afs-web-version

AFS™
Product Brochure
A compact solution for highly parallel single-molecule measurements
Technology
Acoustic Force Spectroscopy
Acoustic Force Spectroscopy, or AFS™, is LUMICKS’ highly parallel single-molecule manipulation method. The AFS™ technology is based
on a powerful yet cost effective lab-on-chip device capable of applying forces on thousands of biomolecules in parallel with high precision.
Acoustic Node
Piezo element
Glass top layer
Y
Acoustic Force
Y
Glass bottom layer
Fluidic layer (channel)
Reference Bead
Antibody - Antigen Affinity
Protein Unfolding
Cell Manipulation
Microrheology
Lipid Membrane
Interactions
DNA-protein Interactions
Glass bottom layer
Piezo elements
Flow channel
Gold electrode
AFS™ Explained
Possibilities
The essence of the AFS™ technology lies in a glass microfluidic chip with two piezo elements that generate resonant acoustic waves
AFS™ enables scientists to investigate the mechanical properties of biological systems by probing them via synthetic microspheres or by
(ultrasound). These resonant acoustic waves are used to exert forces on micrometer sized particles with a different density than the
directly probing the system itself (e.g. a cell). Due to the planar nature of the acoustic waves experiments can be performed in a highly parallel
surrounding medium (e.g. a polystyrene microsphere or a living cell).
fashion. This makes AFS™ an ideal tool to study a wide variety of biological systems, including, but not limited to: Antibody – antigen affinity,
In the compact AFS™ chip the acoustic waves are employed to exert forces from sub-pN to hundreds of pNs on thousands of biomolecules
protein unfolding, cell manipulation, microrheology, lipid membrane interactions and DNA-protein interactions.
(such as DNA, RNA or proteins) or cells in parallel, with sub-millisecond response time and inherent stability [1].
[1] Further reading: Sitters et al. ‘Acoustic Force Spectroscopy’, Nature Methods (2015)
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Applications
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Measuring DNA extension curves in parallel
Measuring RNA polymerase activity
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AFS™ has the powerful and unique capability of performing single-molecule measurements in a highly
With its inherent multiplexing capabilities and force stability AFS™ is the ideal tool to study rare
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parallel fashion. The importance of parallelity lies in the fact that many independent measurements are
DNA processing events on sub-second to hour timescales. RNA polymerase (RNAP) activity
often needed to distinguish heterogeneous behavior and rare events from intrinsic stochasticity caused
is typically of complex nature: Stochastically occurring elongation is frequently interrupted by
by thermal fluctuations.
pausing events of different nature.
In this example we perform a single-molecule experiment with 20 DNA molecules (8.4 kbps in length) in
In this experiment DNA molecules with stalled E.coli RNAP complexes are immobilized on the
parallel. The ability of multiplexing mechanical measurements at the single-molecule level can be readily
AFS™ chip surface. Subsequent attachment of polystyrene microspheres on the E.coli RNAPs
applied to different assays (e.g. RNA or proteins).
enables extension of the DNA molecules. Once transcription and replication are initiated the
Force (pN)
50
20
10
0
1.0
1.5
2.0
2.5
3.0
3.5
DNA Extension (µm)
RNAP Activity (nm)
60
600
400
200
0
0
100
200
300
400
500
Time (s)
activity of many individual E.coli RNAP molecules can be precisely monitored in parallel by measuring the DNA elongation in real time.
As demonstrated here, AFS™ is well suited to resolve the highly stochastic nature of RNAP elongation dynamics that is characterized by
frequently occurring pausing events [1].
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Highly multiplexed antibody-antigen affinity characterization
AFS™ based cell manipulation
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Statistical analysis and high data throughput are of crucial importance to characterize the strength
number of bonds can be probed in parallel, greatly reducing measurement times.
Here we show 251 individual Dig::anti-Dig bonds stretched with a loading rate of 2.3 pN/s (rates
between 10-4 pN/s to 103 pN/s are possible). The rupture force is of each bond is recorded and
plotted into a rupture force histogram. This histogram was obtained in a single experiment under
Counts
of antibody-antigen interactions. Due to the massive multiplexing capabilities of AFS™ a large
AFS™ provides scientists with the unique ability to manipulate any substance that is different in density compared to the liquid medium in
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which it is suspended. This way forces can not only be applied on synthetic microspheres, but also on living matter itself.
