
Imaging.technologies. New integrated analysis features turn voxels into
ResEnhanced™
and Quadfield™
information.
Bruker’s Vutara 352 is at the leading edge of super-resolution microscopy, providing the fastest super-
resolution, Single Molecule Localization (SML) system commercially available. The Vutara 352 enables
During this workshop we will demonstrate new technology for fast 3D Single Molecule
3D SML(SML)
and video-rate
nanoscopy through
Bruker’s
proprietary
ResEnhanced™
and Quadfield™
Localization
and quantitative
analysis
imaging
as well
as multipoint
confocal with
technologies.
New
integrated
analysis
features
turn
voxels
into
information.
Opterra II and correlative imaging between confocal and super resolution.
During this workshop we will demonstrate new technology for fast 3D Single Molecule Localization
(SML)
and quantitative
analysis imaging
as well asasmultipoint
confocal with
Opterra II and in
correlative
Join the Bruker
Vutara workshop
at ELMI
we showcase
achievements
the field of
nanoscopy
andbetween
demonstrate
state-of-the-art
imaging
confocal the
and super
resolution. Vutara 352 and Opterra II. With the ability to
achieve resolutions
20nm
laterally
andas50wenm
axially
at depths in
upthetofield
100ofum,
the Vutara
Join the BrukerofVutara
workshop
at ELMI
showcase
achievements
nanoscopy
and 352
brings multi-color,
high
speed nanoscopy
to aand
wide
array
of biological
demonstrate the
state-of-the-art
Vutara 352
Opterra
II. With
the ability tosamples.
achieve resolutions of
20nm laterally and 50 nm axially at depths up to 100 um, the Vutara 352 brings multi-color, high speed
Topics will include:
nanoscopy to a wide array of biological samples.
Topics will include:
 Sub-second
super-resolution videos
•
Sub-second
super-resolution
 Particle tracking
at speeds videos
up to 3000 frames per second
•
Particle
tracking
at
up to
framesinstead
per second
 Recording of fixedspeeds
images
in3000
seconds
of minutes
•
Recording
of
fixed
images
in
seconds
instead
of
minutes
 Advanced 3D resolution using the patented
biplane
• Advanced
using
biplane and up to 100µm in cleared tissue
 3D
stacking3Dofresolution
of up to
25 the
umpatented
in cell samples
• 3D stackinganalysis
of of up features
to 25 um inofcellthesamples
up to 100µm
in cleared
 Advanced
SRX and
software
to turn
voxelstissue
into information
• Advanced analysis
features
of the for
SRX live-cell
software tomicroscopy
turn voxels into
information
 Multipoint
Confocal
imaging
and
correlative imaging
• Multipoint Confocal imaging for live-cell microscopy and correlative imaging
Correlative confocal-super resolution image showing labeling of synaptic Homer and Bassoon
Correlative confocal-super resolution image showing labeling of synaptic Homer and Bassoon
91
ELMI meeting
european
light microscopy
initiative
elmi
16th international
ELMI meeting
Confocal.nl
Confocal.nl
Re-scan
Confocal Microscopy (RCM) for improved resolution and sensitivity.
(WS1, WS2, WS3,
WS4, WS5, WS6)for researchers.
ABooth
newMB01
microscope
by researchers
Booth 1, Session 1,2,3,4,5 and 6
Re-scan Confocal Microscopy (RCM) for improved resolution
and sensitivity. A new microscope by researchers for
researchers.
The RCM (Re-scan Confocal Microscope) is a standard confocal microscope extended with a
Confocal.nl
The RCM
(Re-scan
Confocal
is a trick
standard
confocal
microscope
extended
re-scan
detection
unit.
With aMicroscope)
simple optical
a lateral
resolution
of 170nm
canwith
be a rescan detection
unit.
WithConfocal
adiameter.
simpleMicroscopy
optical
a lateral
resolution
offor170nm
can be achieved
for any
achieved
for any
pinhole
The trick
pinhole
is only
needed
Z-sectioning.
Since RCM
Re-scan
(RCM)
for
improved
resolution
and sensitivity.
A
new
microscope
by
researchers
for
researchers.
has
also
a
strongly
improved
sensitivity
(4x
better
signal-to-noise
ratio)
here
is
no
need
for
pinhole diameter. The pinhole is only needed for Z-sectioning. Since RCM has also a strongly improved
high
laser
power,
being
more
friendly
for
cells.
At
this
moment
RCM
can
work
in
multiBooth
1,
Session
1,2,3,4,5
and
6
sensitivity (4x better signal-to-noise ratio) here is no need for high laser power, being more friendly
colour
mode
for moment
differentRCM
colour
and ratio-imaging.
But itcolour
is ourcombinations
mission to go
for cells.
At this
can combinations
work in multi-colour
mode for different
and
further
by
extending
the
spectral
range
into
the
IR
for
deep
tissue
imaging
and
real-time
TheitRCM
(Re-scan
Confocal
is aextending
standard confocal
microscoperange
extended
withthe
a IR for deep
ratio-imaging. But
is our
mission
to goMicroscope)
further by
the spectral
into
detection unit. With
a simple
optical trick
a lateral resolution
of 170nm can
be FRET, FRAP,
acquisition speedre-scan
for monitoring
highly
dynamic
processes.
Applications
like
tissue imaging and
real-time
acquisition
speed
for monitoring
highly
dynamic
processes.
Applications
achieved for any pinhole diameter. The pinhole is only needed for Z-sectioning. Since RCM
pH, are Ca2+ are possible and many more biological applications are on the horizon.
has also
strongly
sensitivity
(4x better
here is no are
needon
forthe horizon.
like FRET, FRAP, pH,
are aCa2+
areimproved
possible
and many
moresignal-to-noise
biological ratio)
applications
high laser power, being more friendly for cells. At this moment RCM can work in multi-
In the workshopcolour
we will
how
modeexplain
for different
colour combinations and ratio-imaging. But it is our mission to go
bywill
extending
the spectral
theInsytem
worksfurther
and
itexplain
easily
can
the workshop
wehow
how range into the IR for deep tissue imaging and real-time
acquisition speed for monitoring highly dynamic processes. Applications like FRET, FRAP,
bethe
tuned
specific
biological
sytemforworks
andarehow
easily
canandbemany more biological applications are on the horizon.
pH,
Ca2+itare
possible
application.
We will
also show
that
tuned for specific
biological
application.
In the workshop we will explain how
with
microscope
the sytem
how it
easily can
We this
will RCM
also show
thatworks
withandthis
RCM
be tuned for
specific biological
Confocal.nl
introduces
a
new,
microscope Confocal.nl
introduces
a show
new,that
application.
We will also
affordable confocal
system
that can be
with this
RCM microscope
affordable confocal
system
that
can be
Confocal.nl
new,
plugged in-between
yourintroduces
cameraaand
plugged in-between
your
camera
affordable
confocal
systemand
that can be
microscope.
plugged in-between your camera and
microscope.
microscope.
RCM is a technology that has been developed by Giulia De Luca and Ronald Breedijk in the group of
RCM is a technology
has been
developed
bybyGiulia
DeLuca
Luca
and Ronald
in the
RCM is that
a technology
that has
been developed
Giulia De
and Ronald
Breedijk inBreedijk
the
Erik Manders at the
University
of Amsterdam.
Confocal.nl
is aConfocal.nl
spin-off company
introduces
RCM to
group
of Erik Manders
at the University
of Amsterdam.
is a spin-offthat
company
group of Erik Manders
at the University
of Amsterdam.
Confocal.nl
is a spin-off
company
that
introduces
RCM toit
the market.
Here at the ELMI 2016
it will
be demonstrated for the
theintroduces
market. HereRCM
at the
be demonstrated
for the
that
toELMI
the 2016
market.will
Here
at the ELMI 2016
itfirst
willtime.
be demonstrated for the
first time.
first time.
References
References
Luca, Giulia
M. M.
R.;P.;
Breedijk,
Brandt, Rick
A. J. ; Zeelenberg
Christiaan
De Luca, Giulia M. R.;DeBreedijk,
Ronald
Brandt,Ronald
Rick A.M.
J. ;P.;
Zeelenberg
Christiaan
HC; De Jong
B.E. Timmermans W; Azal
References
HC; De Jong B.E. Timmermans W; Azal Nahidi L; Hoebe RA; Stallinga S.; Manders Erik
Nahidi L; Hoebe RA; Stallinga
S.; Manders Erik M.M. (2013) Re-scan confocal microscopy: scanning twice for better resolution.
M.M. (2013) Re-scan confocal microscopy: scanning twice for better resolution. Biomedical
De
Luca, Giulia
M. R.;
Breedijk,
Ronald M. P.; Brandt, Rick A. J. ; Zeelenberg Christiaan
Biomedical
Optics Express,
4Express,
(11),
2644-2656
Optics
4 (11), 2644-2656
HC; De Jong B.E. Timmermans W; Azal Nahidi L; Hoebe RA; Stallinga S.; Manders Erik
M.M. (2013) Re-scan confocal microscopy: scanning twice for better resolution. Biomedical
Optics Express, 4 (11), 2644-2656
92
16th international
ELMI meeting
FEI Company
european
light microscopy
initiative
elmi
16th international
ELMI meeting
3D cryo-light microscopy enables targeted cryo-electron
tomography
Correlative light and electron microscopy (CLEM) aims at combining the large field of view and
chemical specificity of fluorescence light microscopy (LM) with the high-resolution structural
information revealed by electron microscopy (EM). As a result, correlative approaches can be extremely
powerful in identifying rare events and targeting specific structures in larger volumes for efficient
acquisition of EM data.
Amongst the sample preparation protocols for CLEM, cryogenic approaches are particularly interesting
as they preserve the sample in its near-native, frozen-hydrated state. Potential artifacts caused by
chemical fixation, dehydration and metal contrasting agents are therefore avoided. However, most
biological samples such as cells have to be thinned to a thickness smaller than 500 nm to be accessible
for cryo-electron tomography (cryo-ET) of subcellular structures.
During this workshop, we present the cryo-CLEM workflow described in Arnold, J. et al. (2016)
Biophysical Journal, Volume 110, Issue 4, 860-869. The authors used FEI’s CorrSight light microscopy
platform equipped with a cryo-stage for spinning disk confocal imaging of fluorescently labelled
HeLa cells in cryogenic conditions. The resulting 3D image stacks were used to target a cryo-focused
ion beam (FIB) milling process in order to create an electron transparent lamella, containing the
subcellular structure of interest, for cryo-ET.
Particular emphasis is placed on sample preparation and sample handling as well as the use of
fiducial markers and coordinate transformations for the correlation of LM and FIB.
93
ELMI meeting
european
light microscopy
initiative
elmi
16th international
ELMI meeting
S2, WS4)
FEIimaging
Company
decade
techniques have shifted from two dimensions to
D image data. Amira® is a software solutions co-evolving since 20 ye
chnology
to keepvisualization
pace with and
the advanced
challenging
task of interactive
Interactive
segmentation
workflows data pro
for 3D image data using Amira®
Over the past decade imaging techniques have shifted from two dimensions to the massive collection
will address
recent examples of image processing workflows from the raw i
of 3D image data. Amira® is a software solutions co-evolving since 20 years with the 3D imaging
ons andtechnology
quantitative
Segmentation
is one
of and
the
key problems en
to keep pace analysis.
with the challenging
task of interactive data
processing
analysis.
g. There
bewillaaddress
focus
onexamples
newoftools
for interactive
3Drawimage
Thewill
workshop
recent
image processing
workflows from the
image datasegmenta
®
to
3D
reconstructions
and
quantitative
analysis.
Segmentation
is
one
of
the
key
problems
encountered
f Amira . Another focus will be on advanced segmentation strategies fo
image processing.
will be a structures
focus on new toolsinforainteractive
3D image segmentation
xtract infeatures
likeThere
cellular
semi-automated
way.from the
latest release of Amira®. Another focus will be on advanced segmentation strategies for multiphase
volume data to extract features like cellular structures in a semi-automated way.
94
16th international
ELMI meeting
GE Healthcare Life Sciences
Glass Room IV. (WS4)
european
light microscopy
initiative
elmi
16th international
ELMI meeting
Live cell structured illumination imaging: a new reality
Live cell imaging has long been one of the most challenging applications in fluorescence microscopy.
More recently, with the adoption of super-resolution microscopy, researchers are facing a new complex
imaging challenge. Despite their complexity, both techniques are widely used due to their ability to
answer unique biological questions. Combining these to perform live cell super-resolution imaging
creates additional challenges, but greatly increases the potential scientific reward. Recent advances
in structured illumination microscopy (SIM) have made biologically relevant live cell SIM a reality.
SIM offers researchers a two-fold increase in resolution both laterally and axially, revealing structural
details previously unresolved with conventional microscopy. Of the existing super-resolution
techniques available today, SIM requires the least amount of input light, thus reducing photobleaching
and phototoxicity. In addition, SIM is compatible with standard fluorophores and sample preparation
techniques, requiring minimal sample optimization. For these reasons, SIM is quickly becoming the
most approachable live cell super resolution method.
We will present an overview of structured illumination microscopy including the available SIM
methods (3D SIM, 2D SIM, and 2D TIRF-SIM) and their target applications. The practical considerations
required to utilize SIM will also be discussed with an emphasis on the additional factors critical for
successfully imaging live cells in SIM.
95
ELMI meeting
european
light microscopy
initiative
elmi
16th international
ELMI meeting
Hamamatsu Photonics Deutschland GmbH
A step change in practical usability: Hamamatsu’s simultaneous
two-channel imaging system
Learn how to adjust the W-VIEW GEMINI properly and to use the ORCA –Flash 4.0 LT in GEMINI mode
The W-VIEW GEMINI is an image splitting optics which provides one pair of dual wavelength images
separated by a dichroic mirror onto a single camera. Operating it together with the ORCA-Flash 4.0 LT
in the new W-VIEW Mode allows you to set independent exposure times and readout directions for
your two-channel images. This is the most advanced combined system for your Multicolor TIRF, FRET,
Bifocal imaging or Simultaneous Fluorescence application. This workshop convinces you how easy it is
to adjust and use the GEMINI system.
96
16th international
ELMI meeting
european
light microscopy
initiative
elmi
16th international
Hamamatsu Photonics Deutschland
GmbH
ELMI meeting
Synchronization and triggering the ORCA-Flash 4.0 Scientific
CMOS Camera with peripheral equipment
As in sCMOS technology the readout of the chip is different than in CCD-technology triggering
becomes more challenging. The rolling shutter allows faster and even more precise image acquisition
when it is coupled with the experimental controlling unit. For example spinning disk applications
require a precise synchronization of the camera, light source and spinning disk. In this workshop you
learn which trigger possibilities exist and how to achieve global reset with a rolling shutter camera.
97
ELMI meeting
european
light microscopy
initiative
elmi
16th international
ELMI meeting
ibidi GmbH
Booth MB02
(WS1, WS4)
ibidi
GmbH
Quantitative, real-time oxygen measurement during live cell imaging
Quantitative, real-time oxygen measurement during live cell
ibidi
Booth (WS1, WS4)
imaging
Oxygen
Oxygenplays
playsa acrucial
crucialrole
roleininthethemetabolism
metabolismofofbiological
biologicalmicroorganisms
microorganismsand
andcells.
cells.While
While inin
atmospheric
21 kPa,
kPa, the
the oxygen
oxygen levels
levelseven
eveninside
insideofofhealthy
healthy
atmospheric air
air the
the partial
partial pressure
pressure of
of oxygen
oxygen isis 21
tissues and cells are drastically reduced to roughly 2kPa. Inside of solid tumor cells virtually no
tissues and cells are drastically reduced to roughly 2kPa. Inside of solid tumor cells virtually no oxygen
oxygen is present. The role of hypoxia is becoming of increasing interest in diverse biological
is present.fields
The role
is becoming ofstem
increasing
interest
diverseresearch.
biologicalThereby,
researchitfields
research
suchofashypoxia
tissue engineering,
cell studies
andincancer
is
suchonly
as tissue
stem cell studies
and cancer
research.
it is in
notvitro
onlycell
crucial
to
not
crucialengineering,
to provide physiological
oxygen
conditions
whenThereby,
conducting
culture
experiments,
but
also
to
measure
the
actual
oxygen
levels
in
the
microenvironment
of
the
provide physiological oxygen conditions when conducting in vitro cell culture experiments, but also to
respective
layeroxygen
or tissue.
measure thecell
actual
levels in the microenvironment of the respective cell layer or tissue.
workshop,
introduce
an oxygen
measurement
(OPAL)
InInthisthis
workshop,
we we
willwill
introduce
an oxygen
measurement
systemsystem
(OPAL)
whichwhich
allowsallows
quantitative,
subcellular
microenvironments
quantitative, real-time
real-time monitoring
monitoringofofabsolute
absoluteoxygen
oxygenlevels
levelsin in
subcellular
microenvironments
and
cells.The
Thedevice
deviceis isadaptable
adaptabletotoany
anyfluorescent
fluorescent
microscope
to detect
and even
even inside
inside of
of living
living cells.
microscope
to detect
the
the lifetime of phosphorescence of a oxygen-sensitive reporter sensor that can be incooperated
lifetime
of
phosphorescence
of
a
oxygen-sensitive
reporter
sensor
that
can
be
incooperated
(extraor
(extra- or intracellularly) in cell cultures in a non-invasive manner.
intracellularly) in cell cultures in a non-invasive manner.
98
16th international
ELMI meeting
ibidi GmbH
european
light microscopy
initiative
elmi
16th international
ELMI meeting
ibidi GmbH
New tools to study cell migration with live cell microscopy
New tools to study cell migration with live cell microscopy
Cell migration plays an important role in many physiological and pathological processes such as
ibidi
(WS3, and
WS6)
tissueBooth
generation
repair as well as cancer metastasis. While there are several established methods
to study
cell migration,
chemotaxis
co-culture
in vitro,andinvestigating
processes
under
Cell
migration
plays an important
role and
in many
physiological
pathologicalthese
processes
such as
tissue
generation
and
repair
as
well
as
cancer
metastasis.
While
there
are
several
established
physiological conditions with live cell imaging still remains a challenge.
methods to study cell migration, chemotaxis and co-culture in vitro, investigating these processes
under physiological conditions with live cell imaging still remains a challenge.
We developed several specialized chambers with confined geometries and materials that allow
We
several
specialized
confined
geometries
and materials
that allow
the developed
investigation
of migrating
cellschambers
and evenwith
slowly
migrating
chemotactic
cells towards
a chemical
the
investigation
of migrating
cells andFurther,
even slowly
migrating chemotactic
towards
a
stimulant
with live
cell microscopy.
we developed
a slide that cells
allows
co-culturing
and
chemical
stimulant
withinlive
cell microscopy.
Further,assays,
we developed
a slide that
transmigration
of cells
combination
with perfusion
thereby providing
anallows
ideal inco-vitro model
culturing and transmigration of cells in combination with perfusion assays, thereby providing an
for e.g. endothelial and cancer-interaction studies.
ideal in vitro model for e.g. endothelial and cancer-interaction studies.
In In
this
wound healing,
thisworkshop,
workshop,wewepresent
presentintegrated
integratedmethods
methodsasaswell
wellasasscientific
scientificapplications
applications for
for wound
healing, chemotaxis, co-culture and transmigration in combination with live-cell timelapse
chemotaxis, co-culture and transmigration in combination with live-cell timelapse microscopy.
microscopy.
99
ELMI meeting
european
light microscopy
initiative
elmi
16th international
ELMI meeting
Intelligent Imaging Innovations GmbH
In the workshops we will present our new Lattice Lightsheet Microscope System.
Collecting 3D data from live samples has always been challenging and largely limited by obscuring
out of focus signal and photodamage from repeated whole volume illumination. Lightsheet microscopy
has been used effectively to overcome both of these issues by providing optical sectioning combined
with lower light dose to the sample whilst maintaining signal. Until recently however lightsheet
microscopy has mainly been used for imaging multi-cellular structures. The new Lattice Lightsheet
system offers improvements in speed and resolution such that dynamic intracellular events and even
single molecules can be imaged in 3D over extended periods of time.
Lattice LightSheet uses ultra-thin sheets of light to image 3D cellular dynamics for hundreds of
volumes at dozens of frames per second at diffraction-limited resolution and super-resolution.
Invented by Nobel Laureate Dr. Eric Betzig of the Howard Hughes Medical Institute Janelia Research
Campus, this microscope has been applied to biological systems spanning four orders of magnitude
in space and time. 3D experiments previously limited by phototoxicity in just seconds or minutes can
now be continued safely for hours or days. The combination of high spatiotemporal resolution, speed
and sensitivity make the Lattice LightSheet the ultimate tool in a new era for living cell microscopy.
An extremely sensitive primary objective coupled with a custom-designed illumination system allows
optical sectioning using extremely low light doses for imaging with unprecedented duration. A highspeed spatial light modulator (SLM) in combination with an annular mask allows spatially confined
optical lattices to be projected onto the sample. A galvo mirror controls lattice movement, either
dithering to form a uniform sheet or discretely stepping for super resolution structured illumination
microscopy (SIM).
100
Leica Microsystems CMS GmbH
Am Friedensplatz 3
68165 Mannheim, Germany
16th international
WORKSHOP ROOM 102 (WS1,WS4)
ELMI meeting
european
light microscopy
initiative
elmi
When studying subcellular detail both resolution and sensitivity
of a confocal microsco
16th international
Leicalimiting.
Microsystems
meeting of a confocal microsco
become
Different approaches to improve theELMI
resolution
Seminar Roombut
102 (WS1,
available,
they WS4)
are constantly challenging the sensitivity of the system.
HyVolution
turns
super-sensitivity
into 140thenm
resolution
In
this workshop,
we demonstrate
how we improve
resolution
of the Leica TCS SP8
confocal
microscope
using
super-sensitive
HyD
hybrid
detectors
and
When studying subcellular detail both resolution and sensitivity of a confocal microscope Huygens
can become deconvo
in
our
new
HyVolution
system.
Compared
to
the
usual
diffraction
limit
of around
limiting. Different approaches to improve the resolution of a confocal microscope are available,
but 240 n
achieve sub-diffraction limited resolution of 140 nm laterally and an axial resolution in
they are constantly challenging the sensitivity of the system.
by a factor of 2.
In this workshop, we demonstrate how we improve the resolution of the Leica TCS SP8 confocal
microscope
using super-sensitive
HyD high
hybridsignal-to-noise
detectors and Huygens
deconvolution
in our newBeing a p
This is possible
because of the
ratio of
the HyD detector.
HyVolution
system.
Compared
to
the
usual
diffraction
limit
of
around
240
nm,
we
achieve
subcounting detector, the Leica HyD circumvents noise amplification associated
with tradi
diffraction limited
resolution
140 nm laterally
and an axial resolution
increase bytoa(GaAsP)
factor of 2. photomultip
intensity
averaging.
Itsofsuperior
signal-to-noise
ratio compared
This translates
is possible because
of theresolution
high signal-to-noise
ratio of the with
HyD detector.
a photonthus
into higher
when combined
state ofBeing
the art
deconvolution
counting detector, the Leica HyD circumvents noise amplification associated with traditional intensity
averaging.
Its superior
signal-to-noise
compared
to (GaAsP)
photomultipliers
thus translates
into
Even
single
molecules
such asratio
DNA
origamis
can be
reliably resolved
as demonstrated
when combined
with state of the art deconvolution.
ahigher
140 resolution
nm nanoruler
(see below).
Even single molecules such as DNA origamis can be reliably resolved as demonstrated using a 140
The
main advantage
nm nanoruler
(see below).of using HyVolution over other techniques addressing this resoluti
domain
is
that
youofcan
choose
to acquire
multiple
colors
simultaneously
The main advantage
usingfreely
HyVolution
over other
techniques
addressing
this resolution
domain is at full sp
This
makes
it
ideal
for
fixed
specimen
and
live
cell
imaging
alike.
that you can freely choose to acquire multiple colors simultaneously at full speed.
This makes it ideal for fixed specimen and live cell imaging alike.
XY
Top:
A nanoruler
(DNA origami)
with
Top:
A nanoruler
(DNA
origami) w
a defined
140 nm
spacing spacin
defined
140fluorophore
nm fluorophore
resolved
with HyVolution
is resolved
with HyVolution
(right), but (right),
traditionalconfocal
confocal
imaging (le
not by
by traditional
imaging
Bottom:
Increase
in
axial
resolutio
(left).
Mitochondrial
membranes
Bottom: Increase in axial resolution: labelled
TOM20-GFP.
Sample
courtesy Ur
Mitochondrial
membranes
labelled
Ziegler,
ZMB,
University
with TOM20-GFP. Sample courtesyof Zuric
Switzerland
Urs Ziegler,
ZMB, University of Zurich,
Switzerland
XZ
101
ELMI meeting
european
light microscopy
initiative
elmi
16th international
ELMI meeting
Leica Microsystems
Seminar Room 102 (WS2,WS5)
LAS X – Intuitive, Innovative, Indispensable
Guided Image Acquisition and Analysis in 2D and 3D at its best
The Leica Application Suite X (LAS X) combines the most powerful features available today in
microscope software into one package, focusing on usability in every aspect of the interface,
functionality and workflow.
