OPTICAL MICROSCOPY UEF graduate school 2012

10/22/2012
OPTICAL MICROSCOPY
UEF graduate school 2012
Optical microscopy 2.0 credits
- Practicalities
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Course director: Arto Koistinen, SIB-labs
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phone 044-7163260, email: [email protected]
Course dates: 22.10. – 16.11.2012
Course material:
q Lecture notes
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Downloadable: http://www.uef.fi/siblabs/vm-kurssi/
Bioimaging: current concepts in light and electron microscopy.
Douglas E. Chandler and Robert W. Roberson.
Links from the lecture notes…
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Optical microscopy 2.0 credits
- Intro
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Course aim:
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“To give theoretical and
practical knowledge of optical
microscopy. Basics and usage
of transmitted, phase contrast,
interference and fluorescence
microscopy and imaging
spectroscopy in research are
covered during the course."
http://www.keywordpictures.com/abuse/early%20microscope///
Optical microscopy 2.0 credits
- Practicalities
COURSE PRACTICALITIES
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Optical microscopy 2.0 credits
- Practicalities
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Course programme:
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Lectures 14 h
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Exercices/Demos 12 h
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Exercises are obligatory!
Pair work / Presentation
Imaging of samples (2 h) with the technique you choose
15 min presentation (16.11. at 9-12)
Course evaluation by students is required for UEF graduate school
à FAIL / PASS
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Other questions?
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Exercises/Demos start 15 past (i.e. 8:15, 10:15 etc.) unless adviced
otherwise
Optical microscopy 2.0 credits
- Practicalities
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Lectures:
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L 1: Introduction (Arto Koistinen)
L 2-3: Basic of optics and microscopes (Arto Koistinen)
L 4: Sample preparation (Kirsi Rilla)
L 5: Phase contrast and DIC techniques (Kirsi Rilla)
L 6: Fluorescence techniques (Anita Naukkarinen)
L7: Immunohistochemistry (Anita Naukkarinen)
L 8-9: Confocal microscopy (Kirsi Rilla)
L 10-11: Image analysis methods (Kirsi Rilla)
L 12-13: Imaging spectroscopy (Arto Koistinen)
L 14: Analysis methods for spectroscopy (Lassi Rieppo)
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Optical microscopy 2.0 credits
- Practicalities
Optical microscopy 2.0 credits
- Practicalities
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Exercises / Demos (at Snellmania 3rd floor):
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D1: Sample preparation (ERa/KRi), Histology lab/Anatomy Sne 3245
D2: Basic operation with light microscope (AKo), SIB-labs Sne 3136
D3: Special usage of light microscopy (AKo), SIB-labs Sne 3136
D4: Relief-contrast microscopy (KRi), Anatomy Sne 3156
D5: Confocal microscopy (KRi), Anatomy Sne 3151
D6: Imaging spectroscopies, FTIRi and Raman (LRi); SIB-labs Sne 3146
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Optical microscopy 2.0 credits
- Practicalities
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Groups for exercises
GROUP A
GROUP B
GROUP C
GROUP D
Jawahar Deen Ashik
Mohan Babu
Rieppo Lassi
Pääkkönen Jouni
Siiskonen Hanna
Thota Durga
Huttu Mari
Ryynänen Jussi
Gockert Maria
Ghimire Rajendra
Turunen Mikael
Kaitainen Salla
Hiltunen Minna
Saleem Niyas
Liukkonen Jukka
Viiri Johanna
Optical microscopy 2.0 credits
- Practicalities
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Pair work / imaging, week 45:
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Light microscopy (Arto Koistinen)
Confocal microscopy (Kirsi Rilla)
Fluorescence microscopy (KRi/AKo)
FTIR imaging (Lassi Rieppo)
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Different samples are available, regarding the student’s background
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Technique
Samples
Light microscopy
Confocal microscopy
Fluorescence
FTIR
Biological tissue, material science
Cell culture, tissue?
Tissue (plant or animal)
Tissue (cartilage, bone)
Results and background (from literature) are presented on 16th October
at 9-12 in 1039 (Tietoteknia building) à 15-20 min each presentation
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Optical microscopy 2.0 credits
- Practicalities
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Pair work / imaging
Monday 5.11.
Tuesday 6.11.
Wednesday 7.11.
8-10
Fluorescence
microscopy1
Confocal microscopy1
10-12
Fluorescence
microscopy2
Confocal microscopy2
12-14
Spectroscopy / FTIRi 1
Spectroscopy / FTIRi 2
Thursday 8.11.
Friday 9.11.
Light Microscopy1
Hanna Siiskonen
Light microscopy2
14-16
Microscope reservations for pair imaging at suitable times.
Results and background (from literature) will be presented on 16th October at 9-12 in 1039 (TT).
Optical microscopy 2.0 credits
- Practicalities
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Specimen reservation for pair work
Technique
Students / Group
Light microscopy
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2.
Confocal
microscopy
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2.