20
10
0
0
20
40
60
80
Rupture Force (pN)
identical conditions, ensuring experiment reliability and reproducibility.
50 μm
DNA Extension (µm)
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High precision constant force measurement
2
3
3
2
1
1
2
4
6
Force On
0s
1s
2s
3s
4s
5s
6s
7s
AFS™ is an ideal tool to perform high-precision constant force measurements on single biomolecules.
Here we apply forces on neuroblastoma cells (NE-115) using the AFS™ Module (see page 6) on a commercially available confocal microscope
Here we demonstrate this ability by applying various level of tension on a single DNA molecule (8.4
(Leica TCS SP8). The imaging plane of the confocal scanning system is placed congruent with the acoustic node approximately 40 μm above
kbps in length) tethered to to a polystyrene microsphere (4.5 μm in diameter) while measuring its length
the bottom surface of the AFS™ chip. Once the acoustic field is applied, fluorescently labeled cells are rapidly pushed off the surface into the
using the AFS™ tracking software.
acoustic node and appear in the imaging plane. When the acoustic forces are silenced cells descend by gravitation, gradually disappearing
During the measurement the acoustic force is increased in four steps, increasing the DNA extension
0
0
Force Off
4
8
10
Time (min)
1
Acoustic Force:
Extension Accuracy:
0 pN
205.1 nm
2
Acoustic Force:
Extension Accuracy:
1.3 pN
18.5 nm
3
Acoustic Force:
Extension Accuracy:
8.1 pN
14 nm
4
Acoustic Force:
Extension Accuracy:
15.8 pN
12.7 nm
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from the fluorescent image.
and decreasing the amount of thermal fluctuations. By suppressing the thermal fluctuations a length
By providing the ability to directly apply forces on cells, AFS™ bears great potential for cell biology research, e.g. the study of cell-adhesion,
accuracy of 12.7 nm is achieved (standard deviation of the DNA extension at 60 Hz).
cell motility and stem cell differentiation [2].
The electronic actuation of the piezo element allows for the alteration of constant acoustic forces with
sub-pN precision, stability over hours and sub-ms response time.
Standard deviation at 60 Hz
[1] Courtesy of Anatoly Arseniev, Georgii Pobegalov & Mikhail Khodorkovskii at Peter the Great St. Petersburg Polytechnic University, Russia
[2] Courtesy of Kees Jalink & Bram van der Broek at Netherlands Cancer Institute, The Netherlands.
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AFS™ Stand-alone
AFS™ Stand-alone is LUMICKS’ true turnkey single-molecule tool. It includes various AFS™ chips, a dedicated inverted optical microscope,
fluidics and a workstation with a software package. With the AFS™ Stand-alone you will be able to focus on your experiments right away,
without the need for additional software or imaging tools.
LED Illumination
AFS™ Chip Holder
Manual XY Sample Stage
Gravity, manual or external
syringe pump controlled flow
Features
Monochromatic LED Illumination
ƒ
645 nm wavelength
CFI Achromat Air Objective (upgradable)
ƒ
Nikon 40x, NA 0.65, WD 0.65mm.
ƒ
Corrected for chromatic aberrations, spherical aberrations,
coma and image flatness
Motorized Z Objective Stage
ƒ
High-performance 2-phase stepper micro-translation stage
ƒ
5 mm travel range, 50 nm step size, sub-nm stability
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USB 3.0 CMOS Camera (upgradable)
ƒ
1280 x 1024 pixel (pixel size 5.3 μm), 1.31 MP
ƒ
Real-time parallel measurements up to 60 Hz in full field of
view
Manual XY Sample Stage
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Micrometer precision 20 mm travel range
ƒ
Double-dovetail guide enabling the user to measure at
Acoustic Forces
Workstation & Software
The unique ability of AFS™ to apply forces via an electronically
AFS™ Stand-alone includes a LabView2013 software package
controlled piezo element gives the user unprecedented control
and a powerful workstation. Combined, they are capable of real-
over the forces.