The workflow oriented design and the possibility to recall user defined system settings makes
LAS X a very intuitive imaging platform customized to your needs. LAS X offers innovative software
and hardware options like the Environmental Control module, the Mobile Connection module for
remote access to the imaging station, 3D GSD Wizard, Lambda Square Scan for White-Light Laser
systems or integrated 2D / 3D deconvolution tools. These tools combined with the workflow oriented
user interface makes LAS X an indispensable solution for widefield, confocal and super-resolution
microscope systems.
Obtain reproducible analysis results rapidly and easily with the 3D and 2D image analysis wizards.
Go step by step through the guided workflow: from applying filters, thresholding, and binary image
processing to measurements and classification. Use a binary reference mask in multi-channel image
analysis, for example, to count the number of spots in each nucleus. In addition, you can perform 3D
and 2D tracking experiments or call your ImageJ macros from within the 2D analysis wizard.
The LAS X software goes far beyond simple image acquisition with a host of analysis options. Easily
save and export analysis reports for later review using LAS X or Excel tools. Create user-defined
protocols that can be applied to multiple data sets, or batch process large amounts of data to save time
and ensure compatibility between experiments.
In this workshop, learn the fundamentals of the LAS X software and find out how this powerful
software suite can streamline, enable and enhance your imaging experiments.
Leica Microsystems
LAS X – Intuitive, Innovative, Indispensable
Guided Image Acquisition and Analysis in 2D and 3D at its best
Oliver Schlicker
Leica Microsystems CMS GmbH
Ernst-Leitz-Straße 17-37
35578 Wetzlar, Germany
Workshop room 102 (WS2, WS5)
The Leica Application Suite X (LAS X) combines the most powerful features available today in microscope software into one package, focusing on usability in every aspect of the interface, functionality and workflow.
The workflow oriented design and the possibility to recall user defined system settings makes LAS X a very intuitive imaging platform customized to your needs. LAS X offers innovative software and hardware options like the Environmental Control module, the Mobile Connection module
for remote access to the imaging station, 3D GSD Wizard, Lambda Square Scan for White-Light Laser systems or integrated 2D / 3D deconvolution tools. These tools combined with the workflow oriented user interface makes LAS X an indispensable solution for widefield, confocal and
super-resolution microscope systems.
Obtain reproducible analysis results rapidly and easily with the 3D and 2D image analysis wizards. Go step by step through the guided workflow: from applying filters, thresholding, and binary image processing to measurements and classification. Use a binary reference mask in multichannel image analysis, for example, to count the number of spots in each nucleus. In addition, you can perform 3D and 2D tracking experiments or call your ImageJ macros from within the 2D analysis wizard.
The LAS X software goes far beyond simple image acquisition with a host of analysis options. Easily save and export analysis reports for later review using LAS X or Excel tools. Create user-defined protocols that can be applied to multiple data sets, or batch process large amounts of data to
save time and ensure compatibility between experiments.
In this workshop, learn the fundamentals of the LAS X software and find out how this powerful software suite can streamline, enable and enhance your imaging experiments. 102
Petra Haas, Irmtraud Steinmetz
16th Microsystems
international
Leica
CMS GmbH
ELMI meeting
Am Friedensplatz 3
68165 Mannheim, Germany
european
light microscopy
initiative
Leica Microsystems
Seminar
Room 102 (WS3,
WS6) 102 (WS3, WS6)
Workshop
room
elmi
16th international
ELMI meeting
Light
microscopy
is a gentle
of imaging
light-sensitive sample
Leica
TCSsheet
SP8 DLS
- Lightsheet
on amethod
Confocal
Platform
processes
whole organisms. The specimen is illuminated in a single plane
From
Cells toinOrganisms
phototoxic effects and protects the sample. By moving the sample along the
Lightthree-dimensional
sheet microscopy is a gentle
methodisof realized.
imaging light-sensitive
samplescamera
or fast biological
structures
A high-speed
allows the ima
processes
in
whole
organisms.
The
specimen
is
illuminated
in
a
single
plane,
which
reduces
phototoxic
processes. Light sheet imaging is therefore an ideal tool for observations of
effectsorganisms
and protects the
By moving
sample along the plane, imaging of three-dimensional
insample.
real time
and the
3D.
structures is realized. A high-speed camera allows the imaging of fast cellular processes. Light sheet
Light
sheetanmicroscopy
usually of
requires
dedicated
optical
setup
imaging
is therefore
ideal tool for observations
developinga organisms
in real
time and
3D. on a specia
illumination
andrequires
the detection
objectives
perpendicular
to each
Lightthe
sheet
microscopy usually
a dedicated optical
setup onare
a specialized
system, where
the other
module
DLS
(Digital
Light
fromto Leica
Microsystems
uses DLS
a unique m
illumination
and the
detection
objectives
areSheet)
perpendicular
each other.
The light sheet module
sheet illumination
detection
beam
path the
into
the vertic
(Digitalintegrates
Light Sheet)the
fromlight
Leica Microsystems
uses a uniqueand
mirror
device, which
integrates
light
TCS beam
SP8.path
Once
SP8
into
a TCS
light
sheet inverted
illuminationLeica
and detection
into atheTCS
vertical
axisisof turned
an inverted
Leica
SP8.sheet
Once instrum
microscope
or confocal
functionality.
a TCS compromise
SP8 is turned intoon
a light
sheet instrument
there is no compromise
on microscope or confocal
functionality.
In this workshop, we will show a TCS SP8 DLS system and focus on its fiel
In this workshop, we will show a TCS SP8 DLS system and focus on its fields of application. Sample
Sample variety goes from adherent cells to organisms, e.g. zebrafish embryo
variety goes from adherent cells to organisms, e.g. zebrafish embryos. Sample handling and preparation
preparation
willLearn
be about
discussed
in this
context. that
Learn
about the
new
objective
will beand
discussed
in this context.
new objective
combinations
will broaden
range
will
broaden
the
range
of
possible
applications
even
more.
of possible applications even more.
Image Courtesy of Dr. Meng-Tsen Ke and Dr. Takeshi Imai, Riken Center, Japan
Image Courtesy of Dr. Meng-Tsen Ke and Dr. Takeshi Imai, Riken Center, J
103
ELMI meeting
european
light microscopy
initiative
elmi
16th international
ELMI meeting
Luxendo, Heidelberg
Luxendo, Heidelberg
A practical approach to light-sheet microscopy – focus on the
A
practical approach to light-sheet microscopy – focus on the essential
essential
Seminar
404 Luxendo,
(WS1, WS3,
In this Room
workshop,
the WS5)
light-sheet company, introduces to you the concept of light-sheet
fluorescence microscopy (LSFM) and presents the MuVi-SPIM, the fastest LSFM for 3D imaging of large
In this workshop, Luxendo, the light-sheet company, introduces to you the concept of light-sheet
specimen. microscopy (LSFM) and presents the MuVi-SPIM, the fastest LSFM for 3D
fluorescence
imaging
large specimen.
LSFM isofa non-invasive
optical method ideally suited for the observation of living specimens. It utilizes
a sheet of laser light to illuminate only a thin slice of a fluorescently labeled sample. A wide-field
LSFM is a non-invasive optical method ideally suited for the observation of living specimens. It
fluorescence
microscope,
placed
perpendicular
serves to collect
the fluorescence
utilizes
a sheet
of laser light
to illuminate
onlytoa the
thinlight-sheet,
slice of a fluorescently
labeled
sample. A
signal and fluorescence
to image the observed
region
to a camera.
This side-on
features
wide-field
microscope,
placed
perpendicular
to the illumination
light-sheet, configuration
serves to collect
the
fluorescence
signal
and
to
image
the
observed
region
to
a
camera.
This
side-on
illumination
several advantages: intrinsic optical sectioning, excellent signal-to-noise ratio, high temporal
configuration features several advantages: intrinsic optical sectioning, excellent signal-to-noise
resolution,
and drastically
reduced
and photobleaching
phototoxicity inside
living specimens.
The
ratio,
high temporal
resolution,
andphotobleaching
drastically reduced
and phototoxicity
inside
non-conventional
of LSFM opensgeometry
up a completely
new
wayup
of asample
mounting,
living
specimens. geometry
The non-conventional
of LSFM
opens
completely
new enabling
way of
sample
mounting,
enabling
image
for 3D
imaging
simple
convenient
multi-view
imageconvenient
acquisitionmulti-view
for 3D imaging
by acquisition
simple rotation
of the
samplebywithin
the
rotation of the sample within the medium-filled chamber.
medium-filled chamber.
Theunique
uniqueimplementation
implementationofof Luxendo’s
Luxendo’s MuVi-SPIM
provides
fourfour
simultaneous
orthogonal
views
The
MuVi-SPIM
provides
simultaneous
orthogonal
views
even without
thefor
need
for sample
In combination
with the
latest generation
high
even without
the need
sample
rotation.rotation.
In combination
with the latest
generation
high sensitivity,
sensitivity, high speed sCMOS cameras operated at 100 full frames per second, complete 3D
high speed
sCMOS
cameras
operated
100 full atframes
perillumination
second, complete
3D stacks
be acquired
stacks
can be
acquired
within
a fewatseconds
lowest
intensities.
Thiscan
allows
very
within
a few
seconds
at lowestofillumination
intensities.
ThisObservation
allows very long
time series
long
time
series
observations
fast dynamic
processes.
objective
lensesobservations
with high
numerical
aperture
and smallObservation
magnification
providelenses
sub-micrometer
resolution aperture
on a largeand
fieldsmall
of
of fast dynamic
processes.
objective
with high numerical
view of more than 500 µm. To ensure optimal conditions for the sample, the setup is completed
magnification
provide sub-micrometer
resolution
on 3D
a large
field ofand
viewrotation
of moreofthan
500 µm. To
by
a temperature-controlled
sample stage
for precise
translation
the sample.
ensure optimal conditions for the sample, the setup is completed by a temperature-controlled sample
The
easy-to-useand
design
of of
thetheMuVi-SPIM
ensures the best long-term optostagecompact
for preciseand
3D translation
rotation
sample.
mechanical and thermal stability for your live imaging experiments. Last but not least, with the
The
compact
and
easy-to-use
design
of
the
MuVi-SPIM
ensures
the best long-term opto-mechanical
MuVi-SPIM, Luxendo provides software and hardware tools to handle and access the resulting
and thermal
stability
your
livedelivers
imaging
experiments.
Last
but not least,
withsample.
the MuVi-SPIM,
large
data image
streamforand
thus
to you
the full 3D
information
on your
Luxendo provides software and hardware tools to handle and access the resulting large data image
stream and thus delivers to you the full 3D information on your sample.
104
16th international
ELMI meeting
Luxendo, Heidelberg
european
light microscopy
initiative
elmi
High-speed imaging of larger objects with the MuVi-SPIM
16th international
Luxendo, Heidelberg
Seminar Room
404404
(WS2,
WS4,WS4,
WS6)WS6)
Seminar
Room
(WS2,
ELMI meeting
In this workshop, Luxendo, the light-sheet company, presents to you how to image larger
High-speed
of larger
objects
the MuVi-SPIM
biological
objectsimaging
such as Drosophila
embryos
on thewith
MultiView
(MuVi)-SPIM light-sheet
microscope.
In this workshop, Luxendo, the light-sheet company, presents to you how to image larger biological
objects
as Drosophila
embryos
the MultiView
(MuVi)-SPIM
light-sheet
microscope.
On the such
MuVi-SPIM,
samples
are on
mounted
from below
on a 3D
translation
and rotation stage.
Embedded
in
aqueous
medium,
they
are
illuminated
from
two
opposing
directions
and observed
On the MuVi-SPIM, samples are mounted from below on a 3D translation and rotation
stage.
using two high numerical aperture objective lenses to provide four simultaneous orthogonal
Embedded
in
aqueous
medium,
they
are
illuminated
from
two
opposing
directions
and
observed
views. Speed-optimized multiple view imaging jobs are easily configured in the user interface by
using two high
aperture
objective
lenses to3Dprovide
simultaneous
orthogonal
views.
combining
setsnumerical
of spectral
channel
information,
stackfour
geometry
and timing
parameters.
Subsequently,
they
are
executed
by
the
microscope
without
interaction
from
the
user.
Luxendo
Speed-optimized multiple view imaging jobs are easily configured in the user interface by combining
also
and hardware
handle and
access parameters.
the resultingSubsequently,
large image they
data
sets ofprovides
spectralsoftware
channel information,
3Dtools
stack togeometry
and timing
streams.
are executed by the microscope without interaction from the user. Luxendo also provides software and
hardware
tools to handle
and access
image data
streams.
We
demonstrate
the workflow
howthe
toresulting
get fromlarge
specimen
mounting
to the full 3D microscopic
view
on
your
sample
acquired
with
unprecedented
speed
and
sensitivity.
We demonstrate the workflow how to get from specimen mounting to the full 3D microscopic view
on your sample acquired with unprecedented speed and sensitivity.
Maximum intensity projections of 3D stacks of a Drosophila embryo expressing histone H2AmCherry acquired at two time points (0 min, 215 min) of a 2 min resolution time series (Krzic et
Maximum
intensity 9,
projections
al.,
Nature Methods
2012) of 3D stacks of a Drosophila embryo expressing histone H2A-mCherry
acquired at two time points (0 min, 215 min) of a 2 min resolution time series (Krzic et al., Nature
Methods 9, 2012)
105
ELMI meeting
european
light microscopy
initiative
elmi
16th international
ELMI meeting
Nikon
Glass Room I. (WS1, WS4)
Nikon has launched a new, simplified, N-SIM E system for structured illumination microscopy (SIM).
Our N-SIM E offers equal resolution improvements to conventional, more complicated SIM systems,
namely 110 nm lateral and 270 nm axial resolution for 3D SIM imaging. However, our simpler
hardware and improved reconstruction software module make this system approachable by any user
in a multiuser facility environment.
N-SIM E is based on light diffraction to generate 3D structured illumination, and can mount our
superior, SR grade 100x 1.49 NA (oil immersion) and 60x 1.27 NA (water immersion) objectives, as
well as our 40x 0.95 NA lens (dry objective). This range of objectives catters for multiple applications,
including deep SIM imaging in cleared tissues (up to 50 µm penetration).
The modular nature of our N-SIM E system and the integration with our confocals mean that
this system can be an easy upgrade to any existing Nikon-TiE microscope, expanding its capabilities
and application possibilities. Also, simpler hardware operation and major improvements in the
software suite enable intuitive imaging and image reconstruction for obtaining satisfactory
results beyond the diffraction limit.
During this workshop, we will:
• Show N-SIM E hardware and software improvements
• Demonstrate system operation with a range of biological fluorescent samples, imaging with our
100x 1.49 NA SR grade objective
• Image cleared tissue to illustrate imaging depth
Nikon welcomes you! For more info: [email protected]
Image: Comparison of widefield image (left) and SIM image (right), HeLa cells stained for actin (green)
and tubulin (red), imaged with 100x 1.49 NA objective.
106
16th international
ELMI meeting
Nikon
Glass Room I. (WS2, WS3, WS5)
european
light microscopy
initiative
elmi
16th international
ELMI meeting
Seamless SIM-confocal imaging: extend confocal resolution by
C2+ integration with N-SIM E
In order to provide easy access to super-resolution information to confocal users, Nikon has improved
software tools to enable a smooth transition from confocal imaging to SIM, and viceversa.
Integrated confocal-SIM acquisitions are critical for many experiments, since both techniques
complement each other, and confocal samples are normally usable for SIM.
By using standard “optical configurations” in our software package, where confocal and SIM
imaging settings can be stored, sequential seamless confocal and SIM imaging is possible. Moreover,
our powerful and intuitive software module JOBS enables full acquisition flexibility. Experiments
like large field of view confocal scanning and selection of regions of interest for SIM super-resolution
imaging are possible and, if desired, automatically executed. Full integration of both imaging modes
is now possible in a single experimental run.
In this workshop, we will illustrate system capabilities by presenting the combination of our novel,
simpler N-SIM E system with our robust C2+ confocal. We will:
• Introduce N-SIM E/C2+ confocal hardware and software
100x 1.49 NA objective, performing confocal-SIM seamless acquisition
• Perform imaging by using our JOBS software module, making seamless confocal + SIM image
acquisition fully flexible, even easier
Image: Composition of Nyquist confocal image and SIM image (center) imaged with 100x 1.45 NA lens,
HeLa cells stained ).for tubulin (green) and mitochondria (red).
107
ELMI meeting
european
light microscopy
initiative
elmi
16th international
ELMI meeting
Nikon
Glass Room I. (WS6)
N-SIM E with 40x dry objective, a whole new range of
applications
Nikon has developed a novel approach to doing structured illumination microscopy (SIM) by
implementing an exceptional, 40x Apo λ 0.95 NA air (dry) objective for super-resolution imaging.
This objective lens can be mounted both on our N-SIM and simpler N-SIM E systems.
Our 40x Apo λ 0.95 NA air objective has an exceptional PSF and boasts a collar for aberration
corrections, ensuring optimal super-resolution imaging. 3D SIM imaging with this 40x objective
achieves a lateral resolution (200 nm) that matches and even improves the performance of our 100x
1.49 NA oil immersion lens for conventional imaging.
SIM has traditionally been limited in the range of possible applications due to small field of view
and focus drift issues. However, by imaging with our 40x air objective, users can now perform tiling
and long time-lapse multipoint acquisitions without experiencing immersion oil problems. Moreover,
scanning applications become possible with an unprecedented level of resolution for dry lens
imaging.
During this workshop, we will:
• Show N-SIM E hardware and software
40x 0.95 NA objective
• Perform imaging by using our JOBS software module, making image acquisition fully flexible but
even easier
Image: Comparison of widefield image (left) and SIM image (right) imaged with 40x 0.95 NA dry lens,
HeLa cells stained for actin (green), tubulin (red) and DAPI (blue).
108
16th international
ELMI meeting
Olympus Europa SE & Co. KG
Seminar Room 103 (WS1, WS2, WS4, WS5)
european
light microscopy
initiative
elmi
16th international
ELMI meeting
FLUOVIEW FV3000
- The new confocal laser scanning
FLUOVIEW FV3000 - The new confocal laser scanning microscope
microscope
Room B1, 103(WS1, WS2, WS4, WS5)
quality
of your
live cell imaging
new confocal
laser scanning
Enhance the quality ofEnhance
yourthelive
cell
imaging
withwitha anew
confocal
lasermicroscope
scanning microscope (cLSM)
(cLSM) system allowing you to work with highest speed, outstanding sensitivity and a great
of macro- and micro-confocal imaging capabilities.
system allowing you tocombination
work with
highest speed, outstanding sensitivity and a great combination of
Olympus introduces the FLUOVIEW FV3000 series, the new cLSM which enables you to highspeed imaging
with 438
fps to capture rapid in vivo responses, and offers new levels of total
macro- and micro-confocal
imaging
capabilities.
system transmission efficiency with the TruSpectral detection concept. The fully spectral
FV3000 has improved overall sensitivity and signal-to-noise ratio for excellent multi-color
Olympus introduces the
FLUOVIEW FV3000 series, the new cLSM which enables you to highconfocal imaging.
speed imaging with 438Getfps
capture
rapid
and tooffers
new levels of total system
moreto
details
at resolutions
downin
to vivo
120 nmresponses,
with the latest addition
the Olympus
FLUOVIEW range of laser scanning microscopes. Widen up your possibilities with the new
TruSpectral
detection
concept baseddetection
on patented Volume
Phase Hologram
(VPH)
transmission
transmission efficiency with
the
TruSpectral
concept.
The
fully
spectral
FV3000 has improved
enabling you to select the detection wavelength of each individual channel to 1 nm precision.
overall sensitivity and signal-to-noise
ratio the
forFLUOVIEW
excellent
multi-color
confocal
Olympus invites you to experience
FV3000
cLSM system in this
workshop. imaging.
Get more details at resolutions down to 120 nm with the latest addition to the Olympus FLUOVIEW
range of laser scanning microscopes. Widen up your possibilities with the new TruSpectral detection
concept based on patented Volume Phase Hologram (VPH) transmission enabling you to select the
detection wavelength of each individual channel to 1 nm precision.
Olympus invites you to experience the FLUOVIEW FV3000 cLSM system in this workshop.
109
ELMI meeting
european
light microscopy
initiative
elmi
16th international
ELMI meeting
Omicron-Laserage Laserprodukte GmbH
LedHUB® - Flexible multicolor LED light engines for microscopy
Using high
power multicolor LED systems expands the possibilities in Microscopy and other Life
LedHUB® - The
Flexible
multicolor
light engines
for microscopy
Science
applications.
workshop
shows LED
the advantages
of modular
Multicolor LED light engines
LedHUB® - Flexible multicolor LED light engines for microscopy
in laboratories
microscope
facilities.
Repeatable
results
over
years
is one of the major goals in
Omicron
BoothOmicron
(WS1, WS3, and
WS5)
Booth (WS1, WS3, WS5)
Using
high power
multicolor
LED
systems expands
the possibilities
inthe
Microscopy
and other
research
and
was
Omicron´s
goal
during
development
of
the
LedHUB®
light engine. The realization
Life Science applications. The workshop shows the advantages of modular Multicolor LED light
Using high power multicolor LED systems expands the possibilities in Microscopy and other
engines in laboratories and microscope facilities. Repeatable results over years is one of the
of these
goals,
hands-on
on
or replacing
wavelengths
as well
explaining
major
goals in Life
research
andawas
Omicron´s
goal presentation
during the
development
of theupgrading
LedHUB®
light advantages
Science
applications.
The
workshop
shows the
of modular
Multicolor
LEDas
light
engine. The realization of these goals, a hands-on presentation on upgrading or replacing
in the
laboratories
microscope
Repeatableare
results
over years
is of
onethis
of workshop.
the
wavelengths
asengines
well as explaining
the
integrationand
ofinto
the
LedHUB®
intofacilities.
a microscopy
the
integration
of
LedHUB®
a
microscopy
environment
the
major
topics
environment are the major topics of this workshop.
major goals in research and was Omicron´s goal during the development of the LedHUB® light
wavelengths as well as explaining the integration of the LedHUB® into a microscopy
environment are the major topics of this workshop.
110
16th international
ELMI meeting
european
light microscopy
initiative
elmi
international
Omicron-Laserage Laserprodukte16th
GmbH
ELMI meeting
BrixXps – Versatile picosecond/CW diode lasers for microscopy
BrixXps – Versatile picosecond/CW diode lasers for microscopy
Picosecond
lasers have always been interesting for microscopic applications like Time-Domain FLIM
Booth (WS2, WS4, WS6)
- Flexible
multicolor
LED light
engines
for microscopy
or TCSPCLedHUB®
but Omicron
had a lot
of restrictions
in usability
for other
microscopic
techniques. The new BrixXps
diode lasers
represent
a
new
type
of
laser
which
can
operate
in
various
operating
modes which
Picosecond
lasers
like include
Time-Domain
Omicron
Booth (WS1,
WS3,have
WS5)always been interesting for microscopic applications
FLIMmode
or TCSPC
butmode
had awith
lot of
in digital
usability
for other microscopic
techniques. The
picosecond pulsed
and CW
fastrestrictions
analog and
modulation
of the laser intensity.
Usingnew
high BrixXps
power multicolor
LED represent
systems expands
in Microscopy
and in
other
diode lasers
a new the
typepossibilities
of laser which
can operate
various operating
Applications
like
confocal
FRAPshows
andpulsed
many
kinds of
techniques
can now
Life Science
applications.
The TIRF,
workshop
the advantages
ofsuperresolution
modular
Multicolor
LED
modes
whichimaging,
include
picosecond
mode
and
CW mode
with
fast light
analog
and digital
engines
in
and
microscope
Repeatable
over imaging,
years
one
of the
modulation
the
laser
intensity.
Applications
likeresults
confocal
TIRF,
FRAP
and many
be realized
with
thelaboratories
sameoflaser
source
by justfacilities.
changing
the operating
mode
of
theislaser
with
one click.
major kinds
goals in
and was Omicron´s
goalcan
during
development
of thethe
LedHUB®
light source by just
ofresearch
superresolution
techniques
nowthebe
realized with
same laser
The workshop
will
give
an
overview
on
the
numerous
functions
of
the
BrixXps
lasers
and
their
use
in
changing the operating mode of the laser with one click. The workshop will give an overview on
wavelengths
as
well
as
explaining
the
integration
of
the
LedHUB®
into
a
microscopy
differentenvironment
microscopic
applications.
the numerous
functions
are the major
topicsof
ofthe
thisBrixXps
workshop.lasers and their use in different microscopic applications.
111
ELMI meeting
european