Fluorescence
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2.
FTIR
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2.
Samples
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Optical microscopy 2.0 credits
- Practicalities
INTRODUCTION
Optical microscopy 2.0 credits
- Intro
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We see (only) what we understand !
BUT…
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Do we understand what we see?
We’ll see…
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Human vision is
1. sensitive to differences in contrast.
(We tend to overestimate the amount or size of an object if there is
high contrast vs low contrast.)
2. sensitive to perspective and depth changes.
3. sensitive to orientation of lighting.
(We prefer light to come from above.)
4. And: We fill in what we think should be in the image
SO…
Illustration by Ewald Hering (1861).
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Johann Poggendorff illusion (1860).
The two segments of the diagonal line appear to be slightly offset in this figure.
Johann Zöllner illusion (1860).
The diagonal lines are parallel,
but appear not to be.
The illusion was designed to cause
errors in optical equipment. It did
cause errors, and also great
concern among scientists of the
time, over the validity of all human
observations.
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Trident illusion used to test
populations.
Perspective in this illustration would
not be confusing to a two-dimensional
perceiver.
This illustration was used by
psychologists to study how our
mind copes with conflicting images.
(The figure is either a duck facing left or
a rabbit facing right.)
Modern version
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Reversible Goblet: demonstration of figure-background reversal
(Edgar Rubin 1915)
German postcard from 1888
and
a new version from 1915.
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Mystic Wheel
10/22/2012
Several things happen when you stare at the Mystic Wheel. You might see the illusion shimmer or flutter
slightly. This is because the lenses of your eyeballs are slightly out-of-round. Parts of the illusion move in
and out of focus as your eye scans the image. Looking closer, you get the impression of three dimensions,
where some parts look higher or lower than others. Now, follow the ring of curves nearest the outer edge,
around the wheel and you will see the geometry go from hump to hollow, an impossible trick in three
dimensions.
Artist: Gustave Verbeek
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The Schröder Stairs demonstrates
variation on the spontaneous
perspective reversal illusion.
(In this illustration, the stairs will turn
upside down during a steady gaze.)
The wall with the glass bead will shift
from the foreground to the background
or visa versa.
Maurits Cornelis Escher “Relativity”
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Art by Akiyoshi Kitaoka
Department of Psychology, Ritsumeikan University, Kyoto, JPN
http://www.ritsumei.ac.jp/~akitaoka/index-e.html
Rotating snakes by Akiyoshi Kitaoka
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Scintillating grid
Discovered by E. Lingelbach in 1994
Count the black dots!
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Adelson Illusion
How different are blocks A and B?
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Image from Cardiff
Castle, Cardiff, Wales
Find the “man”
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Joseph Stalin made use of photo
retouching for propaganda
purposes.[4] On May 5, 1920 his
predecessor Vladimir Lenin held a
speech for Soviet troops that Leon
Trotsky attended. Stalin had
Trotsky retouched out of a
photograph showing Trotsky in
attendance. Nikolai Yezhov, an
NKVD leader photographed
alongside Stalin in at least one
photograph, was edited out of the
photograph after his execution in
1940. For more information, see
images altered by Soviet censors.
http://en.wikipedia.org/wiki/Photo_manipulation
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Fraud images in science
http://www.nature.com/news/2009/
091009/full/news.2009.991.html
Manipulating images
The Journal of Cell Biology Volume 166, Number 1, 2004
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Enhancing signals
Image from Purdue University (Jennie Sturgis)
Figure 6. Misrepresentation of
image data.
Cells from various fields have been
juxtaposed in a single image,
giving the impression that they
were present in the same
microscope field. A manipulated
panel is shown at the top. The
same panel, with the contrast
adjusted by us to reveal the
manipulation, is shown at the
bottom.
The Journal of Cell Biology Volume 166, Number 1, 2004
08:58
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Image Publishing
The Journal of Cell Biology
“No specific feature within an image may be
enhanced, obscured, moved, removed,
or introduced. The grouping of images
from different parts of the same gel, or
from different gels, fields, or exposures
must be made explicit by the arrangement
of the figure (e.g., using dividing
lines) and in the text of the figure legend.
Adjustments of brightness, contrast,
or color balance are acceptable if
they are applied to the whole image
and as long as they do not obscure or
eliminate any information present in
the original. Nonlinear adjustments
(e.g., changes to gamma settings) must
be disclosed in the figure legend.”
Cleaning up background
“It is very tempting to use the tool variously known as “Rubber
Stamp” or “Clone Stamp” in Photoshop to clean up unwanted
background in an image. Don’t do it. This kind of manipulation
can usually be detected by someone looking carefully at the
image file because it leaves telltale signs. Moreover, what may
seem to be a background band or contamination may actually
be real and biologically important and could be recognized as
such by another scientist.”
08:58
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Sometimes colors may do the trick…
Human vision more sensitive to color.
Sometimes, pseudocoloring makes it is possible to see slight variations in gray
scales
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