time, nanometer resolution tracking of thousands of microspheres
ƒ
ƒ
ƒ
different locations within the sample
ƒ
Equipped with fine-thread spindles with graduated buttons
ƒ
Without an amplifier forces range from 0 to 40 pN (4.5 μm
in parallel.
diameter polystyrene microspheres)
ƒ
Real-time parallel 3D tracking of 300 microspheres at 25 Hz
With an amplifier force range is extended to 500 pN (4.5 μm
ƒ
Real-time parallel 2D tracking of 1500 microspheres at 25 Hz
diameter polystyrene microspheres)
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Real-time position tracking accuracy (x, y) of 2 nm at 25 Hz
Both static and dynamic force measurements with a 14 bit
ƒ
Real-time height tracking accuracy (z) of 5 nm at 25 Hz
vertical resolution
ƒ
Z-drift correction through reference bead differential tracking
Loading rates ranging from 10-4 pN/s to 103 pN/s
ƒ
Automated force calibration through power spectrum
analysis
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AFS™ Module
In addition to the AFS™ Stand-alone microscope platform, LUMICKS offers the AFS™ Module for use with commercial inverted optical
microscopes. This combination grants you the ability to study mechanical manipulation of biomolecules and cells with the powerful
visualization capabilities of state of the art fluorescence and super-resolution microscopy.
Features
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Compatible with commercial
inverted optical microscopes
ƒ
Built-in amplifier for access to a
larger force spectrum
ƒ
Compatible with external analog
trigger
Force Off
Force On
Fluorescence combined with AFS™
Acoustic Forces
Software
Here we use the AFS™ Module on a commercially available microscope
The unique ability of AFS™ to apply forces via an electronically
A lightweight and easy-to-use software package is included
(Nikon Eclipse Ti). DNA-tethered 4.5 μm polystyrene beads are fluorescently
controlled piezo element gives the user unprecedented control
allowing the application of:
labeled with Atto647 dye and visualized by laser-illuminated epi-fluorescence
over the forces.
microscopy [1].
ƒ
ƒ
ƒ
Constant forces
Forces ranging from 0 to 500 pN can be applied on 4.5 μm
ƒ
Dynamic force ramps
diameter polystyrene microspheres.
ƒ
User-defined time varying force profiles
Both static and dynamic force measurements with 14bit
vertical resolution
ƒ
Loading rates ranging from 10-4 pN/s to 103 pN/s
The AFS™ Module software package does not provide image
analysis tools.
[1] In cooperation with The Department of Physics and Astronomy, the group of Physics and Living Systems, VU University
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History
The field of single-molecule biophysics has radically changed over the past decade, owing to major technological breakthroughs enabling
landmark experiments. Now that the single-molecule methods have reached maturity, they are ready to make a long-lasting impact on the
field of biological research.
Focusing on these new methods, LUMICKS brings to market the revolutionary C-Trap™ Correlative Tweezers-Fluorescence Microscope
(CTFM) and the Acoustic Force Spectroscope (AFS™). LUMICKS is rooted in the research groups of Prof. Gijs Wuite and Prof. Erwin
Peterman at the VU University Amsterdam, and its instruments for single-molecule studies allow the disruptive method of live measurement
and imaging of interactions at the molecular level.
Mission
LUMICKS provides ready-to-use single-molecule instrumentation, allowing you to focus on the science. Understanding complex biological
processes at the single-molecule level is key for prevention and cure of cancer and other diseases. To this purpose, live observation and
measurement have proven game changing, and LUMICKS aims to make these enabling technologies available to biologists to create a better
world.
The LUMICKS team is committed and proud to work on the world’s leading instruments for live measurement
and imaging of interactions at the single molecule level!
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For more information visit
www.lumicks.com
LUMICKS BV
De Boelelaan 1081, 1081HV Amsterdam, The Netherlands
M: [email protected], T: +31 (0) 20 598 79 84
Specifications and equipment are subject to change without
any notice or obligation on the part of the manufacturer.
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