light microscopy
initiative
elmi
16th international
ELMI meeting
Phasics
Booth MB06 (WS1,WS2,WS3, WS4,WS5, WS6)
High definition quantitative phase microscopy combined to
fluorescence imaging using a single camera thanks to a smart
Phasics
plug
& play opto-mechanical module
High definition
phaseismicroscopy
fluorescence
Quantitative
phasequantitative
microscopy (QPM)
a full-fieldcombined
label-freetoimaging
modality. It delivers
imaging
using
a
single
camera
thanks
to
a
smart
plug
&
play
optohighly contrasted images without any labelling and enables extracting a large highly valuable amount
mechanicaldata
module
of quantitative
such as morphological parameters and dry mass. Main applications are cell growth
Section Hall
(WS1,
WS3, WS4, WS5,
WS6)proliferation, tissue imaging…
monitoring,
stem
cellWS2
differentiation,
bacteria
Phasics
offers an innovative QPM method based on a smart optical element placed in front of a
Quantitative phase microscopy (QPM) is a full-field label-free imaging modality. It delivers
detector.
This assembly
is without
mounted
a conventional
microscope
of a classical
highly contrasted
images
anyonlabelling
and enables
extracting instead
a large highly
valuable camera. One
amount of quantitative data such as morphological parameters and dry mass. Main
great
advantage
is
that
no
additional
element
is
introduced
before
the
sample
leading to artifactapplications are cell growth monitoring, stem cell differentiation, bacteria proliferation, tissue
free
images.
Their
segmentation
and
analysis
are
thus
very
robust.
imaging…
During
this workshop, Phasics will be pleased to show its latest innovation: the SID4-Element. This
Phasics offers an innovative QPM method based on a smart optical element placed in front of
opto-mechanical
optical element
for QPM.
It plugs
to a camera chosen
a detector. This module
assemblyintegrates
is mountedPhasics
on a conventional
microscope
instead
of a classical
One great advantage is that no additional element is introduced before the sample
to camera.
optimize
QPM
for
specific
cases
such
as
ultra-fast
phenomena
study,
fluorescence
leading to artifact-free images. Their segmentation and analysis are thus very robust.
combination or large population screening. The presented configuration works with a Andor
workshop, Phasics will be pleased to show its latest innovation: the SID4ZylaDuring
sCMOSthiscamera.
Both hardware and algorithm are optimized to ensure high resolution and
Element. This opto-mechanical module integrates Phasics optical element for QPM. It plugs
to afield
camera
chosen
optimize reaches
QPM forthe
specific
cases suchresolution
as ultra-fast
large
of view.
Thetoassembly
best possible
of phenomena
any objective from 10x
study, fluorescence combination or large population screening.
The presented
to 150x
(down to 300 nm at 1.3 NA). Field of view is up to 850x720 µm² (at 10x). Therefore single cell
configuration works with a Andor Zyla sCMOS camera. Both hardware and algorithm are
optimized to ensure
high resolution
large field The
of view.
The assembly
reaches
best constituents
measurements
are possible
over largeand
population.
low noise
of the system
letsthesmall
resolution of any objective from 10x to 150x (down to 300 nm at 1.3 NA). Field of
suchpossible
as
organelle
or
plasma
membrane
clearly
appear.
Their
dynamics
can
be
studied
at high frame
view is up to 850x720 µm² (at 10x). Therefore single cell measurements are possible over
rate.large population. The low noise of the system lets small constituents such as organelle or
plasma membrane clearly appear. Their dynamics can be studied at high frame rate.
This
system is of high interest for QPM and fluorescence imaging with a single camera. It
This to
system
is ofboth
high interest
for QPM
and fluorescence
a singleillumination.
camera. It The CMOS
is able
acquire
phase and
fluorescence
image byimaging
simplywith
switching
is able to acquire both phase and fluorescence image by simply switching illumination. The
detector
ensures
high
sensitivity
fluorescence
acquisition.
QPM
and
fluorescence
images are acquired
CMOS detector ensures high sensitivity fluorescence acquisition. QPM and fluorescence
images
are
acquired
on
the
same
detector
and
are
then
easy
to
merge.
This
multi-modality
on the same detector and are then easy to merge. This multi-modality set-up will beset-shown during
will be shown during the workshop.
theupworkshop.
112 16th international
ELMI meeting
PicoQuant GmbH
Seminar Room 403 (WS1,WS2, WS3, WS4, WS5, WS6)
european
light microscopy
initiative
elmi
16th international
ELMI meeting
rapid2FLIM: the new and innovative method for ultra-fast FLIM
imaging of biological processes
KEY WORDS: rapid2FLIM, fluorescence lifetime imaging (FLIM), time-correlated single photon
counting (TCSPC), Foerster resonance energy transfer (FRET), confocal fluorescence microscopy
Over the last two decades, time-resolved fluorescence microscopy has become an essential tool
in Life Sciences thanks to measurement procedures such as Fluorescence Lifetime Imaging (FLIM),
lifetime based Foerster Resonance Energy Transfer (FRET), and Fluorescence (Lifetime) Correlation
Spectroscopy (F(L)CS).
Up to now, FLIM data acquisition is considered a somewhat slow process, due to the reduced scan
speed required to collect a sufficient number of photons per pixel for reliable data analysis. This
makes it difficult to use FLIM for following fast FRET processes in biological samples, such as signal
transduction pathways in cells, fast moving sub-cellular structures (e.g., vesicles), or the contraction of
heart muscle cells. We present here a novel and elegant solution to tackle this challenge.
Our approach, named rapid2FLIM, allows imaging with several FLIM images per second for monitoring
e.g., transient molecular interactions as well as fast moving species. The new method exploits recent
hardware developments such as TCSPC modules with ultra short dead times and hybrid photomultiplier
detector assemblies enabling significantly higher detection count rates. Thanks to these improved
hardware components, it is possible to achieve much better photon statistics in significantly shorter
time spans while being able to perform FLIM imaging for fast processes in a qualitative manner
and with high optical resolution. In this way, fast processes such as protein interactions involved in
endosome trafficking can be studied.
We will show the rapid2FLIM approach as part of a complete turn-key upgrade system for a confocal
laser scanning microscope (Nikon A1R) [1]. The data acquisition on this systems is based on TimeCorrelated Single Photon Counting (TCSPC) electronics with ultra short dead time [2] along with
picosecond pulsed diode lasers as excitation sources and highly sensitive, single photon counting
detectors.
REFERENCES:
[1] B. Krämer, V. Buschmann, U. Ortmann, F. Koberling, M. Wahl, M. Patting, P. Kapusta, A. Bülter, R. Erdmann, “Advanced FRET
and FCS measurements with laser scanning microscopes based on time-resolved techniques”, Proceedings of SPIE, 6860,
68601D (2008).
[2] M. Wahl, F. Koberling, M. Patting, H. Rahn, R. Erdmann, “Time-Resolved Confocal Fluorescence Imaging and Spectrocopy
System with Single Molecule Sensitivity and Sub-Micrometer Resolution”, Current Pharmaceutical Biotechnology, 5, 299308 (2004).
113
ELMI meeting
european
light microscopy
initiative
elmi
16th international
ELMI meeting
PicoQuant GmbH
Seminar Room 403 (WS1,WS2, WS3, WS4, WS5, WS6)
Time-Resolved STED Add-ON for the MicroTime 200
confocal Fluorescence lifetime microscope
KEY WORDS: Fluorescence Lifetime Imaging, Förster Resonance Energy Transfer, Pattern Matching,
Fluorescence (Lifetime) Correlation Spectroscopy, Single molecule spectroscopy, Confocal Laser
Scanning Microscopy, Stimulated Emission Depletion (STED), easySTED
The single molecule sensitive confocal fluorescence lifetime microscope MicroTime 200 has been
extended with STED super-resolution capability. The easy STED [1] principle which is employed allows
for easy adjustment and long-term stability. The underlying Time-Tagged Time-Resolved (TTTR)
data acquisition allows to simultaneously record timing and intensity information, both spectrally
and spatially, on a single photon basis and on time scales from sub-nanoseconds to seconds. Pulsed
Interleaved Excitation (PIE) allows for quasi simultaneous confocal and STED imaging.
• The fluorescence lifetime can change, for example, depending on the fluorophore environment (polarity,
pH, temperature, ion concentration, etc.) and thus enables for sensing of the local environment inside
cells. Furthermore, FLIM can be applied to discriminate multiple labels and to eliminate signal artefacts
(e.g., sample background) thereby allowing a higher detection efficiency and more accurate marker
localization. Also, the autofluorescence is characteristic for a certain tissue and can therefore be used, e.g.,
for tumor detection. The lifetime information can further be used for characterization and quality control
of new materials as new fluorescent labels or quantum dots, that are applied in biological imaging.
• STED super-resolution microscopy, which is based on confocal microscopy, allows for optical resolutions in
fluorescence imaging far below the diffraction limit of light. Thus, smaller structures can be resolved which helps
to clarify biological processes and organization. Time-resolved STED using TTTR leads to more flexibility in data
post-processing, and resolution enhancement such as gated STED (gSTED) can be used to improve image quality.
• FCS allows to measure molecular dynamics, interaction and concentration. Using TCSPC, FCS is
significantly improved by weighting the detected photons according to their fluorescence lifetime. FCS
can also be combined with STED super-resolution. The variable size of the detection volume in STED
allows to study molecules at high concentrations, and anomalous diffusion at varying length-scales.
• Single molecule imaging and spectroscopy can reveal properties of molecular behaviour and
interaction that are obscured in ensemble experiments. Single molecule STED microscopy is a great
tool to study the behaviour of STED labels one-by-one. Bleaching and blinking, as well as lifetime
changes due to molecular interaction or conformational changes can be observed.
REFERENCES:
[1] M. Reuss et. al. “Birefringent device converts a standard scanning microscope into a STED microscope that also maps
molecular orientation”, Opt. Exp. 18, 1049-1058 (2010).308 (2004).
114
16th international
ELMI meeting
european
light microscopy
initiative
elmi
16th international
ELMI meeting
Solutions for photo-manipulation, deep-UV microscopy and
fluorescence
life-time imaging
Rapp OptoElectronic provides a broad range of high quality off-the-shelf and customized devices for
photo-manipulation
in microscopy
covering applications
Solutions for
photo-manipulation,
deep-UV like
microscopy and fluorescence
life-time
imaging
- fluorescence
recovery
after photo-bleaching (FRAP)
WS-1 and WS-5 at the AHF
/ Rapp booth
- photo activation/inhibition,
photo-switching/conversion
- fluorescence life-time imaging (FLIM)
- ablationRapp
– laser
nano-surgery
OptoElectronic
provides a broad range of high quality off-the-shelf and customized
devices for photo-manipulation in microscopy covering applications like
- laser-induced
DNA-damage
- fluorescence recovery after photo-bleaching (FRAP)
- laser temperature-jump
- photo activation/inhibition, photo-switching/conversion
- fluorescence life-time imaging (FLIM)
- optogenetics
– laser nano-surgery
- uncaging -- ablation
laser-induced DNA-damage
- laser temperature-jump
- optogenetics
versatile- UGA-42
uncaging series of galvanometer-based
Our
scanning systems find wide-spread use in
research inOur
fields
such
as
electrophysiology,
neuroscience,
cell biology, chemistry, bioengineering and
versatile UGA-42 series of galvanometer-based scanning systems find wide-spread use in
biomaterials.
It
can
be
used
with
lasers
from
194nm
up
to
1,500nm.
research in fields such as electrophysiology, neuroscience, cell biology, chemistry,
bioengineering and biomaterials. It can be used with lasers from 194nm up to 1,500nm.
Besides off-the-shelf
products we provide customized solutions, including deep-UV microscopes and
off-the-shelf
products we provide customized solutions, including deep-UV
objectives Besides
for deep-UV
applications.
microscopes and objectives for deep-UV applications.
For two-photon imaging and simultaneous photo-manipulation we offer the movable objective
For two-photon imaging and simultaneous photo-manipulation we offer the movable
microscopeobjective
(MOM)microscope
from Sutter
together
stimulation
(MOM)
from with
Sutterour
together
with our devices.
stimulation devices.
In this workshop
we
will
present
an
overview
of
our
products
typicalapplications.
applications.
In this workshop we will present an overview of our products and
and typical
The figure
a deep-UV
transmitted
lightimage
image
ofemulsion
an emulsion
using a from
band-pass from
Theshows
figure shows
a deep-UV
transmitted light
of an
using a band-pass
280 –and
380nm
a UV sensitive
camera.
(8x UV-quartz
objective).
280 – 380nm
a UVandsensitive
camera.
(8x UV-quartz
objective).
115
ELMI meeting
european
light microscopy
initiative
elmi
16th international
ELMI meeting
Scientific Volume Imaging
Booth MB05 (WS1,WS2,WS3,WS4,WS5,WS6)
gpu-accelerated image restoration; now also for light sheet
Company: Scientific Volume Imaging
microscopy
Company: Scientific Volum
Title: Huygens gpu-accelerated image restoration; now also for light sheet microscopy
Workshops
Boothbooth
MB05
(allduring
workshop
time slots).
We very much welcome
you at ourat
Huygens
(also
the workshops),
where we will present
our latest developments, including GPU acceleration, floating licenses, and Light Sheet deconvolution.
Title: Huygens
gpu-accelerated
Nowadays, deconvolution is widely accepted
as a fundamental
technique forimage
restoringrestoration;
microscopy now also f
data from widefield, confocal, spinning disk, multiphoton, and
STED imageatdata.
WeMB05
have recently
Workshops
Booth
(all workshop tim
added
a
new
module
to
Huygens
for
the
deconvolution
of
images
from
a
variety
of
Light
Sheet
We very much welcome you at our Huygens booth (also during the workshops), where we will
present
our /
latest developments,
including
GPU acceleration,
floating licenses,
Selective
Plane Ilumination
Microscopy
(SPIM) imaging
setups. and Light Sheet deconvolution.
Nowadays,
deconvolution
widely accepted
as a volumes
fundamental
technique
for restoring
data
Typically, these
systemsisproduce
very large
of data
placing
a heavymicroscopy
load on computer
from widefield, confocal, spinning disk, multiphoton, and STED image data. We have recently added a new
hardware
software
thefrom
years,
the speed
ofSheet
Huygens
deconvolution
has
module to and
Huygens
for theperformance.
deconvolutionDuring
of images
a variety
of Light
/Selective
Plane
Iluminationalong
Microscopy
(SPIM)
imagingWe
setups.
developed
with the
development
ofvery
themuch
central
processing
units
(CPU).
The
new
Huygens
welcome you at our Huygens booth (also during the workshop
Typically,processing
these systems
large
volumes
of data
placing
a heavy
loadacceleration,
on computer
hardware
latest
developments,
including
GPU
floating
licenses, and Ligh
graphics
unitproduce
(GPU) very
acceleration
option
offers
the
same
high-quality
results,
yet even
and software performance. During the years, the speed of Huygens deconvolution has developed along with
deconvolution
is
widely
accepted
as a unit
fundamental
faster!
You can now
realize
deconvolution
results
within
seconds
using
a high-end
NVIDIA
GPU
card technique for
the development
of the
central
processingNowadays,
units
(CPU).
The new
Huygens
graphics
processing
(GPU)
from
widefield,
confocal,
spinning
disk,
and
STED image data.
acceleration
optionHuygens
offers the
same high-quality
results, yet
even
faster!
You can
nowmultiphoton,
realize
deconvolution
and
the powerful
deconvolution
algorithms.
The
unique
brick-splitting,
also
available
in GPU
module GPU
to Huygens
deconvolution
images from a variety of Light S
results within seconds using a high-end NVIDIA
card andfor
thethe
powerful
Huygens of
deconvolution
mode,
enables you to deconvolve very large
files on Microscopy
the
GPU, evenenables
with cards
withsetups.
limited video-RAM.
Ilumination
imaging
algorithms. The unique brick-splitting, also
available in
GPU mode,(SPIM)
you to deconvolve
very large
on the GPU,
even with is
cards
limited
video-RAM.
In
addition,
the
algorithm
is
able
to
Infiles
addition,
the algorithm
ablewith
to accurately
correct
for
spherical
aberration
in
case
of
aaccurately
refractive
Typically, these systems produce very large volumes
of data placing a heavy
correct for spherical aberration in case of a refractive index mismatch.
and software performance. During the years, the speed of Huygens deconvol
index
mismatch.
Multi-user access for Huygens was already
possible
with Remote
and the web-based
Huygens
development
ofwith
the Display
central
processing
unitsthe
(CPU).
The new Huygens gra
Multi-user access for Huygens was the
already
possible
Remote
Display
and
web-based
Remote Manager, which latest version has
a much faster
and more
powerful
OMERO
database
connection.
acceleration
option
offers
the same
high-quality
results,
yet even faster! You
Huygens
Remote
Manager,
latest
version
hasthata much
faster
anda more
OMERO
database
Lately, we
introduced
a newwhich
floating
license
option
extends
the
flexibility
ofpowerful
these
Huygens
multi-user
results
within
seconds
using
high-end
NVIDIA
GPU
card and the powerful
solutions. Free tests licenses for all Huygens
solutionsThe
can unique
be requested
boothalso
or via
[email protected].
algorithms.
brick-splitting,
available
in
connection.
Lately, we introduced a new
floating
license
option
thatat the
extends
the
flexibility
of GPU
thesemode, enables
files on the GPU, even with cards with limited video-RAM. In addition, the a
Huygens multi-user solutions. Free tests
licenses
for
all
Huygens
solutions
can
be
requested
at
the
correct for spherical aberration in case of a refractive index mismatch.
booth or via [email protected].
Multi-user access for Huygens was already possible with Remote Display an
Remote Manager, which latest version has a much faster and more powerful
Lately, we introduced a new floating license option that extends the flexibilit
solutions. Free tests licenses for all Huygens solutions can be requested at the
116
16th international
ELMI meeting
ThermoFisher Scientific
ThermoFisher
Scientific
Seminar Room 405 (WS1,
WS2, WS3, WS4, WS5, WS6)
european
light microscopy
initiative
elmi
16th international
ELMI meeting
Move away from the dark ages- evolve with Invitrogen™ Evos™ Imaging
Move away from the dark ages- evolve with Invitrogen™ Evos™
systems
Imaging systems
Seminar Room B1 Second Floor, #405 (WS1, WS3, WS 4, WS6) The EVOS line of cell imaging systems range from a simple brightfield/phase contrast microscope
through
to fluorescenct,
dual-camera
instruments
capable
of time lapse,contrast
image
The
EVOS
line of cellfully-automated,
imaging systems
range from
a simple
brightfield/phase
microscope
throughand
to fluorescenct,
fully-automated,
of time
stitching, z-stacking
live cell imaging
with incubationdual-camera
on any vesselinstruments
type. They capable
are designed
to
lapse, image stitching, z-stacking and live cell imaging with incubation on any vessel type. They
eliminate
the
complexities
of
microscopy
while
ensuring
you
obtain
excellent,
publication
quality
are designed to eliminate the complexities of microscopy while ensuring you obtain excellent,
images at every
stage.images
They combine
all stage.
aspectsThey
of a digital
inverted
microscope
into a
publication
quality
at every
combine
all aspects
of workstation
a digital inverted
microscope
workstation
intoona compact
device
that No
powers
a single
need
for
compact device
that powers
with a single
switch.
needon
forwith
the dark
roomswitch.
and noNo
more
time
the
darkwaiting
room for
andbulbs
no more
time wasted
waiting
forsystems
bulbs to
be ready.
celltoimaging
wasted
to be ready.
EVOS cell
imaging
make
imaging EVOS
accessible
almost
systems make imaging accessible to almost every lab and budget. Move away from the dark
every evolve
lab and with
budget.
Move away from the dark ages- evolve with Evos!
agesEvos!
Join us at our hands-on workshop to find out about the latest developments in the Invitrogen EVOS FL
Join
us at our
hands-on
find out about
the latest
developments
in up
thea Invitrogen
Auto system,
as well
as our workshop
wide rangetoof Invitrogen™
Molecular
Probes™
reagents. Pick
Molecular
EVOS FL Auto system, as well as our wide range of Invitrogen™ Molecular Probes™ reagents.
Probes
handbook-the
guide
to
all
things
fluorescentand
discuss
your
current
experiments
withyour
our
Pick up a Molecular Probes handbook-the guide to all things fluorescent- and discuss
microscopy
applicationwith
specialists.
current
experiments
our microscopy application specialists.
117
ELMI meeting
european
light microscopy
initiative
elmi
16th international
ELMI meeting
Visitron Systems Gmbh.
Booth MB16 (WS1, WS3, WS5 and WS2, WS4, WS6)
VisiScope-4Elements: Widefield, Confocal, FRAP and TIRF with
enhanced confocal and TIRF illumination capabilities
Combining high-resolution imaging with photomanipulation helps unveiling the dynamics
of molecular, cellular and developmental events. In this seminar, we present the VisiScope
4Elements system, which integrates high resolution confocal and TIRF imaging with rapid 2D-FRAP
photomanipulation. Besides recent advances in optical and electronic design, we will introduce you to
basic aspects of combined confocal, FRAP and TIRF experiments based on our VisiView® Software. Our
seminar will be split inro two parts
Workshop 1, 3 , 5: Confocal & FRAP applications: New Visitron Homogenizer for Confocal
Spinning Disk Scan Heads:
The new Visitron Systems GmbH “VS-Homogenizer” optics are designed to further enhance the laser
illumination of the widely used Yokogawa Confocal Spinning Disk Unit (CSU). This optical component
can be added to already installed CSU confocal scan heads. The existing functionality of the original
confocal head remains. This enhancement offers even illumination of large cell areas and allows highsensitivity imaging of living cells without the need for mathematical shading correction.
Workshop 2, 4 , 6: Optimized for Superresolution: VisiTIRF Condenser combines fiberbased with direct-coupled laser lines:
Localization-based superresolution microscopy methods like STORM, GSDIM or PALM benefit strongly
from an increased laser power available in the sample plane. Our prototype VisiTIRF Condenser gives
the user maximum flexibility in the selection of output power by providing a directly coupled laser in
addition to standard fiber-coupled lasers.
About Visitron Systems GmbH
Systems and Solutions for Biomedical Research, Development and Manufacturing - “Made in Germany”
Visitron Systems GmbH is known as one of the leading companies supplying imaging-solutions in the
field of microscopy for more than 20 years.
Over the last 10 years, Visitron has invested in in-house developments and manufacturing of optics
(TIRF, FRAP, Laser Merge), hardware and imaging software “Made in Germany”. This has moved Visitron
Systems from a microscopy systems integrator to a manufacturer of imaging and manipulation
solutions with a growing range of products.
Supporting our customers in the field, a competent team of highly qualified scientists from the fields
of biology, human genetics, physics, electronic and computer science are responsible for the success of
Visitron Systems GmbH.
118
16th international
ELMI meeting
ZEISS
Seminar Room 105 (WS1,WS3,WS5)
european
light microscopy
initiative
elmi
16th international
ELMI meeting
Speed and sensitivity are major and interrelated parameters, which empower researchers to follow
even more ambitious strategies in analyzing and dissecting static structures and dynamic processes
in living or fixed samples. From a microscope technology perspective, the obsession is clear: in most
applications, we want higher sensitivity, better signal to noise, and faster image acquisition. One
might think that speed or sensitivity have reached a level of saturation beyond which no further
improvement is possible, yet smart new concepts and their intelligent implementation help us break
through this imaginary wall.
In Lightsheet microscopy, the speed of 3D-image acquisition reaches unmatched levels by changing
from the confocal principle to oblique illumination for optical sectioning in the z-dimension. In the
Lightsheet Z.1, this allows switching from scanning to wide field image acquisition with fast and
high speed digital cameras, while still obtaining precise positional information in z. Due to the speed
and low excitation light exposure, very large z-stacks of several thousand sections in z are no longer
a problem. This also opens new routes, not only in fast imaging of living organisms, but also large
samples such as cleared brains.
However, confocal microscopy is not willing to concede at all. In our LSM 880 with Airyscan, new
detector technologies and methods to extract information from the obtained data, enable us to
improve scanning speed to surprising levels, while not giving up the benefits of low noise and high
resolution, and many more tools, which are unique to scanning systems.
In this workshop, we will
explain and demonstrate, how optical sectioning is achieved in Lightsheet
ZEISS
Sensitivity
and confocal microscopyBoosting
andSpeed
howandyou
canin Light
use Microscopy
these technologies to expand the spectrum and
potential of your own research projects.
Section Hall 105 (WS1, WS3, WS5), Monika Marx and Jacques Paysan
Airyscan principle diagram LSM 880
119
Airyscan principle diagram LSM 880
ELMI meeting
european
light microscopy
initiative
elmi
16th international
ELMI meeting
ZEISS
Entering new dimensions of enhanced optical resolution in
combination with full sample flexibility
In light microscopy, optical resolution is limited by diffraction - as formalized in 1873 by Ernst Abbe,
professor of physics at the University of Jena and co-founder of the Carl Zeiss company. Thus, an image
of a point-like object (such as a GFP molecule) is never a point, but an infinite pattern of light: the “Airy
pattern” - which is described by the point spread function (PSF).
Under normal circumstances, the optical resolution of confocal microscopes is limited by the geometry
of the Airy pattern. To obtain structural information beyond this limit, ZEISS has implemented with
Airyscan a new and revolutionary detection technique that can be added as an option to our Laser
Scanning Microscopes (LSM). In simple terms, an array of highly sensitive GaAsP detectors allows
scanning over the Airy disk resulting in a dataset giving an improved resolution down to values of e.g.
140nm laterally and 400nm axially with 488nm illumination. This technique is extremely simple to
use and benefits all typical LSM applications including multiphoton approaches. Thereby no specific
dyes or preparation methods are required. Thus, Airyscan microscopy offers a maximum flexibility of
choosing samples for microscopy beyond the classical resolution limit.
In a Lightsheet microscope, resolution is not only limited by the laws of optics, but also by the
structure of your sample. Photons emitted e.g. by a GFP molecule inside of a living embryo have to
travel through the specimen before they even reach the microscope objective. Nevertheless, subcellular
structures can be observed in living and intact samples. Furthermore, multi-view imaging can enable
nearly isotropic resolution in all dimensions. Combined with sample integrity, flexibility, and minimal
light exposure, the Lightsheet Z.1 therefore allows for experiments, which were unthinkable in the
past.
In this workshop we will discuss and demonstrate how to improve resolution in LSM 880 with
Airyscan, and in Lightsheet Z.1.
ZEISS
Entering new dimensions of enhanced optical resolution in combination with
full sample flexibility
Section Hall 105 (WS2, WS4, WS6), Monika Marx and Jacques Paysan
Lightsheet Z.1 principles diagram
Lightsheet Z.1 principles diagram
120
16th international
ELMI meeting
Poster abstracts
light m
16th internation
ELMI meeting
16th international
ELMI meeting
european
light microscopy
initiative
elmi
16th international
Poster Abstracts Overview
ELMI meeting
P001 Image analysis and data visualization for high-content microscopy
Balint Antal
P002 Signaling connections of urocortin2 in PC12 cells
Bálint Balogh, Alexandra Stayer-Harci, Gergely Berta, Oktávia Tarjányi, Mónika Vecsernyés,
Hajnalka Ábrahám, József Szeberényi, György Sétáló Jr.
P003 In vitro study of periodontal ligament-derived cells subjected to mechanical
stress
Gergely Berta, Eszter Lukács, Eszter Filó, Judit Dobsa, György Sétáló jr., Gyula Szabó, József
Szalma
P004 Providing quantitative fluorescence microscopy in imaging core-facilities:
Example of the Microscopy Rennes Imaging Center (MRic)
Clément Chevalier, Stéphanie Dutertre, Sébastien Huet and Marc Tramier
P005 Hacking, scavenging, and free software: core facility services on a limited
budget.
Jens Eriksson, Stig Ove Bøe
P006 Quantitative phase imaging applied to biological samples using quadri-wave
lateral shearing interferometry
Antoine Federici, Sherazade Aknoun, Pierre Bon, Julien Savatier, Benoit Wattellier, Serge
Monneret
P007 Localization analysis with rainSTORM
Tamás Gajdos, József Németh, József Sinkó, Dániel Varga, Eric J. Rees, Gábor Szabó, Miklós
Erdélyi
P008 High precision spFRET studies on nucleosome transitions on µs-s time scales
Alexander Gansen, Suren Felekyan, Ralf Kühnemuth, Kathrin Tegeler, Katalin Tóth, Claus
Seidel, Jörg Langowski
P009 Phenotyping mouse embryos in the „Deciphering the mechanisms of
developmental disorders” (DMDD) project
Stefan H. Geyer, Lukas Reissig, Julia Rose, Dorota Szumska, Robert Wilson, Timothy Mohun,
Wolfgang J. Weninger
P010 Correlative light and electron microscopy in 3D: new developments and
applications
Christopher J Guérin, Saskia Lippens and Anna Krem
123
ELMI meeting
european
light microscopy
initiative
elmi
16th international
ELMI meeting
P011 Blob diameter and ring thickness: application to measure axons and their myelin
sheath.
Romain Guiet, Sophie Wurth, Olivier Burri, Silvestro Micera, Grégoire Courtine and Arne Seitz
P012 High-throughput measurements of COPII coat turnover with automated FRAP
Aliaksandr Halavatyi, Christian Tischer, Fatima Verissimo, Antonio Z. Politi, Jan Ellenberg,
Rainer Pepperkok
P013 Cell cycle dependent analysis of CENP-A nucleosomes in human kinetochores by
AB-FRET
Christian Hoischen, Sindy Giebe, Shamci Monajembashi, Peter Hemmerich and Stephan
Diekmann
P014 A decade of light-sheet microscopy – already a museum piece?
Wiebke Jahr, Benjamin Schmid, Michael Weber, Jan Huisken
P015 Benchmarking and selection of algorithms and software in bioimage analysis
Michal Kozubek
P016 Optimizing light sheet microscopy for multicolor imaging of variable size samples
on an open imaging facility
Ludovic Leconte, Francois Waharte , Jean Salamero
P017 Detection of fibroblast growth factors diffusion by fluorescence recovery after
photobleaching (FRAP).
Changye Sun, Marco Marcello, Yong Li, David Mason, Raphaël Lévy and David G. Fernig
P018 Assessment of the LSMTech InverterScope® and it’s application to in vivo
2-photon brain imaging.
Joanne Marrison, William Brackenbury, Sangeeta Chawla, Mark Hunt, Miles Whittington,
Peter O’Toole
P019 Analytical model of the Optical Vortex Scanning Microscope
Jan Masajada, Agnieszka Popiołek-Masajada, Łukasz Płocinniczak
P020 MHC I expression regulates co-clustering and mobility of interleukin-2 and -15
receptors in T cells
Gábor Mocsár, Julianna Volkó, Daniel Rönnlund, Jerker Widengren, Péter Nagy, János
Szöllősi, Katalin Tóth, Carolyn K. Goldman, Sándor Damjanovich, Thomas A. Waldmann,
Andrea Bodnár, György Vámosi
P021 PML nuclear body reorganisation in the nucleus using Erythrocyte Mediated
Force Application (EMFA) technique
Shamci Monajembashi, Birgit Perner & Peter Hemmerich
124
16th international
ELMI meeting
european
light microscopy
initiative
elmi
international
P022 Phalloidin binds to MreB from Leptospira interrogans16th
ELMI meeting
Beata Longauer, Szilvia Barko, Emoke Bodis, David Szatmari and Miklos Nyitrai
P023 Challenges in the Labeling and Detection of Viral RNA by Confocal and SuperResolution Fluorescent Microscopy
Olga Oleksiuk, Ji Young Lee, Ralf Bartenschlager
P024 Rapid2FLIM: the new and innovative method for ultra-fast imaging of biological
processes
Sandra Orthaus-Mueller, Ben Kraemer, Astrid Tannert, Tino Roehlicke, Michael Wahl, HansJuergen Rahn, Rainer Erdmann
P025 Multi-scale transport image of the living cell
Szabolcs Osváth, Levente Herényi, Gergely Agócs, Katalin Kis-Petik, Miklós Kellermayer
P026 Analysis of zebrafish kidney development with time-lapse imaging using a
dissecting microscope equipped for optical sectioning
Birgit Perner, Danny Schnerwitzki, Michael Graf,Christoph Englert
P027 Investigation interactions between nuclear receptors using modern biophysical
methods
Bálint Rehó, Péter Brázda, László Nagy, György Vámosi
P028 Dual fluorescent probes for intracellular organelle imaging: focus on rational
design
Silvie Rimpelová, Tomáš Bříza, Zdeněk Kejík, Kamil Záruba, Tomáš Ruml,Vladimír Král
P029 Performance assessment, monitoring and quality control of fluorescence
structured illumination microscopy (SIM) systems
Arnaud Royon
P030 Dual effects of Ras and Rab interactor 1 (RIN1) on filopodial motility and AMPA
receptor endocytosis participate in long-term depression of hippocampal
neurons
Zsófia Szíber, Attila Ignácz, Sven Beyes, Norbert Bencsik, Krisztián Tárnok, Angelika Hausser,
Katalin Schlett
P031 Fluorescence Lifetime correlation spectroscopy (flcs). A powerful tool to measure
concentrations and molecular interactions
Sandra Orthaus-Mueller, Benedikt Kraemer, Steffen Ruettinger, Volker Buschmann, Olaf
Schulz, Felix Koberling, Rainer Erdmann, Mark A. Hink
125
ELMI meeting
european
light microscopy
initiative
elmi
16th international
ELMI meeting
P032 Use of a 690nm cw laser on a confocal microscope for excitation of long
wavelength probes
Owen M. Schwartz and Juraj Kabat
P033 Coaligned Three-Colour STED Nanoscopy Reveals Cytoskeletal Organization at
Synaptic Sites
Sven C. Sidenstein, Elisa D’Este, Marvin J. Böhm, Johann G. Danzl, Vladimir N. Belov, Stefan
W. Hell
P034 Transform your Laser Scanning Microscope to DifferentialPolarization Laser
Scanning Microscope
Gábor Steinbach, Gábor Sipka, András Barta, István Pomozi, Győző Garab
P035 Spectrally resolved fluorescence induction (SRFI) measurements on single cell
level using confocal microscope
Gábor Steinbach, Jiří Liška, Gábor Bernát, Radek Kaňa
P036 Imaging of spatial and temporal lymphatic growth with single-cell resolution by
tissue decolorization
Andrea Styevkóné Dinnyés, Zoltán Jakus
P037 In the footsteps of “intercellular highways”- formation and function of
membrane nanotubes
Edina Szabó-Meleg, Tamás Madarász, Elek Telek, Brigitta Brunner, Henriett Halász, Kinga
Futó, János Matkó, Miklós Nyitrai
P038 Evidence for homodimerization of the c-Fos transcription factor in live cells
revealed by FRET, SPIM-FCCS and MDmodeling
Nikoletta Szalóki, Jan Wolfgang Krieger, István Komáromi, Katalin Tóth, György Vámosi
P039 EGFP oligomers as natural fluorescence and hydrodynamic standards
György Vámosi, Norbert Mücke, Gabriele Müller, Jan Wolfgang Krieger, Ute Curth, Jörg
Langowski, Katalin Tóth
P040 German BioImaging: Recommendations for ALM-CF operations
Nadine Utz 1 and the German BioImaging network
P041 Artifacts analysis in localization based microscopy
Dániel Varga, József Sinkó, Tamás Gajdos, Gábor Szabó, Miklós Erdélyi
P042 Assembly of Interleukin Receptor Subunits
Ádám Kenesei, Julianna Volkó, Péter Várnai, Felix Bestvater, Jörg Langowski, Thomas A.
Waldmann, Katalin Tóth, György Vámosi
126
16th international
ELMI meeting
european
light microscopy
initiative
elmi
international
(P001) Image analysis and data visualization16th
for
high-content
ELMI meeting
microscopy
Balint Antal
University of Debrecen, Faculty of Informatics
[email protected]
High-throughput/high-content microscopy-based screening (HT/HCS) provides an increasingly
powerful tool to discover and functionally annotate genes and biological pathways, which already
led to several important discoveries, like the systematic identification of genes important for mitosis,
endocytosis, and other fundamental processes. Specialised large-scale image and data analysis
methods are needed to produce phenotypic data, limiting such functional genomic annotation
techniques to researchers of groups that possess that expertise. This means that the community at
large is limited in their access to data and their ability to further mine it after publication, reducing the
impact of the expensive HT/HC screens.
First, an approach to the unsupervised segmentation of images using Markov Random Field will be
presented1. The proposed approach is based on the idea of Bit Plane Slicing. We use the planes as initial
labellings for an ensemble of segmentations. We tested our approach on a publicly available database,
where it proven to be competitive with other methods and manual segmentation.
Furthermore, a novel data visualization tool called Mineotaur (http://www.mineotaur.org) will also
be presented2, which will allow the community to mine further the raw multidimensional feature data
and knowledge from published HT/HC screens leading to a better exploitation of experimental results.
The tool is based on a novel data model allowing the visualization and analysis of extremely large
amounts of data. As a demonstrative example, we use phenotypic data extracted from a HT fission
yeast screen.
1. B. Antal, B. Remenyik, A. Hajdu, SIGMAP 2013, pp. 94-99.
2. Bálint Antal, Anatole Chessel, Rafael E Carazo Salas, GENOME BIOLOGY 16:(283) pp. 1-5. (2015)
127
ELMI meeting
european
light microscopy
initiative
elmi
16th international
ELMI meeting
(P002) Signaling connections of urocortin2 in PC12 cells
Bálint Balogh1, Alexandra Stayer-Harci1, Gergely Berta1, Oktávia Tarjányi1, Mónika Vecsernyés1,
Hajnalka Ábrahám1, József Szeberényi1, György Sétáló Jr.1
1) Department of Medical Biology, Medical School, University of Pécs, Pécs, Hungary
[email protected]
Urocortins (Ucn) are ligands of corticotropin-releasing factor (CRF) receptors and have 3 different
types called urocortin 1, 2 and 3. CRF receptors exist as two main isoforms: CRF-R1 and CRF-R2. Ucn1
binds to receptor type 1 but Ucn2 and Ucn3 have higher affinity towards the type 2 receptor. CRF-R1 is
expressed in the brain and in the pituitary gland whereas CRF-R2 predominates in the central nervous
system, the heart, peripheral organs, the brain, epididymis and in the intestinal tract.
Urocortins have important roles in the control of stress response, anxiety, alcohol consumption,
hemodynamic and neuro humoral regulation, in the physiology of the heart and circulation and also in
pathological alterations of these systems. Urocortin can regulate the hypophysis pituitary adrenal axis,
the immune system, behavior and general well-being.
Urocortin2 is produced in the brain and can pass through the blood-brain barrier. It is also expressed
together with its receptor in the adrenal medulla from where the rat pheochromocytoma cell line
(PC12) has been derived. In this cell line system Ucn2 can increase the level of cyclic AMP (cAMP)
that leads to the secretion of noradrenalin. It also causes the activation of proteinkinaseA and the
phosphorylation of ERK proteins that in turn control the biosynthesis of catecholamines.
Upon treatment of PC12 cells with urocortin2 or nerve growth factor (NGF) the phosphorylation of
ERK becomes elevated. However, if both agents are used simultaneously in these cells the activation
of ERK is weaker. To find out the mechanism of these antagonistic effects we used various mutant
cell lines. The activation of ERK was measured by Western blotting and immunocytochemistry.
Densitometry of the Western signals was followed by statistical analysis.
128
16th international
ELMI meeting
european
light microscopy
initiative
elmi
16th international
(P003) In vitro study of periodontal ligament-derived
cells
ELMI meeting
subjected to mechanical stress
Gergely Berta1, Eszter Lukács2, Eszter Filó2, Judit Dobsa2, György Sétáló jr.1, Gyula Szabó2, József Szalma2
1) Pécs University Medical School, Department of Medical Biology, Pécs, Hungary
2) Pécs University Medical School, Department of Prosthodontics, Pécs, Hungary
[email protected]
The aim of this study is twofold: firstly, to investigate the changes in biosynthetic capacity of PGE2
in periodontal ligament (PDL) cells derived from lower third molars (M3) in response to mechanical
compression, and secondly to elucidate the activation kinetics of certain intracellular signal
transduction proteins involved in stress- and survival pathways associated with these conditions.
In the orthodontic-assisted extraction of an M3 the influence of clinical parameters (position of
the tooth or age of the patient) to orthodontic extraction's success rate is unclear. One of our goals
was to investigate whether impaction status of M3s or aging has any influence to compression forcerelated Cyclooxygenase-2 (COX-2) levels and Prostaglandin E2 (PGE2) release in M3s’ PDL fibroblast
cell cultures as the in vitro model of tooth movement. The other aspect of our work is to discover the
signal transduction background by analyzing the short- and long term activity of proteins like the ERK
(Extracellular Signal Regulated Kinase), JNK (Jun N-Terminal Kinase) or p38.
PDL cells obtained from M3 removed for orthodontic reasons from human donors aged 16-55 years
have been subjected to a static compressive load (2-4 g/cm2) for various periods of time (5 min-48
h). Results of Enzyme-Linked Immunosorbent Assay (ELISA), Western blotting methods have shown
that partially impacted third M3s' PDL cells showed significantly higher PGE2 production and COX-2
expression than cells derived from bony impacted M3s, while the age of patients had no such effect.
Immunocytochemistry analysis has also shown ERK, JNK and p38 activation as early as 5 minutes
under load, and also a possible connection between the sustained ERK activation and COX2 production.
129
ELMI meeting
european
light microscopy
initiative
elmi
16th international
ELMI meeting
(P004) Providing quantitative fluorescence microscopy in
imaging core-facilities: Example of the Microscopy Rennes
Imaging Center (MRic)
Clément Chevalier1, Stéphanie Dutertre1, Sébastien Huet2, Marc Tramier2
1) SFR BIOSIT UMS CNRS 3480 / US INSERM 018- MRicphotonicsplatform, Rennes 1 university, 2 avenue du
Professeur Léon Bernard - 35043 Rennes, France
2) IGDR UMR 6290, Rennes 1 university, 2 avenue du Professeur Léon Bernard - 35043 Rennes, France
Corresponding author: [email protected]
With the growing demand of researchersfor new microscopy methods to investigatemolecular
dynamic in living cells, providing user-friendlymicroscopy technologies for such investigation has
become a crucial challenge for imaging core-facilities.
By the technological development ofa unique home-made prototype for fast-FLIM (fluorescence
lifetime imaging microscopy) images acquisition and the provision of a commercial system for FCCS
analysis (Fluorescence cross-correlation spectroscopy),the “Microscopy Rennes Imaging Center”
platformsucceeds in answering researchers’ expectation by bringing two complementary technologies
for the analysis of protein-protein interaction in living cells by FRET (Förster resonance energy transfer)
and/or FCCS.In order to provide “platform-friendly” systems, our core-facility passed through the
critical step of technological transfer to our users by the development and the implementation of both
home-made and commercial softwares that allow users to be easily trained and quickly autonomous.
The presence of these systems in our platform have promoted the emergence of innovative biological
projects such as the development of new FRET biosensors or the analysis of proteins co-diffusion in
nucleus of living cells by FCCS.
To conclude, thanks to our R&D work, our imaging core-facility succeeds in providing two
“user-friendly” imaging systems for quantitative fluorescence imaging. These systems perfectly
answer the demand of researchers for new imaging methods and promote the emergence of new
internationalbiological research projects.
130
16th international
ELMI meeting
european
light microscopy
initiative
elmi
16th international
(P005) Hacking, scavenging, and free software:
core facility
ELMI meeting
services on a limited budget.
Jens Eriksson, Stig Ove Bøe
Advanced Light Microscopy Core Facility Gaustad, Oslo University Hospital, Rikshospitalet, Oslo, Norway.
[email protected]
Running a core facility in imaging can be arbitrarily expensive and you will have to manage your budget
wisely in order to provide the best possible service to your facility’s users. There are many ways to lower
both running costs and the costs of novel equipment investments, if one employs the mindset and tools
of a hacker. Among the hacker’s tools are: social engineering, lateral thinking, and good old-fashioned
tinkering. By incorporating the hacker’s tools into our core facility, we have been able to significantly reduce
running costs, and to expand facility services at the same time. This is exemplified by the repurposing of an
old Zeiss LSM 510 microscope into an extended live-cell system called FrankenScope.
This presentation is meant to both inspire you, and to give you some helpful and cost-saving hacks,
strategies, tips and tricks that you can employ,in order to make ends meet a little bit easier.
131
ELMI meeting
european
light microscopy
initiative
elmi
16th international
ELMI meeting
(P006) Quantitative phase imaging applied to biological
samples using quadri-wave lateral shearing interferometry
Antoine Federici1, Sherazade Aknoun1, Pierre Bon2, Julien Savatier2, Benoit Wattellier1, Serge Monneret2
1) Phasics, Bat Explorer, Espace technologique de Saint Aubin, Saint Aubin, France
2) Institut Fresnel, CNRS UMR 7249, Aix-Marseille Université, Marseille, France
[email protected]
Quantitative phase imaging (QPI) techniques are now commonly used in microscopy for the imaging
of semi-transparent samples as they give access to the optical path difference (OPD) information. In
addition, those techniques are non-invasive imaging modalities and ensure a fast approach.
We propose to use a commercial quadri-wave lateral shearing interferometer (QWLSI) device
(SID4Bio, Phasics SA, Palaiseau, France) directly plugged onto a lateral video port of a classical inverted
microscope using a standard illumination source. Single-shot measurements with sub-nanometric
OPD precision is achieved along with a diffraction-limited lateral resolution and a true video rate
permitting intracellular components detection and dynamic follow-up.
We will introduce several applications and technical improvements of our technique that take
advantage of its high OPD resolution, its achromaticity and its easy implementation on conventional
setups.
We will show that the high-contrast and artifacts-free images produced by our QWLSI device enable
a precise and automatic cellular segmentation from which we can monitor cell cycle over an entire
population with a cellular resolution as well as a quantitative determination of the cell dry mass.
Besides, QWLSI can be easily combined with other microscopic imaging techniques such as
fluorescence or polarization imaging.
Co-localization of OPD and fluorescence signals measured from a single sample provides
complementary information and thus enhances subcellular components identification. While QWLSI
detects cells morphology and can lead to refractive index measurement, fluorescence signal is related
to specific components such as cells skeleton or DNA for example.
In the other hand, QWLSI can be also used for applications such as ordered fibrous structures imaging
in biological samples like cytoskeleton fibers or collagen using polarized light. Anisotropic specificity is
thus detected and leads to contrast-enhanced images.
Three-dimensional samples reconstruction with intracellular resolution combining QWLSI and
low-spatially coherent illumination will be also presented as well as temperature mapping around
nano- and microsources of heat, like gold nanoparticles, via the measurement of the thermal-induced
variation of the refractive index.
132
16th international
ELMI meeting
european
light microscopy
initiative
elmi
(P007) Localization analysis with rainSTORM16th international
ELMI meeting
Tamás Gajdos1, József Németh1,2, József Sinkó1, Dániel Varga1, Eric J. Rees3, Gábor Szabó1,4, Miklós Erdélyi1
1) Department of Optics and Quantum Electronics, University of Szeged, Szeged, Hungary
2) Department of Computer Algorithms and Artificial Intelligence, University of Szeged, Szeged, Hungary
3) Department of Chemical Engineering and Biotechnology, University of Cambridge, Cambridge, UK
4) MTA-SZTE Research Group on Photoacoustic Spectroscopy, Szeged, Hungary
[email protected]
rainSTORM is a software written in MATLAB for evaluating single-molecule localization based
microscopy measurement (PALM, (d)STORM etc.) or simulation (TestSTORM) data. The aim is to generate a
sub-diffraction resolved image by identifying photoswitched single fluorophores in sequences of images.
An image stack usually consists from 5 to 50 thousand frames, which is necessary for nanometre precision.
The current version of the software includes multiple analysis algorithms (1D Gaussian, 2D single
Gaussian, 2D multi Gaussian) for finding blinking fluorophores, and the possibility to set two different
background estimation methods. The precise processing of this huge amount of data is a demanding and
complex computation problem and done by parallel processing each frame.
Thermal and mechanical drift can be a problem during the measurements, which can lead to loss of
precision. The rainSTROM reviewer includes three modes for lateral drift correction, which can overcome
most of the possible scenarios. Before a reconstruction the results can be filtered, and it is also possible
to use the rejected localizations as markers for probable artifacts. Comparison with other packages
(RapidSTORM, QuickPALM, ThunderSTORM etc.) our software has a greater control over the filtering
parameters and the ability to choose the localization algorithm yields in improved results.
The software was developed in collaboration with the Laser Analytics Group at the University of
Cambridge and the Advanced Optical Imaging Group at the University of Szeged. Here, we present the
newest version of rainSTORM (ver 3.1), its user manual and give a short summary about its usage and
opportunities.
References:
References:
• Rees, E. J., Erdelyi,
M., Schierle, G. S. K., Knight, A., & Kaminski, C. F. (2013) Journal of Optics, 15(9), 094012.
Rees, E. J., Erdelyi, M., Schierle, G. S. K., Knight, A., & Kaminski, C. F. (2013) Journal
• Sinkó, J., Kákonyi,
R., Rees, E.,
Metcalf,
D., Knight, A. E., Kaminski, C. F., ... & Erdélyi, M. (2014) Biomedical optics express, 5(3), 778-787.
of Optics,
15(9),
094012.

Sinkó, J., Kákonyi, R., Rees, E., Metcalf, D., Knight, A. E., Kaminski, C. F., ... &
Erdélyi, M. (2014) Biomedical optics express, 5(3), 778-787.
133
ELMI meeting
european
light microscopy
initiative
elmi
16th international
ELMI meeting
(P008) High precision spFRET studies on nucleosome transitions
on µs-s time scales
Alexander Gansen*1, Suren Felekyan*2, Ralf Kühnemuth2, Kathrin Tegeler1, Katalin Tóth1 Claus Seidel2, Jörg
Langowski1
1) DKFZ, Div. Biophysics of Macromolecules, Heidelberg, Germany
2) Heinrich-Heine-Universität, Inst. für Physikalische Chemie, Düsseldorf, Germany
*equal contributions
[email protected]
Nucleosomes play a dual role in compacting the genome and regulating access to specific DNA
sequences. To unravel the underlying mechanism, we characterized structural and dynamic features
of reconstituted mononucleosomes upon NaCl-induced destabilization, using multiparameter singlemolecule FRET. Samples were labeled with donor/acceptor pairs on the DNA and on histone H2B.
Species-selective fluorescence lifetime analysis and dynamic photon distribution analysis allowed
us to identify new intermediates during nucleosome opening, to describe their sub-millisecond
transition kinetics and to develop a structural model. The complete opening pathway proceeds
through a weakening of the interface between the H2A-H2B dimers and the (H3-H4)2 tetramer on
a 100µs time scale, then by a two-step release of the dimers coupled to DNA unwrapping on slower
time scales, extending from several ms to minutes. Nucleosome opening and detachment of histone
dimers proceed in an asymmetric way, determined by the DNA sequence. A mutation (H2AR81A) at the
interface between H2A and H3 facilitates the initial opening, confirming the central role of the dimer :
tetramer interface in nucleosome stability. Partially opened states such as described here might serve
as a convenient nucleation point for DNA-recognizing proteins.
134
16th international
ELMI meeting
european
light microscopy
initiative
elmi
16th international
(P009) Phenotyping mouse embryos in the „Deciphering
the
ELMI meeting
mechanisms of developmental disorders” (DMDD) project
Stefan H. Geyer1, Lukas Reissig, Julia Rose, Dorota Szumska2, Robert Wilson3, Timothy Mohun3,
Wolfgang J. Weninger1
1) Medical University of Vienna, Center for Anatomy and Cell Biology, Austria
2) Wellcome Trust Centre for Human Genetics, Oxford, UK
3) The Francis Crick Institute Mill Hill Laboratory, London, UK
[email protected]
The mouse has become one of the most important model organisms in biomedical research. Its
genome is fully sequenced. The next important step is to characterise the function each mouse gene
plays in normal and diseased mice. For doing this, a number of national and international projects,
which systematically phenotype single knock out mice are conducted under the umbrella of the
international mouse phenotyping consortium (IMPC) (www.mousephenotype.org). Since about
one third of the single knock-out lines produce homozygous offspring that die pre- or perinatally,
projects such as “Deciphering the mechanisms of developmental disorders” (DMDD) were designed to
provide phenotype information on embryos and placentas. One of the central elements of DMDD is to
phenotype E14.5 embryos with the aid of the high resolution episcopic microscopy (HREM) method.
In this presentation we present results from scoring E14.5 embryos in the scope of the DMDD
project. Until now DMDD generated 6807 embryos of 180 newly produced mouse lines. 64 of those
lines produced offspring that managed to survive embryonic day (E) 14.5. The observed phenotypes
are extremely heterogeneous since some lines were obviously delayed in development. Also a broad
spectrum of abnormalities, in various degrees of penetration could be seen in these embryos. The
spectrum ranged from gross abnormalities, which are even detectable with the naked eye, down
to abnormalities in tissue architecture and small structural defects. Such abnormalities, although
potentially able to cause intra-uterine or perinatal death can be only seen with the aid of HREM.
Examples are provided in this presentation.
In summary, DMDD produces and publishes (www.dmdd.org.uk) comprehensive phenotype
information on embryos of knock out lines that produce homozygous per- or perinatally offspring.
It represents an important and growing resource, which aids researching gene function and is freely
accessible for the scientific community.
135
ELMI meeting
european
light microscopy
initiative
elmi
16th international
ELMI meeting
(P010) Correlative light and electron microscopy in 3D: new
developments
and applications
Correlative light and electron microscopy in 3D: new developments and applications Christopher
J Guérin, Saskia Lippens, Anna Kremer
Christopher J Guérin, Saskia Lippens and Anna Kremer, VIB Bio Imaging Core, Ghent, Belgium VIB Bio Imaging
Core,
Ghent, Belgium
[email protected]
Correlative microscopy (CLEM) is an increasingly valuable tool in biomedical research. At the VIB Bisio core valuable
in Belgium are using h
igh resolution Correlative
microscopy
(CLEM)
anImaging increasingly
tool wine biomedical
research.
At the VIB Bio
light microscopy in combination with volume EM (Serial block face and focused Imaging core
in
Belgium
we
are
using
high
resolution
light
microscopy
in
combination
with
volume
ion beam SEM) to correlate functional and structural information in 3D data sets. EM
(Serial block
focused
ioncultured beam SEM)
to correlate
functional and structural information in 3D data
In bface
oth and
tissues and cells. tissues and cultured cells.
sets. In both
Specimen preparation is critical to the success of both the LM and EM imaging, and in a multi-‐user core facility this means adapting protocols for such diverse Specimen
preparation
is critical
the success
both the LMand andcells EM imaging,
andto inma any multi-
‐user core
specimen types from to
single celled oforganisms in culture tissues from both the plant and animal We have investigated any vorganisms
ariations and
facility this
means
adapting
protocols
for suchkingdoms. diverse specimen
types
from singlem
celled
of embedding and staining to optimize both the structural information as well as cells in culture
to many tissues from both the plant and animal kingdoms. We have investigated many
to make the process of reacquiring the region of interest efficient and accurate in variationsvolume of embedding
and staining
to optimize both the structural information as well as to make the
EM imaging. process of reacquiring the region of interest efficient and accurate in volume EM imaging.
Reconstructing and segmenting out structures of interest as well as accurately correlating the structures at different resolution scales remains a challenge. Reconstructing
segmenting out
interest
aspwell
as accurately
structures
Recent and
improvements in sstructures
oftware of
and image rocessing are mcorrelating
aking this the
more at different
resolution
scales
remains asolution challenge.
improvements
software
and
image
efficient but a definitive is Recent
still unavailable. In in
the interim we have processing
developed a workflow to maximize accuracy while minimizing the twe
ime needed are making
this more efficient
but a definitive
solution
is still unavailable.
In the interim
have
developed
for analysis. a workflow
to
maximize
accuracy
while
minimizing
the
time
needed
for
analysis.
3D
CLEM
remains a
challenging
the richness
and precision
of the bresults
makes
it a powerful
3D research
CLEM rtechnique,
emains a cbut
hallenging research technique, ut the richness and tool.
precision of the results makes it a powerful tool. 136
16th international
ELMI meeting
european
light microscopy
initiative
elmi
16th international
(P011) Blob diameter and ring thickness: application
to
ELMI meeting
measure axons and their myelin sheath.
Romain Guiet1, Sophie Wurth2,3, Olivier Burri1, Silvestro Micera2, Grégoire Courtine3,Arne Seitz1.
1) BioImaging and Optics Plateform, École Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland
2) Bertarelli Foundation Chair in Translational Neuroengineering, Center for Neuroprosthetics and Institute of
Bioengineering, École Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland
3) International Paraplegic Foundation Chair in Spinal Cord Repair, Center for Neuroprosthetics and Brain Mind
Institute, École Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland
corresponding author: [email protected]
Neural interfaces enable reading from and writing into the peripheral nervous system. However,
incomplete characterization of the long-term usability and bio-integration of intra-neural electrodes
have restricted their clinical use. Aiming at filling this gap, we assessed the impact of the implanted
electrodes on the nerve by measuring classical markers of nerve “quality”, i.e. the diameter of the fibers
composing the nerve and the thickness of their myelin sheath, and analyzed with respect to their
distance from the electrode and as a function of implantation time.
Analysis was carried out on implanted nerve cross-sections stained to reveal axons and myelin
membrane. The tiled image of the stained nerve section was acquired on a widefield microscope, and
contained between 15000 and 20000 myelinated and unmyelinated axons per image, distributed in
five regions of interest (ROIs). These different ROIs delimitate the three fascicles contained in the rat
sciatic nerve, the interfascicular compartment as well as the implanted electrode and were manually
drawn. This manual segmentation was necessary to accurately identify the limited number of different
fascicles that were hardly distinguishable by an automated procedure. Then, we quantified axon
diameter using an automated workflow composed of a detection of the local maxima, a radial line
profile and a curve fitting that uses a “Super-Gaussian” formula. In order to optimize the workflow
and initialize the fitting, we measured global (image background) and environmental parameters
(distance to closest neighbor). Finally, we measured the myelin thickness for each axon using a similar,
but simpler, line profile and curve fitting approach.
The developed workflow allowed us to automatically and objectively quantify the impact of
intraneural electrode implantation on fiber density and myelination and contributed substantially to
understanding their biointegration over time. Moreover, the image processing was written in ImageJ
macro language and thus could be easily adapted to accurately measure object diameters in a variety
of experiments.
137
ELMI meeting
european
light microscopy
initiative
elmi
16th international
ELMI meeting
(P012) High-throughput measurements of COPII coat turnover
with
automated
FRAP
High-throughput
measurements
of COPII coat turnover with automated
FRAP
Aliaksandr Halavatyi1, Christian Tischer2, Fatima Verissimo1, Antonio Z. Politi1, Jan Ellenberg1,
1
1,2
Aliaksandr
Halavatyi
, Christian Tischer2, Fatima Verissimo1, Antonio Z. Politi1, Jan Ellenberg1,
Rainer
Pepperkok
Rainer Pepperkok1,2
1) Cell Biology and Biophysics Unit, EMBL, Heidelberg, Germany
1) Cell Biology and Biophysics Unit, EMBL, Heidelberg, Germany 2) Advanced Light Microscopy Facility, EMBL,
2)
AdvancedGermany
Light Microscopy Facility, EMBL, Heidelberg, Germany
Heidelberg,
[email protected]
[email protected]
Vesicular protein export from ER is mediated by the turnover of COPII coat at ER-exit sites (ERES).
Vesicularofprotein
fromproteins
ER is mediated
by the
turnover
of COPII
coat characterised
at ER-exit sites
Influence
severalexport
regulatory
on COPII coat
kinetics
has been
previously
with
(ERES). Influence of several regulatory proteins on COPII coat kinetics has been previously
manually
supervised Fluorescence Recovery After Photobleaching (FRAP) experiments. Systematic studies
characterised with manually supervised Fluorescence Recovery After Photobleaching (FRAP)
of
ERES regulatory
mechanisms
require,
however,
substantial
increase ofrequire,
experimental
throughput
that
experiments.
Systematic
studies
of ERES
regulatory
mechanisms
however,
substantial
increase
of experimental
throughput
can be achieved
integrating FRAP with automated
can
be achieved
by integrating
FRAP withthat
automated
feedback by
microscopy.
feedback microscopy.
We developed a set of computational tools for automated FRAP to design and to conduct multistep
acquisition
and analysis
The toolset
Fiji-dependent
framework
contains
We developed
a set ofprotocols.
computational
toolsincludes
for automated
FRAP Java
to design
and that
to conduct
multistep identifying
acquisitiontarget
and analysis
protocols.
toolset includes
Fiji-dependent
Java framework
functions
cells, acquisition
andThe
bleaching
regions. Customisable
workflow
procedures
that contains functions identifying target cells, acquisition and bleaching regions. Customisable
trigger
execution
of
these
functions
on
acquired
images
and
transmit
positions
of
identified
regions
to a
workflow procedures trigger execution of these functions on acquired images and transmit
Zeiss
LSM
780
microscope
via
a
VBA
ZEN
macro.
Additional
set
of
functions
is
suited
to
process
collected
positions of identified regions to a Zeiss LSM 780 microscope via a VBA ZEN macro.
Additional
set recovery
of functions
is suited
process parameters,
collected data:
quantify
recoveryperform
curves,statistical
extract
data:
quantify
curves,
extracttorecovery
control
data quality,
recovery parameters, control data quality, perform statistical analysis and construct analysis
analysis
and construct analysis pipelines from these steps. The toolset can applied for automated FRAP
pipelines from these steps. The toolset can applied for automated FRAP experiments with
experiments
with different
cellular
organelles or compartments
integrating
newpost-acquisition
online and postdifferent cellular
organelles
or compartments
by integrating by
new
online and
image
processing
functions.
acquisition image processing functions.
With our toolset we installed several protocols to envisage COPII dynamics variability between different
With our toolset we installed several protocols to envisage COPII dynamics variability between
ERES
and functions
offunctions
potential COPII
coat regulators.
First,regulators.
we measured
distributions
of turnover
rates for
different
ERES and
of potential
COPII coat
First,
we measured
distributions
of turnover
forand
several
COPII
proteins
and evaluated
changes
of these distributions
upon
several
COPII rates
proteins
evaluated
changes
of these
distributions
upon modulation
of cargo quantities
modulation of cargo quantities and upon siRNA inhibition of protein trafficking regulators.
and
upon siRNA inhibition of protein trafficking regulators. Further on, we installed extended protocols to
Further on, we installed extended protocols to test if cargo quantities at ERES and their positions
test
if
cargo
at COPII
ERES and
their positions
in cells
havequantities
impact on
turnover
rates. in cells have impact on COPII turnover rates.
138
16th international
ELMI meeting
european
light microscopy
initiative
elmi
16th international
(P013) Cell cycle dependent analysis of CENP-A
nucleosomes in
ELMI meeting
human kinetochores by AB-FRET
Christian Hoischen1,2, Sindy Giebe1, Shamci Monajembashi2, Peter Hemmerich2, Stephan Diekmann1
1) Department of Molecular
2) Biology, Imaging Facility
Leibniz Institute on Aging – Fritz Lipmann Institute (FLI), Beutenbergstr. 11, D-07745 Jena, Germany
[email protected]
The human kinetochore is divided into a rather dynamic inner kinetochore[1] present at centromeres
during the whole cell cycle, and an outer kinetochore transiently forming only during mitosis. The inner
kinetochore forms the chromatin structure, whereas the outer kinetochore functions as mechanical
link between inner kinetochore and microtubules and controls mitotic progression. Centromeric
chromatin is epigenetically specified by interspersed regions, in which histone H3 is replaced by the
centromere specific histone variant CenH3 (in humans CENP-A). The CENP-A containing nucleosome is
fundamental for the identity of kinetochores and inheritance of centromere location.
CENP-A and H3 containing nucleosomes have long been considered to be of similar octameric
structure. Surprisingly, however, the presence of tetrameric CENP-A nucleosomes (hemisomes) could
be discovered[2] in interphase cells of Drosophila consisting of one molecule each of CENP-A, H4, H2A,
and H2B. In octameric nucleosomes the DNA is twisted left handed around the proteins, whereas the
DNA is twisted right handed around the tetrameric nucleosomes[3]. Recently, it was observed that
both, octameric and tetrameric CENP-A nucleosomes also exist in human cells[4].
In order to identify the cell cycle time point for this octamer to tetramer transition, we performed
by AB-FRET a cell cycle dependent analysis of structural changes in CENP-A containing chromatin
(collaboration with M. Bui and Y. Dalal, NIH, USA).
(1) Hellwig, D., S. Emmerth, T. Ulbricht, V. Döring, C. Hoischen, R. Martin, C.P. Samora, A.D. McAinsh, C.W. Carroll, A.F. Straight,
P. Meraldi, and S. Diekmann. 2011. Dynamics of CENP-N kinetochore binding during cell cycle.J. Cell Schience 124:38713883
(2) Dalal, Y, H. Wang, S. Lindsay, and S. Henikoff. 2007. Tetrameric structure of centromeric nucleosomes in interphase
Drosophila cells. PlosBiology 5:e218
(3) Furuyama,T. and S. Henikoff. 2009. Centromeric nucleosomes induce positive DNA supercoils. Cell 138:104-113
(4) Dimitriadis, E. K., C. Weber, R. K. Gill, S. Diekmann, and Y. Dalal. 2010. Dynamics of inner kinetochore assembly and
maintenance in living cells. PNAS 107:20317-20322
139
ELMI meeting
european
light microscopy
initiative
elmi
16th international
ELMI meeting
(P014) A decade of light-sheet microscopy – already a museum
piece?
Wiebke Jahr1, Benjamin Schmid1, Michael Weber1, Jan Huisken1
1) Max Planck Institute of Molecular Cell Biology and Genetics (MPI-CBG), Pfotenhauer Str. 108, 01307 Dresden, Germany
[email protected]
Selective Plane Illumination Microscopy (SPIM, [1]) is an increasingly popular imaging technique.
Although SPIM’s working principle can be understood with basic optics knowledge, its concepts are
widely unknown to the non-scientific public. A technical museum in Dresden, Germany, launched an
interactive special exhibition on the occasion of the UNESCO International Year of Light. We have built a
fully functional, educational SPIM (eduSPIM) to demonstrate how developments in microscopy promote
discoveries in biology.
For a better understanding and intuitive operation, we radically reduced a standard SPIM to its essential
A decade of light-sheet microscopy – already a museum piece?
components
without compromising functionality. eduSPIM features one illumination and one detection
Wiebke Jahr , Benjamin Schmid , Michael Weber , Jan Huisken
path
andInstitute
a sealed
sample
chamber.
We
mounted
fixed
zebrafish
embryos with fluorescent vasculature,
1) Max Planck
of Molecular
Cell Biology
and Genetics
(MPI-CBG),
Pfotenhauer
Str. 108,
01307 Dresden,
Germany
because
the
structure
is
meaningful
to
laymen
and
helps
to
visualize
SPIM’s underlying principles. As
[email protected]
Selective Plane
Illumination
Microscopy
(SPIM,
[1]) is an increasingly
popular imagingvia a simplified interface, the data is
visitors
acquire
fluorescence
and
transmission
data
simultaneously
technique. Although SPIM’s working principle can be understood with basic optics knowledge,
its concepts areon-the-fly.
widely unknown to the non-scientific public. A technical museum in Dresden,
visualized
Germany, launched an interactive special exhibition on the occasion of the UNESCO
International
Year
Light. We have
built a fully
functional,
(eduSPIM) tointuitive usage. In case of a hardware
We designedofdedicated
control
software
foreducational
eduSPIMSPIM
to maximize
demonstrate how developments in microscopy promote discoveries in biology.
outage,
software
to a fall-back
function. Email notifications
For a better the
understanding
and switches
intuitive operation,
we radically mode
reduced asimulating
standard SPIM microscope
to its
essential components without compromising functionality. eduSPIM features one illumination
and one
detection
paththe
and aerror
sealed can
sample
We mounted
zebrafish
with log-in. On a dedicated website, we
are
sent
so that
bechamber.
resolved,
eitherfixed
on-site
or embryos
via remote
fluorescent vasculature, because the structure is meaningful to laymen and helps to visualize
SPIM’s underlying
principles. and
As visitors
acquire of
fluorescence
transmission
collect
usage statistics
a snapshot
the mostandrecent
data todataevaluate the sample quality and the
simultaneously via a simplified interface, the data is visualized on-the-fly.
reach
of our
microscope.
We designed
dedicated
control software for eduSPIM to maximize intuitive usage. In case of a
hardware outage, the software switches to a fall-back mode simulating microscope function.
Thenotifications
universalareconcepts
presented
alsoeitherhelp
toorcommunicate
Email
sent so that the
error can be will
resolved,
on-site
via remote log-in. other scientific concepts to laymen
On a dedicated website, we collect usage statistics and a snapshot of the most recent data to
in
interactive
The
specific
eduSPIM design is adapted easily for various outreach and teaching
evaluate
the sample settings.
quality and the
reach
of our microscope.
The universaland
concepts
presented
also useful
help to communicate
other scientific
concepts to
activities
it may
evenwill
prove
for labs looking
for a simple
SPIM.
laymen in interactive settings. The specific eduSPIM design is adapted easily for various
outreach
and teaching
activitiesPhysik
and it may
even prove useful
for labs looking
a simple
We thank
Thorlabs,
Instrumente,
Toptica,
Zeissforand
AHFSPIM.
for generous support.
We thank Thorlabs, Physik Instrumente, Toptica, Zeiss and AHF for generous support.
References
References
[1][1]
Huisken,
J. et al.
Sectioning Deep
Inside Live
Embryos
by Selective
Plane
Huisken,
J. “Optical
et al. “Optical
Sectioning
Deep
Inside
Live Embryos
by Selective Plane Microscopy,”
Microscopy,” Science, 305, 1007-1009 (2004).
Science, 305, 1007-1009 (2004).
1
1
1
1
Fig 1: Computer model
(a) and photograph
(b) of the eduSPIM setup.
Fig 1: Computer model (a) and photograph (b) of the eduSPIM setup.
140
16th international
ELMI meeting
european
light microscopy
initiative
elmi
16th international
(P015) Benchmarking and selection of algorithms
and software
ELMI meeting
in bioimage analysis
Michal Kozubek
Centre for Biomedical Image Analysis, Faculty of Informatics, Masaryk University, Brno, Czech Republic
Benchmarking and selection of algorithms and software in bioimage analysis
[email protected]
Michal Kozubek1
Computer analysis of microscopy images has been an indispensable part of biological research for
1) Centre
for in
Biomedical
Analysis,
Faculty of Informatics,
Masaryk
Czech Republic
decades.
While
the lastImage
century
semi-automatic
methods
wereUniversity,
sufficientBrno,
for many
applications, it is
no longer
true
for
the
21st
century
due
to
the
tremendous
increase
of
the
amount
of
acquired
data that
[email protected]
can be processed only using fully automatic methods tuned for a particular application. Biologists have
Computer analysis of microscopy images has been an indispensable part of biological research
thus to
rely on the correctness of results obtained by computer analysis. This in turn requires paying
for decades. While in the last century semi-automatic methods were sufficient for many
properapplications,
attention toitthe
quality
control
of the
opendue
source
as well
as commercial
is no
longer
true for
theavailable
21st century
to the
tremendous
increasesoftware
of the for
amount
of
acquired
data
that
can
be
processed
only
using
fully
automatic
methods
tuned
for a
bioimage analysis.
particular application. Biologists have thus to rely on the correctness of results obtained by
Thecomputer
common way
of checking
of image
analysis
software
to use benchmark
image
analysis.
This inthe
turnperformance
requires paying
proper
attention
to theisquality
control of the
available
openproperties
source as well
commercial
software
formethods
bioimageon
analysis.
datasets
of known
and asevaluate
various
analysis
the same data. Unfortunately,
in biological
imaging
community
there
has been a lack
of publicly
available
reference
images
for a long
The common
way
of checking
the performance
of image
analysis
software
is to use
benchmark
datasets ofwith
known
properties
andground
evaluate
various
on the same
data.
time,image
both simulated
precisely
known
truth
as wellanalysis
as realmethods
ones annotated
by experts.
Unfortunately, in biological imaging community there has been a lack of publicly available
Nevertheless,
inspired
medical
community,
also bioimaging
community
reference images
forby
a long
time,imaging
both simulated
with precisely
known ground
truth ashas
wellrecently
as
realthe
onesstrong
annotated
realized
needbyforexperts.
benchmarking various image analysis methods and software packages
in order
to compare their performance and assess their suitability for specific applications. This
Nevertheless, inspired by medical imaging community, also bioimaging community has recently
presentation
the recent
progress invarious
this respect,
benchmarks
realized summarizes
the strong need
for benchmarking
image reviews
analysis available
methods and
software and
packages in order to compare their performance and assess their suitability for specific
describes
the
general
process
of
designing
a
bioimage
analysis
benchmark
or
challenge
(benchmarking
applications. This presentation summarizes the recent progress in this respect, reviews available
effortbenchmarks
associated toand
a particular
including
selection
of reference
datasets
describes conference),
the general process
of proper
designing
a bioimage
analysisimage
benchmark
or and
challenge
(benchmarking
effort associated
to a applications
particular conference),
including
proper
selection
evaluation
metrics.
Finally, examples
of biological
will be shown
for which
benchmarking
of reference image datasets and evaluation metrics. Finally, examples of biological applications
of relevant
methods
have already
been performed
including
thehave
latest
comparison
will beimage
shownanalysis
for which
benchmarking
of relevant
image analysis
methods
already
been of
performed
including
the
latest
comparison
of
34
software
packages
for
localization
microscopy.
34 software packages for localization microscopy.
M. Kozubek
(2016)
Challenges
and and
Benchmarks
in Bioimage
Analysis.
Advances
AnatomyAENATOMY
mbryology and
M. Kozubek
(2016)
Challenges
Benchmarks
in Bioimage
Analysis.
ADVANCES
MBRYOLOGY
AND C9,
ELL
IOLOGY 219, Chapter 9, in press
Cell BEiology
219, Chapter
in Bpress
30
7
Bioimage and Medical Image Analysis Challenges
5
20
Other
15
ISBI
MICCAI
10
4
Other
ISBI
3
MICCAI
2
5
0
Bioimage Analysis Challenges
6
25
1
2007 2008 2009 2010 2011 2012 2013 2014 2015
0
2007
2008 2009
2010
2011
2012 2013
2014
2015
141
ELMI meeting
european
light microscopy
initiative
elmi
16th international
ELMI meeting
(P016) Optimizing light sheet microscopy for multicolor imaging of
variable size samples on an open imaging facility
Ludovic Leconte, Francois Waharte , Jean Salamero
PICT-IBiSA, UMR144 CNRS-Institut Curie 26 rue d’Ulm 75248 Paris, France
[email protected]
Our instrumental setup is optimized for fast multicolor (4 laser lines from 405 to 638 nm, 2 cameras)
3D imaging. Careful choice of optical components reduces chromatic aberrations to improve image
quality and acquisition speed. Biological samples are made transparent and opaque areas which lead
to the formation of shadow stripes. To reduce illumination artefacts, we implemented a scanning
system at the entry of the cylindrical lens, driven by a home-made controller based on an Arduino
system. We also designed a bright field illumination module, controlled by Arduino and Metamorph.
To image large samples, we have designed a new incubation chamber which allows to make a quick
assembly on any type of objective using 3D printing technology. Moreover, it allows a fine control of
the temperature and CO2 concentration and is easy to maintain.
Light sheet is shaped using a cylindrical lenses and an excitation objective 10X NA 0.3. Fluorescence
is collected by a water-dipping objective (16X NA 0.8 or 40X NA 0.8) and detected simultaneously on
two channels with 2 sCMOS cameras fast-linked to a high-speed storage PC (SSD hard drives) for highspeed and high spatial resolution acquisitions.
Since one of our main goals is to make the whole system easy to handle and reliable, the whole
system is driven by a user-friendly software based on Metamorph, and integrates accurate image
processing algorithms. For easy motion of the sample in 4 axes, we use a Xbox One controller.
Last but not least, dynamic imaging in SPIM produces large data sets to handle and multiple
detection channels require accurate 3D reconstruction and image registration software. Our project
will benefit of the expertise in bioimaging informatics from both Curie Institute and INRIA-Rennes
(Serpico’s team).
142
16th international
ELMI meeting
european
light microscopy
initiative
elmi
Detection of fibroblast growth factors diffusion by fluorescence recovery
16thdiffusion
international by
(P017)
of fibroblast
afterDetection
photobleaching
(FRAP). growth factors
ELMI meeting
fluorescence recovery after photobleaching (FRAP).
1
2
1
Changye Sun , Marco Marcello , Yong Li , David Mason2, Raphaël Lévy1
1
Changye
, Marco
andSun
David
G.Marcello
Fernig12., Yong Li1, David Mason2, Raphaël Lévy1, David G. Fernig1
1) Department
of Biochemistry,
Integrative
1) Department
of Biochemistry,
Institute of Institute
IntegrativeofBiology,
and Biology, and 2) Centre for Cell
Integrative Biology, University of Liverpool, Liverpool L69 7ZB, UK.
2) Centre
for Cell Imaging, Institute of Integrative Biology, University of Liverpool, Liverpool L69 7ZB, UK.
[email protected]
Imaging, Institute
[email protected]
Fibroblast growth factors are crucial effectors in the processes of signal transduction, proliferation
Fibroblast growth factors are crucial effectors in the processes of signal transducti
and morphogenesis
of a wide variety of cells and tissues. The range of biological outcomes generated
proliferation and morphogenesis of a wide variety of cells and tissues. The range of biologi
by many
signalling
proteins
in development
and homeostasis
increased by their
interactions with
outcomes generated by
many signalling
proteins in isdevelopment
and homeostasis
is increased
glycosaminoglycans,
particularly
heparan sulfate (HS). This
interactionheparan
controls sulfate
the localisation
and interact
their interactions
with glycosaminoglycans,
particularly
(HS). This
controls
the localisation
and movement
these
signalling
but whether
movement
of these
signalling proteins,
but whetherofsuch
control
dependsproteins,
on the specificity
of thesuch cont
depends
on known.
the specificity
interactions
not known.
We used five
fibroblast
grow
interactions
is not
We used of
fivethe
fibroblast
growthisfactors
with an N-terminal
HaloTag
for
factors with an N-terminal HaloTag for fluorescent labelling (Halo-FGFs), with we
fluorescent
labelling and
(Halo-FGFs),
well-characterised
andand
distinct
HS binding
and diffusion
characterised
distinct with
HS binding
properties,
measured
their properties,
binding and
measured
their
binding
and
diffusion
in
pericellular
matrix
of
fixed
rat
mammary
27
fibroblasts.
Halopericellular matrix of fixed rat mammary 27 fibroblasts. Halo-FGF1, Halo-FGF2 and Halo-FG
to HS,
interacted
with also
chondroitin
FGF1,bound
Halo-FGF2
andwhereas
Halo-FGF6Halo-FGF10
bound to HS,also
whereas
Halo-FGF10
interactedsulfate/dermatan
with chondroitin sulfate, a
FGF20
did
not
bind
detectably.
The
distribution
of
bound
FGFs
in
pericellular
sulfate/dermatan sulfate, and FGF20 did not bind detectably. The distribution of bound FGFsmatrix
in was
homogenous, and for FGF10 exhibited striking clusters. Fluorescence recovery af
pericellular matrix was not homogenous, and for FGF10 exhibited striking clusters. Fluorescence
photobleaching showed that FGF2 and FGF6 diffused faster, whereas FGF1 diffused mo
recovery
after photobleaching
showed
that FGF2
FGF6 diffused
faster,
whereas FGF1
slowly,
and FGF10 was
immobile.
Theand
software
written
in MATLAB
fordiffused
image more
analysis is fre
slowly,
and
FGF10
was
immobile.
The
software
written
in
MATLAB
for
image
analysis
is
freely
available (Sun C, Marcello M, Li Y., Mason D, Lévy R and Fernig D (2016)available
OPEN BIOLOG
10.1098/rsob.150277).
results
demonstrate
that the
of the interactions
(Sun C,DOI:
Marcello
M, Li Y., Mason D, Lévy RThe
and Fernig
D (2016)
OPEN BIOLOGY
DOI:specificity
10.1098/rsob.150277).
proteins
with glycosaminoglycans
their binding
and with
diffusion.
Moreover, extracellu
The results
demonstrate
that the specificity ofcontrols
the interactions
of proteins
glycosaminoglycans
matrix has long-range structures, since cells regulate independently the distribution of differ
controls
their binding and diffusion. Moreover, extracellular matrix has long-range structures, since
protein sites in glycosaminoglycans.
cells regulate independently the distribution of different protein sites in glycosaminoglycans.
143
ELMI meeting
european
light microscopy
initiative
elmi
16th international
ELMI meeting
(P018) Assessment of the LSMTech InverterScope® and it’s
application to in vivo 2-photon brain imaging.
Joanne Marrison1, William Brackenbury2, Sangeeta Chawla2, Mark Hunt2, Miles Whittington2, Peter O’Toole1
1) Imaging and Cytometry Laboratory, Bioscience Technology Facility, Department of Biology, University of York,
York,UK
2) Department of Biology, University of York, York, UK
[email protected]
We use an inverted multiphoton microscope in a multi-disciplined Department of Biology for in vivo
liver and spleen imaging. However, some sample types cannot be readily addressed using an inverted
microscope and we now have a need to use the same microscope for in vivo brain imaging.
To address this problem we recently purchased an LSM Tech InverterScope® objective inverter to
convert our inverted system to an upright configuration. After testing we found this was not optimized
for 2-photon imaging of green fluorescent protein (GFP). LSM Tech was very proactive and further
customized the InverterScope® to maximize both the excitation and emission transmission. After
testing the customized InverterScope® we found the excitation power exiting the objective for five
wavelengths (405, 488, 561, 633 and 920nm) was minimally affected and there was also no impact on
evenness of illumination across the field. Using a standard fluorescent sample there was only a 15%
loss in emission at the internal detectors after 488nm excitation and a 7% loss in emission at the nondescanned detectors after 920nm excitation compared to without the InverterScope®.
On the basis of these results we used the customised InverterScope® for in vivo imaging of microglia
through a cranial window of a sacrificed CX3CR1-GFP mouse. We successfully imaged to depths of
400μm using laser powers between approximately 3mW-16mW (as measured at the objective)
dependent on imaging depth. This was comparable to settings used without the InverterScope®
but easier and quicker to set up, meaning contact with the animal was minimized, an important
consideration for imaging an anaesthetised animal.
In conclusion our data shows the customised LSM Tech InverterScope® to be an effective alternative
to purchasing a new system, at least in the preliminary proof of concept phases of this type of work
and has enabled us to progress work that would classically have required an upright/fixed stage
multiphoton system. For a multiuser facility, this approach is proving highly effective.
144
16th international
ELMI meeting
european
light microscopy
initiative
alytical model of the Optical Vortex Scanning Microscope
elmi
16th
international
(P019) Analytical model of the Optical Vortex
Scanning
ELMI meeting
2
Masajada , Agnieszka Popiołek-Masajada , Łukasz Płocinniczak2,3
1Microscope
Jan Masajada1, Agnieszka Popiołek-Masajada2, Łukasz Płocinniczak2,3
artment of Fundamental Problems of Technology, Wroclaw University of Science and Technology
1) Department of Fundamental Problems of Technology, Wroclaw University of Science and Technology
analytical [email protected]
model of our current optical vortex scanning microscope (OVSM) will b
ussed (Fig. 1). The OVSM works with optical vortices which are difficult but very sensitiv
The analytical
model of
our current
vortexsimple scanning microscope
(OVSM) will be discussed
(Fig.
ctures in the light filed. We hope optical
to win and superresolving instrument aft
1).
The
OVSM
works
with
optical
vortices
which
are
difficult
but
very
sensitive
structures
in
the
light
filed.
king some technical problems. The other strong points of the OVSM design (large worki
We hope to win simple and superresolving instrument after breaking some technical problems. The other
nce, simple optical system, fast measurements) will be indicated. Presently the most important ta
strongeffective points of theprocedures OVSM design (large
distance,
simple optical
system, fast measurements)
o develop the for working
surface topography reconstruction. For this goal th
will be indicated. Presently the most important task is to develop the effective procedures for surface
ytical model of the OVSM optical system is very helpful. topography reconstruction. For this goal the analytical model of the OVSM optical system is very helpful.
Fig. 1. Basic optical setup of the optical vortex scanning microscope
Fig. 1. Basic optical setup of the optical vortex scanning microscope
model is based on solving the theFresnel diffraction integral for the OVSM Our model
is based
on solving
Fresnel diffraction
integral for
the basic OVSM
system,basic including
three system
uding three possible
possible sample scanning: standard by (i.e.
sample ways ofways sampleof scanning:
standard
(i.e. by sample
moving),(i.e. internal
by vortexmoving), lens shift) intern
by vortex lens shift) and vertical. The model includes the presence of simple phase objec
and vertical. The model includes the presence of simple phase object. The model will be discussed in the
context
of superresolution.
The conclusions
willof be superresolution. also supported by experimental
results.
model will be discussed in the context The conclusions will be al
ported by experimental results. 145
ELMI meeting
european
light microscopy
initiative
elmi
16th international
ELMI meeting
(P020) MHC I expression regulates co-clustering and mobility of
interleukin-2 and -15 receptors in T cells
Gábor Mocsár1,#, Julianna Volkó1,#, Daniel Rönnlund2, Jerker Widengren2, Péter Nagy1, János Szöllősi1,3, Katalin
Tóth4, Carolyn K. Goldman5, Sándor Damjanovich1, Thomas A. Waldmann5, Andrea Bodnár1, György Vámosi1,*
1) Department of Biophysics and Cell Biology, Faculty of Medicine, University of Debrecen, Debrecen, Hungary
2) Royal Institute of Technology, Stockholm, Sweden
3) MTA-DE Cell Biology and Signaling Research Group, University of Debrecen, Debrecen, Hungary
4) German Cancer Research Center, Biophysics of Macromolecules, Heidelberg, Germany
5) Lymphoid Malignancies Branch, Center for Cancer Research, National Cancer Institute, Bethesda, USA
# G. M. and J. V. contributed equally to this work
* Corresponding author
email: [email protected]
MHC glycoproteins form supramolecular clusters with interleukin-2 and -15 receptors in lipid rafts
of T cells. The role of highly expressed MHC I in maintaining these clusters is unknown. We knocked
down MHC I in FT7.10 human T cells, and studied protein clustering at two hierarchic levels: molecular
aggregations and mobility by FRET and fluorescence correlation spectroscopy, and segregation into
larger domains or superclusters by superresolution STED microscopy. FCS based molecular brightness
analysis revealed that the studied molecules diffused as tight aggregates of several proteins of a kind.
Knockdown reduced the number of MHC I containing molecular aggregates and their average MHC
I content, and decreased the heteroassociation of MHC I with IL-2Rα/IL-15Rα. The mobility of not
only MHC I but also that of IL-2Rα/IL-15Rα increased, corroborating the general size decrease of tight
aggregates. A multifaceted analysis of STED images revealed that the diameter of MHC I superclusters
diminished from 400-600 to 200-300 nm, whereas those of IL-2Rα/IL-15Rα hardly changed. MHC I
and IL-2Rα/IL-15Rα colocalized with GM1 ganglioside-rich lipid rafts, but MHC I clusters retracted
to smaller subsets of GM1- and IL-2Rα/IL-15Rα-rich areas upon knockdown. Our results prove that
changes in expression level may significantly alter the organization and mobility of interacting
membrane proteins.
146
16th international
ELMI meeting
european
light microscopy
initiative
elmi
16th international
(P021) PML nuclear body reorganisation in the
nucleus using
ELMI meeting
Erythrocyte Mediated Force Application (EMFA) technique
Shamci Monajembashi1, Birgit Perner1,Peter Hemmerich1
1) Leibniz Institute for Aging Research - Fritz-Lipman-Institute, Jena, Germany
[email protected]
Optical tweezers are ideal tools for handling biological objects (1). Erythrocyte Mediated Force
Application (EMFA) technique is a variant of optical tweezers, which can be used to exert a vertical
pressure on cells using erythrocytes as pure force transducers. EMFA is now offering a new approach
to apply a vertical force. Previously, we have used EMFA as a tool for in vitro blood pressure simulation
and other applications (2).
We have now started to employ EMFA to analyze assembly mechanisms of PML (Promyelocytic
leukemia) nuclear bodies. PML bodies are common in almost all mammalian cells, with 1 to 30
per nucleus having a size range of 0.2-1 µm. Functionally, PML bodies are mainly involved in stress
response pathways. In the nucleus, the PML bodies have extensive contact with chromatin, thus
stabilizing their position.
PML bodies react to stress in two ways: Either by dissociation or disruption into PML body components
(PML microstructures) under environmental stress such as heat/heavy metal shock or they disrupt into
PML microbodies, if the DNA is damaged (3).
In order to examine the response of PML bodies to mechanical stress, we employed EMFA technique
to exert opto-mechanical forces on cells. Using mechanical stress on chromatin; local fission of PML
nuclear bodies as well as local de novo formation of PML bodies (MSIPs) was observed. The formation
of MSIPs appeared to occur either through assembly from smaller bodies or assembly from free PML
molecules.
Our data suggest that PML bodies may act as sensors of mechanical stress on chromatin.
References:
(1) Greulich KO. Et.al. (2000) J.Microsc. 198. 182-187
(2) Grigaravicˇius P. et al. (2009) ChemPhysChem. 10. 79-85
(3) Eskiw C.H. et al. (2004) JBC. Vol.279. 9577–9585
147
ELMI meeting
european
light microscopy
elmi
initiative
epartment of Biophysics, Medical
School, University of Pécs, Pécs, Szigeti str. 12, H-7624, Hungary;
aculty of Sciences, University of Pécs, Ifjúság str. 8, H-7624, Hungary;
16th international
ELMI meeting
nos Szentágothai
Center, Pécs, H-7624, Hungary; 3 MTA-PTE Nuclear-Mitochondrial Interactions
24-27 MayResearch
2016, Debrecen, Hungary
earch Group.
(P022) Phalloidin binds to MreB from Leptospira interrogans
[email protected]
Beata Longauer1,2, Szilvia Barko1,3, Emoke Bodis1,3, David Szatmari1, Miklos Nyitrai1,3
eB (Murein
regionofB)
is a bacterial
actin-like
protein,
key player
1) Department
Biophysics,
Medical School,
University of
Pécs, Pécs,which
Szigeti str.is12,a H-7624,
Hungary;in the
intenance
of cell
shapeUniversity
and essential
coordinator
the cell-wall synthesis. Although its
2 )Faculty
of Sciences,
of Pécs, Ifjúság
str. 8, H-7624,ofHungary;
3 )János Szentágothai
Center, Pécs,
H-7624,crystallography
Hungary; 3 MTA-PTE Nuclear-Mitochondrial
quence identity
is low asResearch
compared
to actin,
studies showedInteractions
that the MreB
Research
Group.
d actin share a similar three-dimensional crystal structure with a conserved [email protected]
nding domain.
The exact localisation of MreB is still unkonwn in the bacterial cell because
the modified
protein.
MreBdistribution
(Murein regionof
B) istagged
a bacterial
actin-like protein, which is a key player in the maintenance of
shape
andpurified
essential and
coordinator
of the cell-wall
synthesis.
its sequence
identity using
is low
our workcellwe
have
characterised
MreB
from Although
Leptospira
interrogans
as
compared
to
actin,
crystallography
studies
showed
that
the
MreB
and
actin
share
a
similar
threenaturing type of purification which solved the previously described problems in the
crystal structure
with a conserved
nucleotide-binding domain. The exact localisation of
rificationdimensional
of the soluble
and functional
protein.
MreB is still unkonwn in the bacterial cell because of the modified distribution of tagged protein.
is MreB can
biochemical
properties
to the
special,
In ourcarry
work novel
we havestructural
purified andand
characterised
MreB from
Leptospiraattributed
interrogans using
denaturing
rkscrew type
shaped
cell typewhich
of Spirochetes.
We have
found
that by
reason
of itsof actin-like
of purification
solved the previously
described
problems
in the
purification
the soluble
ucture MreB
can bind
phalloidin, the specific actin-filament labelling toxin. Therefore
and functional
protein.
MreB can carry
novel structural
biochemical
properties
attributed
to the special,
corkscrew
orescentlyThis
conjugated
phalloidin
can and
serve
as a versatile
tool
to visualise
the prokaryotic
shaped
cell
type
of
Spirochetes.
We
have
found
that
by
reason
of
its
actin-like
structure
MreB
can bind
oskeleton in vitro or even in vivo.
phalloidin, the specific actin-filament labelling toxin. Therefore fluorescently conjugated phalloidin
can serve as a versatile tool to visualise the prokaryotic cytoskeleton in vitro or even in vivo.
B. megaterium cells labelled with Alexa488-phalloidin, and investigated by SIM (Structured
Illumination
Microscope).
ScaleAlexa488-phalloidin,
bar: 20um.
megaterium cells labelled
with
and investigated by SIM (Structured
umination Microscope). Scale bar: 20um.
148
16th international
ELMI meeting
european
light microscopy
initiative
elmi
16th international
(P023) Challenges in the Labeling and Detection
of Viral RNA by
ELMI meeting
Confocal and Super-Resolution Fluorescent Microscopy
Olga Oleksiuk1, Ji Young Lee1, Ralf Bartenschlager1
1) Department of Infectious Diseases, Molecular Virology, University of Heidelberg, Germany
[email protected]
Hepatitis C virus (HCV) and Dengue virus (DV) belong to the same family of plus-strand RNA viruses,
the Flaviviridae. For productive propagation, the viral RNA (vRNA) genome has to be translated,
replicated and assembled into virus particles. By using electron microscopy, we found that both viruses
rearrange intracellular membranes that serve as scaffold for the assembly of replication factories. In
case of DV, these correspond to endoplasmic reticulum derived vesicular invaginations (diameter 80120 nm). The detection of these structures, the presumed sites of vRNA replication, and vRNA itself by
fluorescent microscopy is challenging due to specificity in labeling and low copy number of vRNA per
cell.
To study the fate of DV RNA in infected cells, we employ two different approaches: immunofluorescence
by double-strand RNA-specific antibody and branched DNA-based FISH (Affymetrix), the latter being
suitable to visualize positive or negative strand DV RNA, with the possibility to improve optical
resolution by SIM microscopy. In addition, newly synthesized DV RNA was metabolically labeled by
feeding of cells with Bromouridine (BrU). Infected cells were analyzed by confocal microscopy and
single-cell 3D-quantitative imaging analysis (ImarisXT, Bitplane AG).
In DV-infected cells the dsRNA signal partly colocalizes with the positive strand RNA signal obtained
by FISH as well as the signal for de novo synthesized RNA and slightly overlaps with negative RNA. The
high specificity of BrU-labeling of de novo synthesized DV RNA was proven by colocalization analysis
with viral proteins and DV positive RNA strand. Most negative-strand RNA-containing structures
accumulated in the perinuclear region and their number per cell correlated with virus-induced
membrane rearrangements. Within less than one hour newly synthesized DV RNA was incorporated
into new virions, concomitant with an increase of total amounts of positive-strand RNA by 4%.
149
ELMI meeting
european
light microscopy
initiative
elmi
16th international
ELMI meeting
(P024) Rapid2FLIM: the new and innovative method for ultrafast imaging of biological processes
Sandra Orthaus-Mueller, Ben Kraemer, Astrid Tannert, Tino Roehlicke, Michael Wahl, Hans-Juergen Rahn,
Rainer Erdmann
PicoQuant GmbH, Rudower Chaussee 29, 12489 Berlin, Germany
[email protected]
KEY WORDS: rapid2FLIM, Fluorescence Lifetime Imaging (FLIM), Time-Correlated Single Photon
Counting (TCSPC), Foerster Resonance Energy Transfer (FRET), confocal fluorescence microscopy
Over the last two decades, time-resolved fluorescence microscopy has become an essential tool in Life
Sciences thanks to measurement procedures such as Fluorescence Lifetime Imaging (FLIM), lifetime based
Foerster Resonance Energy Transfer (FRET), and Fluorescence (Lifetime) Correlation Spectroscopy (F(L)
CS) down to the single molecule level. Today, complete turn-key systems are available either as standalone units [1] or as upgrades for confocal laser scanning microscopes (CLSM) [2]. Data acquisition on
such systems is typically based on Time-Correlated Single Photon Counting (TCSPC) electronics along with
picosecond pulsed diode lasers as excitation sources and highly sensitive, single photon counting detectors.
Up to now, TCSPC data acquisition is considered a somewhat slow process, due to the slow scan speed
required to collect a sufficient number of photons per pixel for reliable data analysis. This makes it difficult
to use FLIM for following fast FRET processes in biological samples, such as signal transduction pathways
in cells, fast moving sub-cellular structures (e.g., vesicles), or the contraction of heart muscle cells. We
present here a novel and elegant solution to tackle this challenge.
Our approach, named rapid2FLIM, exploits recent hardware developments such as TCSPC modules with
ultra short dead times and hybrid photomultiplier detector assemblies enabling significantly higher
detection count rates. Thanks to these improved hardware components, it is possible to achieve much
better photon statistics in significantly shorter time spans while being able to perform FLIM imaging for
fast processes in a qualitative manner and with high optical resolution. These shorter acquisition times
allow imaging with several FLIM images per second for monitoring, e.g., transient molecular interactions
as well as fast moving species. In this way, it would be possible to study fast processes such as protein
interactions involved in endosome trafficking.
REFERENCES:
[1] M. Wahl, F. Koberling, M. Patting, H. Rahn, R. Erdmann, “Time-resolved Confocal Fluorescence Imaging and Spectrocopy
System with Single Molecule Sensitivity and Sub-Micrometer Resolution”, Current Pharmaceutical Biotechnology, 5, 299-308
(2004).
[2] B. Krämer, V. Buschmann, U. Ortmann, F. Koberling, M. Wahl, M. Patting, P. Kapusta, A. Bülter, R. Erdmann, “Advanced FRET and
FCS measurements with laser scanning microscopes based on time-resolved techniques”, Proceedings of SPIE, 6860, 68601D
(2008).
150
16th international
ELMI meeting
european
light microscopy
initiative
elmi
16th international
(P025) Multi-scale transport image of the living
cell
ELMI meeting
Szabolcs Osváth1,2, Levente Herényi1, Gergely Agócs1, Katalin Kis-Petik1, Miklós Kellermayer1,2
1) Department of Biophysics and Radiation Biology, Semmelweis University, Budapest, Hungary
2) MTA-SE Molecular Biophysics Research Group, Hungarian Academy of Sciences, Budapest, Hungary
[email protected]
The living cell is a highly non-equilibrium system in which trafficking via active- and passivetransport is of vital importance. Selectively labeled intracellular motions have been studied before,
but how these motions fit into the broader context of all cellular transports is unclear. We used phase
contrast microscopy to detect virtually all intracellular transport phenomena. Explicit relationship
was found between motional fluctuations and image brightness fluctuations. Fourier analysis of
movies of HEP2 cells revealed scale-independent transports in the interphase cell, exhibiting selfsimilar temporal behavior in all intracellular locations. The locally observed Hurst coefficient of the
self-similar temporal pattern was used to construct a transport image of the cell. The cell showed
spatially differentiated transport phenomena. The cytoplasm of the cell was found to be dominated
by superdiffusion driven by active transport. Brownian diffusion and subdiffusion was also observed,
mostly inside the nucleus. We believe that the abundance of subdiffusion reports in the literature
arises from observing movements which are not integral part of the cellular physiology. We propose
the use of label-free imaging methods and Hurst coefficient transport imaging as an effective tool to
visualize physiological transport processes of living cells.
151
ELMI meeting
european
light microscopy
initiative
elmi
16th international
ELMI meeting
(P026) Analysis of zebrafish kidney development with timelapse imaging using a dissecting microscope equipped for
optical sectioning
Birgit Perner1, Danny Schnerwitzki1, Michael Graf1,3,Christoph Englert1,2
1) Molecular Genetics, Leibniz Institute on Aging – Fritz Lipmann Institute (FLI), Jena, Germany
2) Faculty of Biology and Pharmacy, Friedrich Schiller University of Jena, Jena, Germany
3) Carl Zeiss Microscopy GmbH, Jena, Germany
[email protected]
In order to understand organogenesis, the spatial and temporal alterations that occur during
development of tissues need to be recorded.
The method described here allows time-lapse analysis of normal and disturbed kidney development
in zebrafish embryos by using a fluorescence dissecting microscope equipped for structured
illumination and z-stack acquisition. The advantage of the experimental setup is the combination of
a zoom microscope with simple strategies for re-adjusting movements in x, y or z direction without
additional equipment.
To apply this method, nephrogenesis was visualized by using transgenic zebrafish (Tg(wt1b:GFP))
with fluorescently labeled kidney structures. Additionally, renal defects were triggered by injection
of an antisense morpholino oligonucleotide against the Wilms tumor gene wt1a, a factor known
to be crucial for kidney development. This technique can also be applied to investigate normal and
impaired morphogenesis of other zebrafish organs such as heart or liver or to observe wound healing
and regeneration.
152
16th international
ELMI meeting
european
light microscopy
initiative
elmi
16th international
(P027) Investigation interactions between nuclear
receptors
ELMI meeting
using modern biophysical methods
Bálint Rehó1, Péter Brázda2, László Nagy2, György Vámosi1
1) Department of Biophysics and Cell Biology, University of Debrecen
2) Department of Biochemistry and Molecular Biology, University of Debrecen
[email protected]
Nuclear receptors are transcription factors that regulate gene expression in a ligand dependent
manner. They play an important role in cell differentiation, growth, and death. We investigated
interactions and dynamics of retinoic acid receptor (RAR) and retinoid X receptor (RXR) acting in a
dimer in living cells. Their operation is described by the molecular switch model. In the absence of
ligand receptors are bound to DNA associated with a corepressor complex, and repress transcription.
Upon binding an agonist, receptors change their conformation, and the corepressor complex is
replaced by coactivator complexes resulting in gene transcription. This model is being changed for a
more dynamic one due to intense investigations in the field.
In our studies we aimed to determine the affinity of the receptors to chromatin and to each other
in the absence and presence of ligand. We transfected HeLa or AD 293Tcells with nuclear receptors
tagged with fluorescent proteins (EGFP, mCherry). We monitored changes in mobility by fluorescence
correlation spectroscopy (FCS), and dimerization by Förster resonance energy transfer (FRET).
Agonist treatment decreased the mobility of GFP-RAR and GFP-RXR molecules transfected alone.
Co-transfection of the two receptors decreased their mobility even in the absence of ligand. This is
probably due to dimerization and the increased affinity of the RAR-RXR complex to the chromatin as
compared to that of the monomers. FRET results showed that RAR-RXR heterodimerization as well as
RXR homodimerization increased in the presence of RAR and RXR ligand.
Our combined FCS and FRET measurements reveal that both receptor-receptor and receptorchromatin interactions are ligand dependent and change dynamically during activation.
153
ELMI meeting
european
light microscopy
initiative
elmi
16th international
ELMI meeting
(P028) Dual fluorescent probes for intracellular organelle
imaging: focus on rational design
Silvie Rimpelová1, Tomáš Bříza1,2, Zdeněk Kejík1,2, Kamil Záruba1, Tomáš Ruml1,Vladimír Král1,2
1) University of Chemistry and Technology Prague; Prague, Czech Republic
2) First Faculty of Medicine, Charles University in Prague, Prague, Czech Republic
[email protected]
Intracellular organelle probes for real-time fluorescence microscopy in living cells are powerful
tools in order to obtain important information about a biological system and its complexity in a
cellular context. Mostly, such small organelle fluorescent probes can be used for visualization of
only one cellular target (organelle). Here, taking the advantage of the inherent dual fluorescence
emission phenomenon (with good spectral separation, based on the environment), we have focused
on rational design of such probes employing structural variability. These intracellular probes show
selective localization in different organelles, fluorescence emission of which is detected at different
wavelengths. The relationship between the chemical structure, photo-physical properties, localization
and cytotoxicity of the probes was explored in a great detail using a panel of cell lines. A series of 12
compounds with structural variability was tested. Probes from low to high photostability of cyan to
deep red fluorescence emission were obtained together with probes with dual fluorescence emission
maxima, which depended on their localization site. These probes enable to study morphological
changes and physiology of cell organelles by wide-field, confocal and structured illumination
fluorescence microscopy in living cells. The major advantages of these probes are: specificity,
applicability in low nanomolar concentrations, high photostability and feasible synthetic accessibility.
This work was supported by the Technical agency of the Czech Republic (TE01020028) and by the
Charles University (UNCE 204011/2012 and P24/LF1/3).
154
16th international
ELMI meeting
european
light microscopy
initiative
elmi
international
(P029) Performance assessment, monitoring16th
and
quality control
ELMI meeting
of fluorescence structured illumination microscopy (SIM) systems
Arnaud Royon
Argolight SA
[email protected]
Structured illumination fluorescence microscopes are of particular interest in cell biology, as they
provide lateral and axial resolutionPerformance
that permitassessment,
to resolve most
of the cell features with a low photomonitoring and quality control of fluorescence
toxicity, they allow live-cell imaging,
and
they
are
compatible
standard
structured illumination microscopywith
(SIM)
systems dyes and staining
protocols. However, SIM systems are complex instruments. In that sense, they need to be assessed
Arnaud Royon
routinely so that they can provide their full potential.
1
1) Argolight SA
[email protected]
We have engineered a new evaluation
slide dedicated to SIM systems, the Argo-SIM, which consists of
a custom glass substrate, the Argoglass®,
on a stainless
steelmicroscopes
carrier. Different
fluorescent
Structuredset
illumination
fluorescence
are of particular
interest patterns,
in cell biology, as
they provide
lateral
and
axial
resolution
that permit to are
resolve
most of the inside
cell features
in 2D and in 3D, with typical dimensions
that
are
less
than
the
resolution,
embedded
the with a
low photo-toxicity, they allow live-cell imaging, and they are compatible with standard dyes
and
staining
protocols.
However,
SIM
systems
are
complex
instruments.
In
that
sense, they
glass. Each fluorescent pattern is designed for one or several performance assessments.
need to be assessed routinely so that they can provide their full potential.
have engineered a new evaluation slide dedicated to SIM systems, the Argo-SIM, which
Non-exhaustively, the Argo-SIMWe
allows
assessglass
andsubstrate,
monitor
the following
of SIM
consists
of to
a custom
the Argoglass®,
set oncharacteristics
a stainless steel carrier.
Different
patterns,
in 2D
andof
in view,
3D, with
typical dimensions
that are less chromatic
than the resolution,
systems: Evenness of illumination,fluorescent
distortion
of
the
field
parcentrality,
parfocality,
are embedded inside the glass. Each fluorescent pattern is designed for one or several
performance
assessments.
shifts, co-localization issues, stitching
performance,
stage repositioning accuracy, intensity response of
the system, spectral response of the
system, lateral
power,to objective
issues,the3Dfollowing
reconstruction
Non-exhaustively,
theresolving
Argo-SIM allows
assess and monitor
characteristics of
SIM systems: Evenness of illumination, distortion of the field of view, parcentrality,
precision, distances in XY and Z, and
algorithms
reconstruction
accuracy.
parfocality, chromatic shifts, co-localization issues, stitching performance, stage repositioning
accuracy, intensity response of the system, spectral response of the system, lateral resolving
power, objective issues, 3D reconstruction precision, distances in XY and Z, and algorithms
reconstruction accuracy.
For example, a pattern containing the three meridians of a sphere, as shown in the image below,
For example,
pattern
containing
meridians of a sphere,
as shown
the image
allows to assess the chromatic aberrations
of athe
system
in 3D,thethethree
3D reconstruction
precision,
asinwell
below, allows to assess the chromatic aberrations of the system in 3D, the 3D reconstruction
as lateral and axial resolutions. In the
poster,
we will
showandother
examplesInofthe
performance
precision,
as well
as lateral
axial resolutions.
poster, we willassessments
show other examples
of performance assessments of SIM systems.
of SIM systems.
10 µm
155
ELMI meeting
european
light microscopy
initiative
elmi
16th international
ELMI meeting
(P030) Dual effects of Ras and Rab interactor 1 (RIN1) on
filopodial motility and AMPA receptor endocytosis participate
in long-term depression of hippocampal neurons
Zsófia Szíber1, Attila Ignácz1, Sven Beyes1,3, Norbert Bencsik1, Krisztián Tárnok1, Angelika Hausser3,
Katalin Schlett1,2
1) Dept. Physiology and Neurobiology, Eötvös Loránd University, Budapest, Hungary
2) MTA-ELTE-NAP B - Neuronal Cell Biology Research Group, Budapest, Hungary
3) Institute of Cell Biology and Immunology, University Stuttgart, Stuttgart, Germany
[email protected]
Ras and Rab interactor 1 (RIN1) is predominantly expressed in neurons. RIN1 is a Ras effector, and
signals through two downstream effectors: Abl non-receptor tyrosine kinases that control actin
cytoskeleton remodeling and Rab5 GTPases that control receptor endocytosis and trafficking. RIN1 has
a guanine nucleotide exchange factor (GEF) activity for Rab5, promoting Rab5-mediated endocytosis
of cell-surface receptors. So far, RIN1’s role has been indicated to inhibit the acquisition of fear
memories, with a critical role for RIN1 in gating the acquisition and persistence of cue-dependent fear
conditioning. Thus, RIN1 may act as an inhibitor of plasticity and learning.
We aimed to clarify the dual effects of RIN1 in embryonic hippocampal neurons. Several fluorescently
labelled RIN1 mutants were created and expressed in RIN1 knockout cultured neurons. The S351A point
mutation inhibits 14-3-3 binding and the sequestration of active RIN1 from the cytoplasm, rendering a
constitutively active form of RIN1. The E574A point mutation blocks the interactions with Rab5 while
the Y36F (QM) point mutant selectively impairs Abl kinase activation.
Live cell imaging of motile dendritic filopodia, anti-GluA1 antibody feeding and transferrin
uptake together with morphological analyses of fluorescently labelled neurons revealed that RIN1
orchestrates its downstream pathways to enhance long-term depression in hippocampal neurons.
Its Rab5 GEF activity is responsible for increasing GluA1 endocytosis and the removal of postsynaptic
AMPA receptors upon chemically induced depression (cLTD) in hippocampal cultures, while the Abl
kinase binding is responsible for regulating dendritic filopodial motility in order to stabilize future
synaptic connections.
Research was supported by the KTIA_NAP_13-2014-0018 grant and by the Hungarian Scientific
Research Foundation grant K81934 to KS, by the HA 3557/11-2 DFG project to AH and KS and by the
MÖB-DAAD and Erasmus travel exchange programs.
156
16th international
ELMI meeting
european
light microscopy
initiative
elmi
16th international(flcs).
(P031) Fluorescence Lifetime correlation spectroscopy
ELMI meeting
A powerful tool to measure concentrations and molecular
interactions
Sandra Orthaus-Mueller1, Benedikt Kraemer1, Steffen Ruettinger1, Volker Buschmann1, Olaf Schulz1,
Felix Koberling1, Rainer Erdmann1, Mark A. Hink2
1) PicoQuant GmbH, Rudower Chaussee 29, 12489 Berlin, Germany
2) Dept. of Molecular Cytology, University of Amsterdam, Sciencepark 904, Amsterdam, The Netherlands
[email protected]
Keywords: Fluorescence Correlation Spectroscopy (FCS), Fluorescence Lifetime Correlation
Spectroscopy (FLCS), Fluorescence Cross-Correlation Spectroscopy (FCCS), diffusion, binding studies.
Fluorescence Correlation Spectroscopy has become a standard tool in biophysics to study diffusion
properties and molecular interactions in solution. In recent time, it is used more frequently also in
complex environments like cells and multi-label applications. Common problems complicating these
experiments include detector afterpulsing and spectral crosstalk. Looking at the nanosecond arrival
time of the detected photons after pulsed excitation can, in a straightforward way, identify artifact
signals and help to distinguish and separate photons coming from species with different emission
lifetime properties.
We will show current results for absolute concentrations measurements of diffusing proteins in live
cells as well as dual color FCCS binding studies. Especially in dual color applications when two pulsed
lasers are not available the decay pattern analysis allows quantitatively to separate the pulsed laser
excited fluorescence from the CW excited one to overcome spectral bleed through problems.
We will present an universal approach on how to use the fluorescence lifetime information to improve
and extend Fluorescence Correlation Spectroscopy, especially in order to simplify cross-correlation
measurements.
157
ELMI meeting
european
light microscopy
initiative
elmi
16th international
ELMI meeting
(P032) Use of a 690nm cw laser on a confocal microscope for
excitation of long wavelength probes
Owen M. Schwartz, Juraj Kabat
National Institute for Allergy and Infectious Disease, NIH, Bethesda, Maryland 20892 USA
[email protected]
Since the earliest days of commercial confocal systems, the longest excitation wavelength has always
been 633nm. This was primarily determined by the output lines of the Kr-Ar mixed gas lasers first
used on confocal systems, and later by the 633nm HeNe which was widely available. More recently
many fluorescent dyes have become available with excitations maxima ranging from 700 to 800nm.
Investigators who use these dyes for flow cytometry studies have asked if they can be used on our confocal
systems.
We have coupled a 690nm CW laser to our Leica SP8 confocal microscope to allow for imaging in the farred region of the spectrum. Control of the laser power is done with an AOTF as with any other laser line.
Detection via the spectrophotometer and internal PMTs or HyDs works well with a high signal to noise
ratio. Background fluorescence with these longer wavelength dyes appears to be minimal. Alexa 700
gives a very bright signal with very low laser power while generating little auto fluorescence. Crosstalk
between the traditional dye Alexa 633 and Alexa 700 is minimal.
Longer wavelength dyes such as Alexa 750 and Indocyanine Green (Ex 780nm Em 820nm) are also
excited well by the 690nm laser. Detection of emitted fluorescence beyond 750nm is better accomplished
by use of external APD detectors with selective barrier filters.
By using the full range of the 5 internal detectors and the external ADP detectors we have found that
samples labeled with 5 and 6 different fluorophores can be imaged with little crosstalk between channels.
158
16th international
ELMI meeting
european
light microscopy
initiative
elmi
16th international
(P033) Coaligned Three-Colour STED Nanoscopy
Reveals
ELMI meeting
Cytoskeletal Organization at Synaptic Sites
Sven C. Sidenstein, Elisa D’Este, Marvin J. Böhm, Johann G. Danzl, Vladimir N. Belov, Stefan W. Hell
Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077
Göttingen, Germany
[email protected]
Far-field fluorescence superresolution techniques like stimulated emission depletion (STED)
nanoscopy allow the separation structures below the diffraction limit of visible light. Especially for life
science applications, simultaneous superresolution imaging of multiple species is demanded. Here we
present a multicolour STED nanoscope scheme based on a single depletion beam at 620 nm providing
down to ~35 nm resolution and up to three, intrinsically co-aligned superresolution channels with low
crosstalk. The performance of our arrangement is demonstrated for imaging of both living and fixed
samples. In particular, the subcortical cytoskeletal organization of cultured hippocampal neurons was
analyzed. We identified a ~190 nm periodic actin/spectrin lattice along mature dendrites which can
also enter into the spines. We believe that our multicolour nanoscope system and in particular the 620
nm STED line can become a viable option for routine STED applications.
159
ELMI meeting
european
light microscopy
initiative
elmi
16th international
ELMI meeting
(P034) Transform your Laser Scanning Microscope to
Differential-Polarization Laser Scanning Microscope
Gábor Steinbach1,2, Gábor Sipka1, András Barta3, István Pomozi4, Győző Garab1,5
1) Institute of Plant Biology, Biological Research Center, Szeged, Hungary
2) Centre ALGATECH, Institute of Microbiology, CAS, Třeboň, Czech Republic
3) Estrato R&D Ltd., Budapest, Hungary
4) Drem Ltd., Budapest, Hungary
5) Biofotonika R&D Ltd., Szeged, Hungary
[email protected]
Most biological samples contain large, hierarchically organized, complex molecular structures, for
example: cellulose or actin fibers, stacked membranes, macromolecular chains, condensed DNA, protein
aggregates. Differential polarization (DP) techniques provide important information about highly
organized microscopic samples [1-2]. The first DP-LSM designed and constructed by us was based on
a Zeiss LSM410 [3-4]; an easy-to-install DP attachment was used to equip an Olympus Fluoview 500,
essentially without changing its optical and electronic units [5]. These DP-LSMs, using high-frequency
modulation and demodulation units, similar to those in dichrographs, and some passive polarization
optical elements, allow fast and precise pixel-by-pixel measurements of the following DP quantities:
linear (LD) and circular dichroism (CD), anisotropy of the fluorescence emission upon non-polarized
excitation (r, confocal) and circularly polarized luminescence (CPL), fluorescence detected linear and
circular dichroism (FDLD and FDCD, confocal), the degree of polarization of the fluorescence emission (P,
confocal), and linear birefringence (LB). These DP-LSMs have been used for the mapping of anisotropic
molecular organization of e.g. plant cell walls and cellulose fibers, amyloids, cell membranes, artificial
chlorosomes and thylakoid membranes [5 and references therein]. Via appropriately adopting the DP
attachment, most LSMs can readily be transformed into DP-LSM, which can thus be used to obtain
unique structural information on the anisotropic molecular organization of biological samples and
intelligent materials.
KEY WORDS: anisotropic molecular macroassemblies, differential-polarization laser scanning
microscope (DP-LSM), photoelastic modulator (PEM), polarized light,
REFERENCES:
[1] Savic A. et al., Microsc Microanal. 2016 22(2):361-367.
[2] Chappaz-Gillot C. JACS 2012 134:944-954
[3] Garab G. et al., Patent US8451446 B2
[4] Steinbach G. et al., Acta Histochem. 2009;111(4):316-25
[5] Steinbach G. et al., Methods Appl. Fluoresc. 2 (2014) 015005 (9pp)
160
16th international
ELMI meeting
european
light microscopy
initiative
elmi
16th international
(P035) Spectrally resolved fluorescence induction
(SRFI)
ELMI meeting
Spectrally resolved fluorescence induction (SRFI)
measurements on single ce
measurements on single cell level using confocal microscope
level using confocal microscope
1
1
1
1
Gábor Steinbach
, Jiří
Liška1,2,3, 1Gábor
, Radek
KaňaBernát
Gábor
Steinbach
, Jiří Bernát
Liška1,2,3
, Gábor
, Radek Kaňa1
1) Centre
ALGATECH,
Institute
of Třeboň,
Microbiology,
CAS, Třeboň, Czech Republic 2) Institute of Botanics, CA
1) Centre ALGATECH,
Institute
of Microbiology,
CAS,
Czech Republic
3) University of South Bohemia, Faculty of Science, Česke Budejovice, Czech Republic
2) Institute ofTřeboň,
Botanics,Czech
CAS, Republic
Třeboň, Czech
Republic
3) University [email protected]
South Bohemia, Faculty of Science, Česke Budejovice, Czech Republic
[email protected]
The induction of chlorophyll fluorescence is a well-known method in photosynthesis researc
The induction
of chlorophyll
fluorescence
is a well-known
method
in photosynthesis
research. Usually,
Usually,
fluorescence
induction
measurements
on microalgae
and cyanobacteria
are perform
liquidmeasurements
cultures, and on
themicroalgae
signal recorded
represents theareaverage
of potentially
heterogeneo
fluorescenceusing
induction
and cyanobacteria
performed
using liquid
signals of
individual.
from single cells
– or cell particles
cultures, andfluorescence
the signal recorded
represents
the Gathering
average of information
potentially heterogeneous
fluorescence
can reveal the inherent heterogeneity behind these mean values. The available spectral detecto
signals of individual.
Gathering information
from single cells
– ora cell
particles –high
can reveal
the inherent
of the up-do-date
confocal microscopes
have
sufficiently
temporal
resolution for su
heterogeneityinvestigations.
behind these mean values. The available spectral detectors of the up-do-date confocal
microscopes have a sufficiently high temporal resolution for such investigations.
We are developing a new system based on a Zeiss LSM880 confocal microscope (equipped w
We are developing
a new system based on a Zeiss LSM880 confocal microscope (equipped with 8.9
8.9 nm resolution spectral detector) and an attached microcontroller driven illumination syste
nm resolution
spectral
detector)flashes
and an(intensity,
attached microcontroller
driven illumination
The with t
The programmable
duration and wavelength)
are fullysystem.
synchronized
imaging
the communication
trigger ports
of the
microscope.
system will
programmable
flashesusing
(intensity,
duration and wavelength)
are fully
synchronized
withThe
the imaging
usingapply flash
between
acquiring
– this
thatapply
the images
are taken
under the
the same lig
the communication
trigger
ports ofthe
theframes
microscope.
Theensures
system will
flashes between
acquiring
conditions from the first pixel to the last.
frames – this ensures that the images are taken under the same light conditions from the first pixel to
the last. The resulting 2+1D dataset (images with spectral information) with the metadata (markers
certain
times)
information with
aboutthethe
fluorescence
of t
The resulting
2+1Dillumination
dataset (images
withprovides
spectral information)
metadata
(markersresponse
for
photosynthetic microorganisms with a reasonable spatial resolution.
certain illumination times) provides information about the fluorescence response of the photosynthetic
microorganisms with a reasonable spatial resolution.
Figure: custom-developed data analyzer software for SRFI measurements
Figure: custom-developed
data analyzer software for SRFI measurements
161
ELMI meeting
european
light microscopy
initiative
elmi
16th international
ELMI meeting
(P036) Imaging of spatial and temporal lymphatic growth with
single-cell resolution by tissue decolorization
Andrea Styevkóné Dinnyés1,2, Zoltán Jakus1,2
1) Semmelweis University, Budapest, Hungary
2) Hungarian Academy of Sciences, Budapest, Hungary
[email protected]
Novel and unexpected roles of altered lymphatic fuction have recently been implicated in the
Imaging of spatial and temporal lymphatic growth with single-cell
pathogenesis of hypertension, atherosclerosis,
myocardial
infarction, obesity and metabolic diseases,
resolution by tissue
decolorization
but the molecular mechanisms Andrea
regulating
lymphatic
growth
remain not fully understood due to
Styevkóné Dinnyés , Zoltán Jakus
the great limitations of the available
experimental
systems.
In
our studies we aimed to develop an
1) Semmelweis University, Budapest, Hungary 2) Hungarian Academy of Sciences, Budapest, Hungary
effective approach to monitor lymphatic growth.
[email protected]
In different experimental systems, lymphatic growth was shown by fluorescent, confocal and twoNovel and unexpected roles of altered lymphatic fuction have recently been implicated in the
photon microscopy. First, paraffin-based
histology
was performed
followed
by immunohistochemistry.
pathogenesis
of hypertension,
atherosclerosis,
myocardial
infarction, obesity and metabolic
diseases, but the molecular mechanisms regulating lymphatic growth remain not fully
In the second set of the experiments,
whole-mount
immunostaining
or
lymphatic
reporter strains were
understood due to the great limitations of the available experimental systems. In our studies
we
aimed
to
develop
an
effective
approach
to
monitor
lymphatic
growth. approach
utilized to visualize the lymphatic vessels. Third, a recently described tissue decolorization
In different
experimental
lymphatic
growth
shownalcohol
by fluorescent,
confocal and
was optimized, in which embryonic
and adult
tissuessystems,
was made
using
thewas
amino
containing
two-photon microscopy. First, paraffin-based histology was performed followed by
CUBIC cocktails followed by the immunostaining
of
the
lymphatic
vessels.
immunohistochemistry. In the second set of the experiments, whole-mount immunostaining or
lymphatic reporter strains were utilized to visualize the lymphatic vessels. Third, a recently
Paraffin based histology combined
with immunohistochemistry appeared to be efficient to visualize
described tissue decolorization approach was optimized, in which embryonic and adult tissues
was greatly
made using
the by
amino
containing
followed by the
the lymphatic vessels, but it was
limited
thealcohol
imaging
plane CUBIC
in the cocktails
two-dimensional
immunostaining of the lymphatic vessels.
approach. The whole-mount and lymphatic reporter systems made possible the rapid visualization of
Paraffin based histology combined with immunohistochemistry appeared to be efficient to
the lymphatics, but only on the surface
the lackvessels,
of organ
Thebytissue
decolorization
visualizedue
the to
lymphatic
but ittransparency.
was greatly limited
the imaging
plane in the twodimensional approach. The whole-mount and lymphatic reporter systems made possible the
approach allowed us to make both
developing
embryonic
and
adult
organs
completely
transparent.
rapid visualization of the lymphatics, but only on the surface due to the lack of organ
transparency.
The tissue decolorization
us immunostaining
to make both developing
Although tissue decolorization resulted
in undetectable
fluorescentapproach
reporterallowed
signals,
embryonic and adult organs completely transparent. Although tissue decolorization resulted in
undetectable
fluorescent
reporter
appeared to be efficient to show the
appeared to be efficient to show the
lymphatic
vessels
withsignals,
singleimmunostaining
cell resolution.
lymphatic vessels with single cell resolution.
We demonstrated that whole body tissue decolorization allows us to visualize the lymphatic
that whole body tissue decolorization allows us to visualize the lymphatic
vessels with single cell resolutionWe
indemonstrated
developing
organs
andinadult
tissues.
Imaging
vessels
vessels
with single cell
resolution
developing
organs
and adultlymphatic
tissues. Imaging
lymphatic
vessels in decolorized tissues provides new perspectives for the studies focusing on spatial
in decolorized tissues provides new
perspectives
for
the
studies
focusing
on
spatial
and
temporal
and temporal lymphatic growth, which are essential for the development of novel therapeutic
approaches modulating
growth and
lymphatic growth, which are essential
for the lymphatic
development
of function.
novel therapeutic approaches
modulating lymphatic growth and function.
1,2
162
1,2
16th international
ELMI meeting
european
light microscopy
initiative
elmi
16th international
(P037) In the footsteps of “intercellular highways”formation
ELMI meeting
and function of membrane nanotubes
Edina Szabó-Meleg1,2, Tamás Madarász1, Elek Telek1, Brigitta Brunner3, Henriett Halász3, Kinga Futó1, János
Matkó4, Miklós Nyitrai1,2
1) Department of Biophysics, Medical School, University of Pécs, Hungary
2) Szentágothai Research Center, University of Pécs, Hungary
3) Faculty of Sciences, University of Pécs, Hungary
4) Department of Immunology, Faculty of Science, Eötvös Loránd University, Hungary
[email protected]
Direct cell-cell communication is crucial for multicellular organisms to crosstalk and to pass information
from one cell to another. Until recently direct cell-cell communication was only described via gap junctions
and synaptic signaling. They were assumedIn to
the only
way of passing
information
between
eukaryotic
thebe
footsteps
of “intercellular
highways”formation and
function of
membrane nanotubes
cells. Membrane nanotubes – as filopodium-like
cell-cell
bridges
–
were
identified
in
2004
as
a
new form
Edina Szabó-Meleg , Tamás Madarász , Elek Telek , Brigitta Brunner , Henriett Halász ,
,
János
Matkó
,
Miklós
Nyitrai
Kinga
Futó
of intercellular communication and matter transport. These thin membrane protrusions that physically
Department of Biophysics, Medical School, University of Pécs, Hungary
connect two cells were found to be intercellular
highways
forofcalcium
Szentágothai Research
Center, University
Pécs, Hungaryions, different cell organelles (e.g.
Faculty of Sciences, University of Pécs, Hungary
Department of Immunology, Faculty of Science, Eötvös Loránd University, Hungary
mitochondria), lipid molecules, various
proteins,
prions,
vesicles,
DNA and RNA molecules. They have
[email protected]
role in the effective propagation of bacteria and viruses (HIV) among cells. Membrane nanotubes show
cell-cell communication is crucial for multicellular organisms to crosstalk and to pass
similarity in several properties with Direct
membrane
protrusions
surface
of thewascells,
information
from one
cell to another.appearing
Until recently on
directthe
cell-cell
communication
only but
described via gap junctions and synaptic signaling. They were assumed to be the only way of
possess characteristic differences frompassing
theminformation
(they differ
instance
in their nanotubes
length –and
diameter).cellbetweenfor
eukaryotic
cells. Membrane
as filopodium-like
cell bridges – were identified in 2004 as a new form of intercellular communication and
matter transport.
Theseisthin
membrane
protrusions that
physically connect two cells
were
A proposed way of membrane nanotube
formation
that
they develop
as actin-dependent
membrane
found to be intercellular highways for calcium ions, different cell organelles (e.g.
mitochondria),
lipid molecules,
various proteins,
prions,substrate
vesicles, DNA53
andprotein
RNA molecules.
protrusions from directed, filopodium-like
structures.
As insulin
receptor
(IRSp53)
They have role in the effective propagation of bacteria and viruses (HIV) among cells.
Membrane
nanotubes show similarity
in and
severalsuperresolution
properties with membrane
protrusions(SIM)
promotes filopodia formation, in this work
laser-scanning
confocal
microscopy
appearing on the surface of the cells, but possess characteristic differences from them (they
for instance in their length and diameter).
were applied to investigate the effectdiffer
of IRSp53
protein on the formation and morphology of membrane
A proposed way of membrane nanotube formation is that they develop as actin-dependent
nanotubes. The effect of latrunculinmembrane
A wasprotrusions
studiedfromindirected,
filopodia
and nanotubes
in the
absence
filopodium-like
structures. As insulin
receptor
substrate and
protein (IRSp53) promotes filopodia formation, in this work laser-scanning confocal and
presence of the overexpressed IRSp53.53superresolution
Mitochondrial
and(SIM)
vesicular
transport
was
also ofexamined
through
microscopy
were applied
to investigate
the effect
IRSp53 protein
on
the formation and morphology of membrane nanotubes. The effect of latrunculin A was
membrane nanotubes.
studied in filopodia and nanotubes in the absence and presence of the overexpressed IRSp53.
Mitochondrial and vesicular transport was also examined through membrane nanotubes.
Our results suggest that albeit membrane nanotubes show similarity in several aspects with filopodia
Our results suggest that albeit membrane nanotubes show similarity in several aspects with
show differences
them in the Prototypical
basic mechanism ofvesicles
formation.tracked
Prototypicalwithin
also show differences from them in thefilopodia
basicalso
mechanism
offrom
formation.
vesicles tracked within nanotubes illustrating bidirectional traffic of microvesicles within
nanotubes illustrating bidirectional traffic
of microvesicles
within thick membrane nanotubes.
thick membrane
nanotubes.
1,2
1
1
1
4
3
3
1,2
1
2
3
4
This work was supported by grant K104971 This
sponsored
by thebyHungarian
Science
FundNational
(OTKA).
work was supported
grant K104971National
sponsored by
the Hungarian
Science Fund (OTKA).
163
ELMI meeting
european
light microscopy
initiative
elmi
16th international
ELMI meeting
(P038) Evidence for homodimerization of the c-Fos transcription
factor in live cells revealed by FRET, SPIM-FCCS and MDEvidence for homodimerization of the c-Fos transcription factor in live cells
modeling
revealed by FRET, SPIM-FCCS and MD-modeling
Nikoletta Szalóki1, Jan Wolfgang Krieger2, István Komáromi3, Katalin Tóth2, György Vámosi1
Nikoletta Szalóki1, Jan Wolfgang Krieger2, István Komáromi3, Katalin Tóth2, György Vámosi
1) Department of Biophysics and Cell Biology, Faculty of Medicine, University of Debrecen, Hungary
1
Department
of Biophysics
and Cell
Biology,
Faculty of Medicine,
University
2) German
Cancer Research
Center (DKFZ),
Biophysics
of Macromolecules,
Heidelberg,
Germanyof Debrecen, Hungary
2
German Cancer Research Center (DKFZ), Biophysics of Macromolecules, Heidelberg, Germany
3) HAS-UD
VascularVascular
Biology, Thrombosis
and Haemostasis
Research Group,Research
Hungarian
Academy
of Sciences,
3
HAS-UD
Biology, Thrombosis
and Haemostasis
Group,
Hungarian
Academy of Sciences,
Debrecen,
Hungary
DivisionDivision
of ClinicalofLaboratory
Science, Department
Laboratory Medicine
Debrecen,
Hungary
Clinical Laboratory
Science,ofDepartment
of Laboratory Medicine
Faculty
of Medicine,
University
of Debrecen,
Debrecen, Hungary
Faculty
of Medicine,
University
of Debrecen,
Debrecen, Hungary
[email protected]
[email protected]
The c-Fos and c-Jun transcription factors, members of the activator protein-1 (AP-1) complex
c-Fos and
c-Junto transcription
members
of thethe
activator
form The
heterodimers
and bind
DNA via a basic factors,
leucine zipper,
and regulate
cell cycle,protein-1
apoptosis,(AP-1) com
form heterodimers
to zipper
DNA fragments
via a basic
zipper,
regulate the cell cy
differentiation,
etc. Purified and
c-Junbind
leucine
couldleucine
also form
stable and
homodimers,
apoptosis,
differentiation,
etc.
Purified
c-Jun
leucine
zipper
fragments
whereas c-Fos leucine zipper homodimers were found to be much less stable in earlier in vitrocould
studies.also form st
homodimers, whereas c-Fos leucine zipper homodimers were found to be much less stabl
The importance
of c-Fos overexpression in tumors and the controversy in the literature concerning
earlier in vitro studies. The importance of c-Fos overexpression in tumors and the controvers
c-Fos the
homodimerization
prompted us toc-Fos
investigate
Fos homodimerization.
FRET andusmolecular
literature concerning
homodimerization
prompted
to investigate
brightness
analysis of fluorescence
correlation
spectroscopy data
from live HeLa
cells transfected
with
homodimerization.
FRET
and molecular
brightness
analysis
of fluorescence
correla
spectroscopy
data c-Fos
fromindicated
live HeLa
cells
transfected
with
fluorescent
protein-tagged cfluorescent
protein-tagged
that c-Fos
formed
homodimers.
We developed
a method
indicated
c-Fos
formed homodimers.
method
to determine
to determine
thethat
absolute
concentrations
of transfectedWe
and developed
endogenous ac-Fos
and c-Jun,
which the abso
concentrations
transfected
and endogenous
c-Fos(Kd=7.8±2
and c-Jun,
allowed us to determ
allowed
us to determineof
dissociation
constants
of c-Fos homodimers
μM)which
and c-Fos–c-Jun
dissociation constants of c-Fos homodimers (Kd=7.8±2 μM) and c-Fos–c-Jun heterodimers
heterodimers
(on the order of 10-100 nM) from FRET titrations. Imaging fluorescence cross-correlation
the order of 10-100 nM) from FRET titrations. Imaging fluorescence cross-correla
spectroscopy
and molecular
modeling modeling
simulationssimulations
confirmed that
c-Fos homodimers
stably
spectroscopy
and molecular
confirmed
that c-Foswere
homodimers
were st
associated
and
could
bind
to
the
chromatin.
Our
results
establish
c-Fos
homodimers
as
a
novel
form
of
associated and could bind to the chromatin. Our results establish c-Fos homodimers
as a n
the AP-1
complex,
which
may complex,
be an autonomous
in c-Fos overexpressing
tissues, factor in cform
of the
AP-1
whichtranscription
may be factor
an autonomous
transcription
overexpressing
tissues,
and could contribute to tumor development.
and could
contribute to tumor
development.
215
215
Fos -EGFP + Fos -mRFP1
N /N
K =7.8 ± 2.0 μM
d
E =9.5 ± 0.8%
0
A
D
2.0±0.1
1.15±0.05
0.45±0.05
Diffusi
on
coeffici
ent
(D )
cross
2
[μm /s]
215
215
EGFP-P30Fos -EGFP+
Fos -EGFP +
mRFP1Jun-mRFP1 215
Fos -mRFP1
164
16th international
ELMI meeting
european
light microscopy
initiative
elmi
16th international
(P039) EGFP oligomers as natural fluorescence
and
ELMI meeting
hydrodynamic standards
György Vámosi1, Norbert Mücke2, Gabriele Müller2, Jan Wolfgang Krieger2, Ute Curth3, Jörg Langowski2,
Katalin Tóth2
1) Department of Biophysics and Cell Biology, Faculty of Medicine, University of Debrecen, Debrecen, Hungary
2) DKFZ, Biophysics of Macromolecules, Heidelberg, Germany
3) Institute for Biophysical Chemistry, Hannover Medical School, Hannover, Germany
[email protected]
EGFP oligomers are convenient standards for experiments on fluorescent protein-tagged
biomolecules. In this study, we characterized their hydrodynamic and fluorescence properties. Diffusion
coefficients D of EGFP1-4 were determined by analytical ultracentrifugation with fluorescence detection
and by fluorescence correlation spectroscopy (FCS), yielding 83.4…48.2 μm2/s and 97.3…54.8 μm2/s
from monomer to tetramer. A “barrels standing in a row” model agreed best with the sedimentation
data. Oligomerization red-shifted EGFP emission spectra without any shift in absorption. Fluorescence
anisotropy increased, indicating homoFRET between the subunits. Fluorescence lifetime decreased
only slightly (4%) indicating insignificant quenching by FRET to subunits in non-emitting states.
FCS-measured D, particle number and molecular brightness depended on dark states and lightinduced processes in distinct subunits, resulting in a dependence on illumination power different for
monomers and oligomers. Since subunits may be in “on” (bright) or “off” (dark) states, FCS-determined
apparent brightness is not proportional to that of the monomer. From its dependence on the number
of subunits, the probability of the “on” state for a subunit was determined to 96% at pH8 and 77% at
pH6.38, i.e., protonation increases the dark state. These fluorescence properties of EGFP oligomeric
standards can assist interpreting results from oligomerized EGFP fusion proteins of biological interest.
165
ELMI meeting
elmi
rman BioImaging, University of Konstanz, Germany
european
light microscopy
initiative
[email protected]
16th international
ELMI meeting
Facilities (CF) for advanced light microscopy (ALM) have become indispensable suppo
for research
the life sciences.
Their Recommendations
organizational structure
technical characteristi
(P040)inGerman
BioImaging:
forand
ALM-CF
operations
quite diverse,
although the tasks they pursue and the services they offer are quite simila
efore, throughout
Europe, scientists of ALM-CF are forming networks to promo
Nadine Utz 1 and the German BioImaging network
actions and discuss best practice models. Here, we present recommendations for ALM-C
ations by
the German
ALM-CFs,
1) German
BioImaging,network
University of of
Konstanz,
Germany German BioImaging (GerBI), which have bee
[email protected]
ished in Microscopy Research and Technique [1]. Special emphasis is given
nizational,
management
matters,
thebecome
training
of CF support
staff and
Coretechnical,
Facilities (CF)and
for advanced
light microscopy
(ALM)tohave
indispensable
unitsusers, to th
research intopic
the lifeof
sciences.
organizationaland
structure
and technical
are quite
easinglyforpressing
data Their
management
analysis,
and characteristics
to the aspect
of profession
diverse,
although
the
tasks
they
pursue
and
the
services
they
offer
are
quite
similar.
Therefore,
lopment and careers in CFs. While we discuss these issues mainly in relation to Germa
throughout Europe, scientists of ALM-CF are forming networks to promote interactions and discuss
M-CFs, most of the content is of interest to CFs for the life sciences in general.
best practice models. Here, we present recommendations for ALM-CF operations by the German
network of ALM-CFs, German BioImaging (GerBI), which have been published in Microscopy Research
Elisa Ferrando-May*,
Hellaemphasis
Hartmann,
Reymann,
Nariman
Ansari,matters,
Nadine
and Technique [1]. Special
is givenJürgen
to organizational,
technical,
and management
to Utz, Han
ch Fried,theChristian
Christian
Liebig,
Terjung,
training of CFKukat,
staff andJan
users,Peychl,
to the increasingly
pressing
topic ofStefan
data management
andVibor
analysis,Laketa, An
and to theWeidtkamp-Peters,
aspect of professional development
and careers inWerner
CFs. WhileZuschratter,
we discuss these Sergiy
issues mainly
bert, Stefanie
Astrid Schauss,
Avilov and th
in
relation
to
German
ALM-CFs,
most
of
the
content
is
of
interest
to
CFs
for
the
life
sciences
in
general.
man BioImaging network (2016) Advanced light microscopy core facilities: Balancin
[1] Elisaand
Ferrando-May*,
Hartmann, Jürgen Reymann,
Nariman Ansari, Nadine
Hans-Ulrich
ice, science
career,HellaMICROSCOPY
RESEARCH
ANDUtz,TECHNIQUE
DO
Fried, Christian Kukat, Jan Peychl, Christian Liebig, Stefan Terjung, Vibor Laketa, Anje Sporbert, Stefanie
002/jemt.22648 2016
Weidtkamp-Peters, Astrid Schauss, Werner Zuschratter, Sergiy Avilov and the German BioImaging
network (2016) Advanced light microscopy core facilities: Balancing service, science and
career, MICROSCOPY RESEARCH AND TECHNIQUE DOI: 10.1002/jemt.22648 2016
166
16th international
ELMI meeting
european
light microscopy
initiative
elmi
16thmicroscopy
international
(P041) Artifacts analysis in localization based
ELMI meeting
Artifacts analysis in localization based microscopy
Dániel Varga1, József Sinkó1, Tamás Gajdos1, Gábor Szabó1,2, Miklós Erdélyi1
Dániel Varga1, József Sinkó1, Tamás Gajdos1, Gábor Szabó1,2, Miklós Erdélyi1,
2) MTA-SZTE Research
Group onResearch
Photoacoustic
Szeged,
Hungary Szeged, Hungary
2) MTA-SZTE
Group Spectroscopy,
on Photoacoustic
Spectroscopy,
[email protected]
[email protected]
Since the development of localization based super-resolution microscopy, this technique is in the
the development
of localization
based
super-resolution
technique is in the
focus ofSince
attention,
inter alia it exceeds
about one
magnitude
the spatialmicroscopy,
resolution ofthisconventional
focus
of
attention,
inter
alia
it
exceeds
about
one
magnitude
the
spatial
resolution
of conventional
light microscopes. However, such high resolution requires at least the same degree of precision.
The
light microscopes. However, such high resolution requires at least the same degree of precision.
minor effects
are
typically
neglected
in
traditional
microscopes
and
start
to
play
a
key
role
in
The minor effects are typically neglected in traditional microscopes and start to play athe
key role in
interpretation
of the final image.
the interpretation
of the final image.
Here we studied both theoretically and experimentally the errors, aberrations, and their effects.
we studied both theoretically and experimentally the errors, aberrations, and their effects.
BecauseHere
experiments
are expensive and time consuming, we speed up the investigation and
Because experiments are expensive and time consuming, we speed up the investigation and
optimization
by
TestSTORM,
a powerfulaopen
sourceopen
simulator.
investigations
showed thatshowed
the that
optimization by TestSTORM,
powerful
source The
simulator.
The investigations
the
implementation
of
the
image
is
a
challenge
due
to
imaging
artefacts.
implementation of the image is a challenge due to imaging artefacts.
We categorized the possible imaging artefacts by their origin, namely, what is the causative factor:
We categorized the possible imaging artefacts by their origin, namely, what is the causative
the optical
components,
thecomponents,
sample or the the
algorithms.
showed
how monochromatic
factor:
the optical
sample We
or the
algorithms.
We showedand
howchromatic
monochromatic
and chromatic
in focus and
defocus
arrangements,
and off-axis
position of
aberrations,
in focus andaberrations,
defocus arrangements,
on-axis
and off-axis
positionon-axis
of the molecules
affected
the
molecules
affected
the
final
image
quality
and
made
difference
between
static
and
stochastic
the final image quality and made difference between static and stochastic factors. We also give a clue,
factors. We also give a clue, how these effects can be eliminated or reduced.
how these effects can be eliminated or reduced.
Fig. 1.Fig.
The1.effect
of deeper
sample
imaging,
(A) the
position
of four
vesicles
on on
thethe
frame
The effect
of deeper
sample
imaging,
(A) centre
the centre
position
of four
vesicles
frame (sized
256
×
256
CCD
pixels,
1
CCD
pixel
equals
160
nm
in
the
focal
plane),
(B)
and
(C)
super-resolved
(sized 256 × 256 CCD pixels, 1 CCD pixel equals 160 nm in the focal plane), (B) and (C) super-resolved
of the four vesicles in case of 80 nm and 5 m deep imaging respectively.
picturespictures
of the four
vesicles in case of 80 nm and 5 μm deep imaging respectively.
References:
References:  M. Erdélyi, J. Sinkó, R. Kákonyi, A. Kelemen, E. Rees, D. Varga, G. Szabó (2015)
• M. Erdélyi, J. Sinkó,
R. Kákonyi,88,
A. Kelemen,
E. Rees, D. Varga, G. Szabó (2015) Methods, 88, 122–132.
Methods,
122–132.
• J. Sinkó, R. Kákonyi,
E. Rees,R.
D. Metcalf,
A. E.E.Knight,
F. Kaminski,
Szabó,
and M. Erdélyi
Biomedical
Optics and
 J. Sinkó,
Kákonyi,
Rees,C. D.
Metcalf,G.A.
E. Knight,
C. F.(2014)
Kaminski,
G. Szabó,
Express, 5(3), 778-787.
M. Erdélyi (2014) Biomedical Optics Express, 5(3), 778-787.
167
ELMI meeting
european
light microscopy
initiative
elmi
16th international
ELMI meeting
(P042) Assembly of Interleukin Receptor Subunits
Ádám Kenesei1, Julianna Volkó1, Péter Várnai2, Felix Bestvater3, Jörg Langowski3, Thomas A. Waldmann4,
Katalin Tóth3, György Vámosi1
1) Department of Biophysics and Cell Biology, University of Debrecen, Hungary
2)Department of Physiology, Semmelweis University, Budapest, Hungary
3) German Cancer Research Center, Heidelberg, Germany
4) Lymphoid Malignancies Branch, Center for Cancer Research, National Cancer Institute, Bethesda, USA
[email protected], [email protected]
Interleukin-2 and -15 cytokine growth factors play pivotal roles in the regulation of the immune
system bycontrolling life and death of the lymphocytes: activate similar signal transduction pathways
and also have contrasting roles in adaptive immune responses. IL-2 is used therapeutically as an
immune adjuvant in certain types of lymphoproliferative diseases and cancers, IL-2 antagonists can
prevent organ transplant rejection.Their heterotrimeric receptors have two subunits (beta and gamma)
in common and may exist in the membrane of immunecellsindifferent receptor forms having different
ligand binding affinity.Preassembly of the receptor chains has already been characterized at the cell
surface in humans, but their formation inside the cell (prior to reaching the cell membrane) remains to
be clarified. Our aim was to investigate the associations of receptor subunits after their synthesis in the
endoplasmic reticulum (ER) and the Golgi of living cells.
We created plasmid constructs expressing different receptor chains tagged with EGFP or mCherry and
optimized their transient co-transfection inHeLa cells. To assess dimer formation, we measuredintensity
based Förster resonance energy transfer (FRET) between the FP tagged subunits by a Leica TCS SP5
confocal microscope. As a third label,BFP-tagged ER and Golgi markers were used to evaluate FRET
data in an organelle specific manner. In addition to using wild type forms of the receptor subunits, we
also created a truncated beta chain to reduce the distance between the C termini and thereby enhance
the FRET signal.Pixel by pixel evaluation of the confocal microscopic images was achieved using
FiJi ImageJ software and the RiFRET plugin. Interleukin receptor subunits showed low but positive
intracellular FRET efficiency during their trafficking: the energy transfer between both thebeta and
alpha subunits and between the beta andgamma subunits was higher in the ERthan in the Golgi.These
data suggest that the subunits associate after their synthesis in the endoplasmatic reticulum, then
their interactions are weakened in the Golgi. In IL-2 producing T cells, signaling might take place in
the cell before receptor subunits are expressed in the plasma membrane. Our results may have clinical
importance in antibody therapies against lymphoma targeting receptor subunits.
168
16th international
ELMI meeting
Participants list
e
light mi
16th internation
ELMI meeting
24-27 May 2016, Debrecen,
Kölcsey Center - Hotel Lyciu
16th international
ELMI meeting
A
Anderson, Kurt The Francis
Crick Institute
United Kingdom
[email protected]
Arnaud, Rehel
Phasics
France
[email protected]
Ankerhold, Richard
Carl Zeiss Microscopy GmbH
Germany
[email protected]
Arndt-Jovin, Donna
Max Planck Institute for
Biophysical Chemistry
Germany
[email protected]
Antal, Miklós
University of Debrecen
Hungary
[email protected]
european
light microscopy
initiative
elmi
16th international
ELMI meeting
Atkinson, Benjamin
Intelligent Imaging Innovations
GmbH
United Kingdom
[email protected]
Auer, Herbert
iLab Solutions, LLC
USA
[email protected]
Artacho, José
Ecole Polytechnique Fédérale de
Lausanne (EPFL)
Switzerland
[email protected]
Aswani, Kavita
Excelitas Technologies
Canada
[email protected]
Aumayr, Karin
Research Institute of Molecular
Pathology
Austria
[email protected]
Bacso, Zsolt
Hungary
[email protected]
Balogh, Bálint
PTE ÁOK
Hungary
[email protected]
Barkó, Szilvia
University of Pécs
Hungary
[email protected]
Bailly, Almut
Chroma Technology
Germany
[email protected]
Bardia, Lidia
Institute for Research in
Biomedicine - IRB Barcelona
Spain
[email protected]
Barna, Laszlo
Institute of Experimental Medicine,
Hungarian Academy of Sciences
Hungary
[email protected]
Antal, Bálint
Hungary
[email protected]
Antz, Chistoph
LUXENDO
Germany
[email protected]
Ayadin, Ferhan
Biological Research Centre of the
Hungary
[email protected]
B
171
ELMI meeting
european
light microscopy
initiative
elmi
16th international
ELMI meeting
Bauer, Christoph
University of Geneva
Switzerland
[email protected]
Biocco, Michel
Bruker
France
[email protected]
Brom, Manuela
DKFZ
Germany
[email protected]
Bayer, Harald
Nikon GmbH
Austria
[email protected]
Birngruber, Konstantin
TOPTICA Photonics AG
Germany
[email protected]
Browne, Mark
Andor Technology
USA
[email protected]
Belyaev, Yury
University of Bern
Switzerland
[email protected]
Borkowski, Krzysztof
Olympus Czech Group, s.r.o. Czech
Republic
[email protected]
Brutkowski, Wojciech
Olympus Polska Sp. z o.o.
Poland
[email protected]
Bosset, Jérôme
University of Geneva
Switzerland
[email protected]
Bulkescher, Jutta
University of Copenhagen - Center
for Protein Research
Denmark
[email protected]
Bender, Carola
Arivis AG
Germany
[email protected]
Berger, Martin
Laboratory Imaging, spol. s.r.o.
Czech Republic
[email protected]
Berta, Gergely
University of Pécs
Hungary
[email protected]
Bozzo, Luigi
Lausanne (EPFL)
Switzerland
[email protected]
Braunstein, Thomas
University of Copenhagen
Denmark
[email protected]
Bundschuh, Sebastian
Max Planck Institute of Molecular
Cell Biology and Genetics
Germany
[email protected]
Burger, Gabriele
Leica Microsystems
Germany
[email protected]
C
Call, Peter
CoolLED Limited
United Kingdom
[email protected]
172
Chevalier, Clément
SFR Biosit UMS CNRS 3480 / US
INSERM 018
France
[email protected]
Christensen, Nynne
Copenhagen University
Denmark
[email protected]
16th international
ELMI meeting
Ciceri, Ferdinando
Mad City Labs GmbH
Switzerland
[email protected]
Coppey-Moisan, Maïté
Institute Jacques Monod
France
[email protected]
Colombelli, Julien
Spain
[email protected]
Cordelières, Fabrice
Bordeaux Imaging Centre
France
[email protected]
Combs, Christian
NIAID-NIH
USA
[email protected]
Crivaro, Marko
University of Helsinki
Finland
[email protected]
european
light microscopy
initiative
elmi
Cullen,
Patrick
16th
international
ELMI
meeting
Lumenera
Corporation
Canada
[email protected]
Csatorday, Karoly
Horiba Scientific
USA
[email protected]
Csucs, Gabor
ETH Zurich
Switzerland
[email protected]
D
de Vries, Anthony
Germany
[email protected]
Dienes, Beatrix
Hungary
[email protected]
Demchenko, Alexander
Palladin Institute of Biochemistry
Ukraine
[email protected]
Dietzel, Ralf
Omicron-Laserage Laserprodukte
GmbH
Germany
[email protected]
d'Herouel, Aymeric
University of Luxembourg
Luxembourg
[email protected]
Draude, Georg
Chroma Technology
Germany
[email protected]
Drent, Peter
Confocal.nl
Netherlands
[email protected]
E
Ebeling, Carl
Bruker
USA
[email protected]
Eich, Florian
Germany
[email protected]
Eisler, Stephan
Central Facility For Advanced
Microscopy
Germany
[email protected]
173
ELMI meeting
european
light microscopy
initiative
elmi
16th international
ELMI meeting
Enyedi-Kolhász, László
UNICAM Magyarország Kft.
Hungary
[email protected]
Eriksson, Jens
Oslo University Hospital
Norway
[email protected]
Erdelyi, Miklos
University of Szeged
Hungary
[email protected]
Eulitz, Stefan
Miltenyi Biotec GmbH
Germany
[email protected]
F
Fernandez-Rodriguez, Julia
University of Gothenburg
Sweden
[email protected]
Francois, Liliana
DKFZ
Germany
[email protected]
Fried, Hans
DZNE
Germany
[email protected]
Fischer, Joachim
Abberior Instruments GmbH
Germany
[email protected]
Freisinger, Tina
ibidi GmbH
Germany
[email protected]
Friedrich, Martin
Wiley-VCH Verlag GmbH
Germany
[email protected]
Gelman, Laurent
Friedrich Miescher Institute for
Biomedical Research
Switzerland
[email protected]
Girod, Andreas
University of Luxembourg Campus Belval
Luxembourg [email protected]
G
Gadella, Theodorus
University of Amsterdam
Netherlands
[email protected]
Gajdos, Tamás
Hungary
[email protected]
Gansen, Alexander
DKFZ
Germany
[email protected]
Gehrig, Jochen
Acquifer AG
Germany
[email protected]
174
Geyer, Stefan
Medical University of Vienna
Austria
[email protected]
Giesebrecht, Jan
FEI Munich GmbH
Germany
[email protected]
Glombik, Michael
Germany
[email protected]
Glösmann, Martin
University of Veterinary Medicine
Vienna
Austria
[email protected]
16th international
ELMI meeting
Gotzmann, Josef
Max F. Perutz Laboratories GmbH
Austria
[email protected]
Gröger, Marion
Core Facility Imaging - Medical
University Vienna
Austria
[email protected]
Graewe, Walter
Hamamatsu Photonics Deutschland GmbH Grunwald, David
Germany
University of Massachusetts
[email protected]
Medical School
USA
Gregor, Ingo
[email protected]
Georg-August-University
Germany
[email protected]
european
light microscopy
initiative
elmi
Guérin,
Christopher
16th
international
ELMI
meeting
Vlaanderen
Institute of
Biotechnology (VIB)
Belgium
[email protected]
Guiet, Romain
Lausanne (EPFL)
Switzerland
[email protected]
H
Haas, Petra
Leica Microsystems
Germany
[email protected]
He, Jiaye
MPI-CBG
Germany
[email protected]
Hess, Lord
Acquifer AG
Germany
[email protected]
Halavatyi, Aliaksandr
EMBL Heidelberg
Germany
[email protected]
Hell, Stefan
Germany
Heusermann, Wolf
University of Basel
Switzerland
[email protected]
Hansen, Michael
Denmark
[email protected]
Hemmerich, Peter
Leibniz Institute On Aging - FritzLipmann-Institute
Germany
[email protected]
Hapek, Anna
IST Austria
Austria
[email protected]
Henrich, Matthias
Germany
[email protected]
Hauschild, Robert
IST Austria
Austria
[email protected]
Hernandez-Varas, Pablo
Holtackers, René
Nikon Instruments Europe B.V.
University of Zürich
Netherlands
[email protected] Switzerland
[email protected]
Hirokawa, Haruka
Tokai Hit Co., Ltd.
Japan
[email protected]
Hoischen, Christian
Leibniz Institute On Aging - FritzLipmann-Institute
Germany
[email protected]
175
ELMI meeting
european
light microscopy
initiative
elmi
16th international
ELMI meeting
Horn, Thomas
ETH Zurich
Switzerland
[email protected]
Hubbard, Andrew
Andor Technology
United Kingdom
[email protected]
Horvath, Peter
Biological Research Centre of the
Hungary
[email protected]
Hufnagel, Lars
LUXENDO
Germany
[email protected]
Huisken, Jan
Germany
[email protected]
I
Imreh, Gabriela
Karolinska Institutet
Sweden
[email protected]
Igaz, Antal
Carl Zeiss Technika Kft.
Hungary
[email protected]
J
Jahr, Wiebke
MPI CBG
Germany
[email protected]
Jennings, Lisa
ThermoFisher Scientific
United Kingdom
[email protected]
Ji, Na
Howard Hughes Medical Institute
USA
[email protected]
Kabat, Juraj
NIAID-NIH
USA
[email protected]
Kalkhoven, Carla
Netherlands
[email protected]
Katona, István
MTA KOKI
Hungary
[email protected]
Kaiser, Peter
Visitron Systems GmbH
Germany
[email protected]
Karsai, Attila
Microtrade Kft.
Hungary
[email protected]
Kenesei, Ádám
Hungary
[email protected]
Jaiswal, Jyoti
The George Washington University
USA
[email protected]
K
176
16th international
ELMI meeting
european
light microscopy
initiative
elmi
Kenkkilä, Jussi
Finland
[email protected]
Konzack, Sven
Leica Microsystems GmbH
Germany
[email protected]
Krunic,
Damir
16th
international
ELMI
DKFZ meeting
Keppler, Antje
EMBL
Germany
[email protected]
Kovács, András
Zenon Bio Kft.
Hungary
[email protected]
Keresztúri, Péter
Carl Zeiss Technika Kft.
Hungary
[email protected]
Kozubek, Michal
Masaryk University
Czech Republic
[email protected]
Kukat, Christian
Max Planck Institute for Biology
of Ageing
Germany
[email protected]
Koch, Marc
Bruker
France
[email protected]
Krens, Gabriel
IST Austria
Austria
[email protected]
Germany
[email protected]
Kumar, Abhishek
Austria
[email protected]
Kun, András
Auro-Science Consulting Kft
Hungary
[email protected]
L
Laketa, Vibor
Heidelberg University Hospital
Germany
[email protected]
Langowski, Jörg
DKFZ
Germany
[email protected]
Liebel, Urban
Acquifer AG
Germany
[email protected]
Lampe, Marko
EMBL Heidelberg
Germany
[email protected]
Leconte, Ludovic
Institut Curie
France
[email protected]
Llado, Anna
Biomedicine - IRB Barcelona Spain
[email protected]
Langlois, Eric
Lumencor Inc.
USA
[email protected]
Liebe, Susanne
Leica Microsystems
Germany
[email protected]
Lohmüller, Bertram
Hamamatsu Photonics
Deutschland GmbH
Germany
[email protected]
177
ELMI meeting
european
light microscopy
initiative
elmi
16th international
ELMI meeting
M
Macco, Romina
Bruker
Italy
[email protected]
Marx, Monika
Germany
[email protected]
Manders, Erik
University of Amsterdam
Netherlands
[email protected]
Masajada, Jan
Wrocław University of Science and
Technology
Poland
[email protected]
Manuelian, Tamara
Arivis AG
Germany
[email protected]
Matkó, János
Eotvos Lorand University
Hungary
[email protected]
Marawske, Stefan
McKechnie, James
Photometrics
Germany
[email protected] United Kingdom
United Kingdom
Marcello, Marco
Melzer, Volker
University of Liverpool
Qioptiq Photonics GmbH & Co. KG.
United Kingdom
Germany
[email protected]
[email protected]
Marosvoelgyi, Maria
Milius, Doreen
Arivis AG
IST Austria
Germany
Austria
[email protected]
[email protected]
Marrison, Joanne
Mitkovski, Mišo
University of York
United Kingdom
Experimental Medicine
[email protected]
Germany
[email protected]
178
Miyazono, Yuya
Germany
[email protected]
Mocsár, Gábor
Hungary
[email protected]
Monajembashi, Shamci
Leibniz Institute on Aging – Fritz
Lipmann Institute (FLI)
Germany
[email protected]
Monks, Colin
GmbH
Germany
[email protected]
Morrison, Ian
University of York
United Kingdom
[email protected]
Mueller, Tobias
Gregor-Mendel-Institute
Austria
[email protected]
Munck, Sebastian
VIB
Belgium
[email protected]
16th international
ELMI meeting
N
Nagy, Peter
Hungary
[email protected]
Niedetzky, Csaba
Supertech Kft.
Hungary
[email protected]
Nitschke, Roland
Albert Ludwigs University of
Freiburg
Germany
[email protected]
european
light microscopy
initiative
elmi
16th international
ELMI meeting
Noll, Florentine
Germany
[email protected]
O
Oberdiek, Hans-Jürgen
Germany
[email protected]
Oleksiuk, Olga
University of Heidelberg
Germany
[email protected]
O'Toole, Peter
University of York
United Kingdom
[email protected]
Ogg, Stephen
University of Alberta
Canada
[email protected]
Orthaus-Müller, Sandra
PicoQuant GmbH
Germany
[email protected]
Owe, Simen
Ortomedic AS
Norway
[email protected]
Ohlenschläger, Ingo
Nikon GmbH
Austria
[email protected]
Osváth, Szabolcs
Semmelweis University
Hungary
osvath.szabolcs@med.
semmelweis-univ.hu
P
Pantazis, Periklis
ETH Zurich
Switzerland
[email protected]
Parashuraman, Raman
Institute of Protein Biochemistry
Italy
[email protected]
Paszulewicz, Anna
Bitplane AG
Switzerland
[email protected]
Papon, Gautier
Argolight
France
[email protected]
Pasierbek, Pawel
IMBA - Institute of Molecular
Biotechnology
Austria
[email protected]
Pawley, James
University of Wisconsin
USA
[email protected]
179
ELMI meeting
european
light microscopy
initiative
elmi
16th international
ELMI meeting
Paysan, Jacques
Germany
[email protected]
Peters, Matthias
TOPTICA Photonics AG
Germany
[email protected]
Plantard, Laure
Denmark
[email protected]
Peker, Bülent
Germany
[email protected]
Petri, Gabriele
Research Institute of Molecular
Pathology
Austria
[email protected]
Pongor, Csaba
Institute of Experimental Medicine,
Hungary
[email protected]
Pfuhl, Andreas
LUXENDO
Germany
[email protected]
Potcoava, Mariana
GmbH
Germany
[email protected]
Pepperkok, Rainer
EMBL Heidelberg
Germany
[email protected]
Perner, Birgit
Leibniz Institute on Aging – Fritz
Lipmann Institute (FLI)
Germany
[email protected]
Peterbauer, Thomas
Max F. Perutz Laboratories GmbH
Austria
[email protected]
Pham, Trung
ibidi GmbH
Germany
[email protected]
Pike, Jeremy
Cancer Research UK Cambridge
Institute
United Kingdom
[email protected]
Prats, Clara
Denmark
[email protected]
Pylvänäinen, Joanna
Åbo Akademi University
Finland
[email protected]
R
Rabis, Claudia
Lasos Lasertechnik GmbH
Germany
[email protected]
Rauscher, Sabine
Austria
[email protected]
180
Rehó, Bálint
University of Debrecen Department of Biophysics and Cell
Biology
Hungary
[email protected]
Reichart, Ursula
University of Veterinary Medicine
Vienna
Austria
[email protected]
Reisen, Daniel
Bitplane AG
Switzerland
[email protected]
Reither, Sabine
EMBL Heidelberg
Germany
[email protected]
16th international
ELMI meeting
Reuss, Matthias
Germany
[email protected]
Richards, Owen
GmbH
Germany
[email protected]
Rimpelova, Silvie
University of Chemistry and
Technology
Czech Republic
[email protected]
Ritz, Sandra
Institute for Molecular Biology
(IMB)
Germany
[email protected]
Rockel, Thomas
Miltenyi Biotec GmbH
Germany
[email protected]
european
light microscopy
initiative
elmi
Roussel,
Julien
16th
international
ELMI
meeting
FEI Munich
GmbH
Germany
[email protected]
Royon, Arnaud
Argolight
France
[email protected]
Rózsa, Balázs
Femtonics Kft.
Rodighiero, Simona
Hungary
ETH Zurich
Switzerland
[email protected]
[email protected]
S
Saari, Markku
Turku Centre for Biotechnology
Finland
[email protected]
Sage, Daniel
Biomedical Imaging Group
Switzerland
[email protected]
Sampaio, Paula
I3S/IBMC
Portugal
[email protected]
Sandholm, Jouko
Turku Centre for Biotechnology
Finland
[email protected]
Sapuppo, Paolo
Leica Microsystems
Italy
[email protected]
Scarpellini, Alessandra
Netherlands
[email protected]
Schauss, Astrid
CECAD
Germany
[email protected]
Schlett, Katalin
Eötvös Loránd University
Hungary
[email protected]
Schlicker, Oliver
Leica Microsystems GmbH
Germany
[email protected]
Schmied, Christopher
Germany
[email protected]
Schmitt, Michael
Jena University - Institute of
Physical Chemistry
Germany
[email protected]
Schoonderwoert, Vincent
Scientific Volume Imaging B.V.
Netherlands
[email protected]
Schön, Christoph
Olympus Austria GmbH
Austria
[email protected]
181
ELMI meeting
european
light microscopy
initiative
elmi
16th international
ELMI meeting
Schroth-Diez, Britta
MPI-CBG
Germany
[email protected]
Self, Tim
University of Nottingham
United Kingdom
[email protected]
Sommerauer, Michael
Germany
[email protected]
Schulz, Olaf
PicoQuant GmbH
Germany
[email protected]
Sétáló, György
PTE ÁOK
Hungary
[email protected]
Schwartz, Owen
NIAID-NIH
USA
[email protected]
Sidenstein, Sven
Germany
[email protected]
Spitaler, Martin
Max Planck Institute of
Biochemistry
Germany
[email protected]
Schwarz, Tobias
ETH Zurich
Switzerland
[email protected]
Schwarz, Vera
University of Münster
Germany
[email protected]
Sebestyén, Árpád
BioMarker Kft.
Hungary
[email protected]
Seitz, Arne
Lausanne (EPFL)
Switzerland
[email protected]
Selchow, Olaf
LUXENDO
Germany
[email protected]
182
Sinko, Jozsef
Hungary
[email protected]
Sipka, Gábor
University of Szeged - Institute of
Plant Biology
Hungary
[email protected]
Steinbach, Gábor
Centre ALGATECH
Czech Republic
[email protected]
Steinmetz, Irmtraud
Leica Microsystems
Germany
[email protected]
Strasser, Christine
Switzerland
[email protected]
Sisamakis, Evangelos
PicoQuant GmbH
Germany
[email protected]
Strinagri, Chiara
Ecole Polytechnique
France
[email protected]
Smedh, Maria
University of Gothenburg
Sweden
[email protected]
Styevkóné Dinnyés, Andrea
MTA-SE
Hungary
dinnyes.andrea@med.
semmelweis-univ.hu
16th international
ELMI meeting
european
light microscopy
initiative
elmi
Swedlow, Jason
University of Dundee
USA
[email protected]
Szabó-Meleg, Edina
University of Pécs
Hungary
[email protected]
Székely-Bata,
Anikó
16th
international
ELMI
meeting
Microtrade
Kft.
Szabo, Gabor
Hungary
[email protected]
Szalóki, Nikoletta
University of Szeged - Department
of Biophysics and Cell Biology
Hungary
[email protected]
Szentesi, Péter
Hungary
[email protected]
Szabó, Ágnes
Hungary
[email protected]
Szalóki, Gábor
Hungary
[email protected]
Hungary
[email protected]
Szöllősi, János
Hungary
[email protected]
T
Tanhuanpaa, Kimmo
Finland
[email protected]
Tischer, Christian
EMBL Heidelberg
Germany
[email protected]
Tsuchiya, Takanori
Tokai Hit Co., Ltd.
Japan
[email protected]
Tarapcsák, Szabolcs
University of Debrecen - Faculty of
Medicine
Hungary
[email protected]
Tosi, Sébastien
Spain
[email protected]
Tuengerthal, Frank
Lasos Lasertechnik GmbH
Germany
[email protected]
Terjung, Stefan
EMBL Heidelberg
Germany
[email protected]
Tóth, Katalin
DKFZ
Germany
[email protected]
U
Ujlaky-Nagy, László
Hungary
[email protected]
Utz, Nadine
University of Konstanz
Germany
[email protected]
183
ELMI meeting
european
light microscopy
initiative
elmi
16th international
ELMI meeting
V
Vámosi, György
Hungary
[email protected]
Vida, László
UNICAM Magyarország Kft.
Hungary
[email protected]
Varga, Daniel
Hungary
[email protected]
Vidos, Ana
Ruđer Bošković Institute
Croatia
[email protected]
Volkó, Julianna
Hungary
[email protected]
W
Wachsmuth, Malte
LUXENDO
Germany
[email protected]
Weber , Igor
Ruđer Bošković Institute
Croatia
[email protected]
Wisniewski, Jan
Howard Hughes Medical Institute
USA
[email protected]
Wadel, Kristian
FEI Munich GmbH
Germany
[email protected]
Wagner, Thomas
Photometrics/ QImaging
Germany
[email protected]
Weidtkamp-Peters, Stefanie
University Duesseldorf
Germany
[email protected]
Wurm, Christian
Germany
[email protected]
Wheeler, Ann
University of Edinburgh
United Kingdom
[email protected]
Wurm, Helmut
Visitron Systems GmbH
Germany
[email protected]
Z
Zimmermann, Timo
Zarda, Boris
Center for Genomic Regulation
Leica Microsystems Switzerland
[email protected] Spain
[email protected]
Zambo, Kristóf
Zobiack, Nicole
Carl Zeiss GmbH
Austria
GmbH
[email protected]
Germany
[email protected]
Ziegler, Urs
University of Zurich
Switzerland
[email protected]
184
Zorloni, Alberto
Bruker
Italy
[email protected]
Zsebik, Barbara
University of Debrecen - Medical
and Health Science Center
Hungary
[email protected]
16th international
ELMI meeting
Notepad
light m
16th internation
ELMI meeting
16th international
ELMI meeting
Notepad
european
light microscopy
initiative
elmi
16th international
ELMI meeting
187
ELMI meeting
european
light microscopy
initiative
16th international
ELMI meeting
188
elmi
16th international
ELMI meeting
european
light microscopy
initiative
elmi
16th international
ELMI meeting
189
ELMI meeting
european
light microscopy
initiative
16th international
ELMI meeting
190
elmi
16th international
ELMI meeting
european
light microscopy
initiative
elmi
16th international
ELMI meeting
191
ELMI meeting
european
light microscopy
initiative
16th international
ELMI meeting
192
elmi
16th international
ELMI meeting
european
light microscopy
initiative
elmi
16th international
ELMI meeting
193
ELMI meeting
european
light microscopy
initiative
16th international
ELMI meeting
194
elmi
16th international
ELMI meeting
european
light microscopy
initiative
elmi
16th international
ELMI meeting
195
ELMI meeting
european
light microscopy
initiative
16th international
ELMI meeting
196
elmi
16th international
ELMI meeting
european
light microscopy
initiative
elmi
16th international
ELMI meeting
197
ELMI meeting
european
light microscopy
initiative
16th international
ELMI meeting
198
elmi
16th international
ELMI meeting
european
light microscopy
initiative
elmi
16th international
ELMI meeting
199
ELMI meeting
european
light microscopy
initiative
16th international
ELMI meeting
200
elmi
16th international
ELMI meeting
european
light microscopy
initiative
elmi
16th international
ELMI meeting
201
ELMI meeting
european
light microscopy
initiative
16th international
ELMI meeting
202
elmi
16th international
ELMI meeting
european
light microscopy
initiative
elmi
16th international
ELMI meeting
203
ELMI meeting
european
light microscopy
initiative
16th international
ELMI meeting
204
elmi
16th international
ELMI meeting
european
light microscopy
initiative
elmi
16th international
ELMI meeting
205
ELMI meeting
european
light microscopy
initiative
16th international
ELMI meeting
206
elmi
csád
i csoport
hadház
tMap.hu
ntyú
dalunk:
szentmárton
E4U
s térkép
sszerdahely
ojekt
dy hiányzik egy
ozzáadhatod a
l a hiányzó
lénagárd
lákat, éttermeket
at.
ger
essz valamit!
keszi
esztése
ek
egy világtérkép,
sonló emberek
ad licenc alatt
ський
krajna
×

-
+
100 m
 Útvonalterv
énye
u 1
sés
 Helyek
198
Leaflet | © OpenStreetMap közreműködők
Debrecen
Tetszik
| Rólunk | Hírek | Letöltés | Wiki | OpenStreetMap.org

european light microscopy initiative

Transcription

Similar documents

Light-sheet microscopy

I`m helping to end MS by participating in the 2016 Jayman BUILT MS

Emeryville Advanced Imaging Referral Form Back

Lightsheet Z.1 - Micron Optical

The Board of Directors of Qatar Fuel (WOQOD) is pleased to invite

RIMI Capabilities Patient Brochure

5 `S` Training Program at ABV-IIITM, Gwalior on 21.07.2016

Imaging Unlimited

Archiving 2010 Call for Papers

Kodály Zoltán Ifjúsági Világzenekar