Stimulation ofliving neurons

E. coli bacteria

Hela cell

Application Note #138
Combined Optical andAtomic Force Microscopy
Both Inverted Optical Microscopy (IOM) and Atomic
Force Microscopy can be operated in a very wide rangeof specialty modes, and both have proven to be essential
to the study of biological specimens in near-physiologicalenvironments. Where IOM, particularly fluorescencemicroscopy, is based on the staining and tracking ofmolecules inside the cells or tissues to allow direct
observation of of cell structure and dynamic processes,
atomic force microscopy is primarily a surface investigation
technique where the tip physically interacts with thesample to enable topographical, electrical, mechanicalor chemical characterization. To monitor both types of
information simultaneously, fully integrated optical andatomic force microscope (AFM) systems, such as the
Bioscope CatalystTM, have been developed. This applicationnote reviews a few representative applications that can becovered by this type of AFM/IOM approach.

Combining Atomic Force Microscopy and Optical
Information in a Single Image
All atomic force microscopy techniques are based on thesame principle: a sharp tip mounted on a reflective-backed
cantilever is scanned over the sample surface. A laser

Huntingtin aggregates

beam is focused onto the cantilever and each time the tip
gets deflected by the local change in topography, the laser
spot position on the photodiode is changed. This is how aheight profile of the surface can be generated. Historically,
contact mode atomic force microscopy was developed
to get topographical information from the sample, as well
as data concerning the friction forces between the tip
and the surface.1 In the early nineties, force spectroscopy
was developed, which brings the tip into contact withthe surface and retracts. This technique has been widelyused to determine the specific interaction force between amolecule attached to the AFM tip and the complementary
molecule present on the surface.2 Around the same time,
TappingModeTM was developed, where the AFM tip onlyinteracts intermittently with the surface and thus negligible
shear and friction forces are applied to the sample. 3 Most
recently, Peak Force Tapping® Mode has been developed,
which is based both on the oscillation of the cantilever andthe fact that a force curve is captured each time the probe
contacts with the surface.4 It’s the first technique thatprovides direct nanoquantification of the AFM signals athigh resolution. This mode has been proven to provide very
valuable results on a wide range of samples.5-7

Figure 1: Required setup for IOM/AFM combination. Most of existing condensors (1) (0.5, 0.52. 0.55) should fit the AFM head (2). The
cantilever holder (3) should contain a transparent part to let the incident light go through. The sample itself should lay on a substrate whichenables both optical and AFM measurements (4), like glass-bottom Petri dishes. The sample insert (5) should be large enough to allow
using high magnification objectives and have some heating capabilities to keep the specimens at 37°C. The x,y stage (6) should move thesample in x,y, have good feedback capabilities and allow navigating on a large optical window. The turret should be equipped with large range
objectives for easier tip/sample positioning.

Any of the above-mentioned atomic force microscopy
modes can be combined with most of the existing opticaland fluorescence techniques, although the most commonlyused setup is the combination of atomic force microscopy
and epifluorescence.8,9 In fluorescence microscopy
techniques, a sample is illuminated with a light of a specificwavelength that is absorbed by the fluorophores. As aconsequence, those molecules emit a light of a longer
wavelength, detected through a microscope objective.
Unlike phase contrast or differential contrast microscopy
techniques, which are based on the transmission orreflection of the white light, these techniques allow thelabeling of any kind of organelle and other subcellularcomponents, from nucleic acids to proteins or lipids.
Figure 1 shows a schematic setup detailing how such a
combination is possible. A key to success in this type ofexperiments is to have enough space around the sample
to analyze so that the AFM head fits with any type ofcondensor and also allows compatibility with any type ofobjective. Keeping cells alive for an extended period oftime is made possible by the use of features such as the2

Figure 2: Atomic force microscopy/epifluorescence overlay of Lovocells. The atomic force microscopy image (100x100μm) is a mix of
topography and amplitude error channels. Automatic overlays canbe achieved by making a quick calibration and registration. Theneach objective will have a specific calibration file that can be loaded
and re-used for further experiments. If the tip has to be changed,
the user will only have to capture an image of the new tip to update
the file.

Perfusing Stage Incubator, which maintains the sample
in a fully closed environment and under constant gas andfluid flow.10
Overlaying atomic force microscopy and optical images
is possible by using the Bruker’s Microscopy Image
Registration Overlay (MIRO ®) option. An example achievedon Lovo cells is shown in figure 2. On the fluorescencepart of the image, the cell nuclei have been labeled withDAPI and the actin cytoskeleton with α-phalloidin. The
AFM image has been acquired in TappingMode and is a mixof topography and amplitude error channels. This type ofsoftware can be used to overlap atomic force microscopy
and fluorescence images and also to target specificlocations to perform force curves directly from the opticalimage.11 This feature is of particular importance for pointand-shoot experiments carried out with functionalized tips.

Correlating Fluorescence Data and Measurementof Specific Unbinding Events
The combination of atomic force microscopy andspectroscopy can be used to detect specific interaction
forces between two molecules with piconewton
sensitivity.2,12,13 The experiment described in figure 3
illustrates how in situ fluorescence measurements can beassociated to the presence of specific force measurements
detectable by an AFM tip. In this instance, an AFM tip wasfunctionalized with a pore-forming toxin called Pro-Aerolysin(PA), which has the ability to recognize any inositol grouppresent at the surface of living cells.14,15 On the other hand,cells were transfected with a specific plasmid so that they
over-express GlycosylPhosphatidylInositol (GPI)-anchoredproteins at their surface. The plasmid also contains a
GFP-coding sequence so that all cells over-expressing

Figure 3: Correlation between fluorescence intensity and specific AFM force measurements on living Hela cells. A tip was functionalized witha toxin able to target any inositol-containing molecule. Fluorescent cells over-express GPI-anchored proteins and exhibit a high number of
specific unbinding events (yellow retraction curve) whereas for most of the non-fluorescent cells, no event (red retraction curve), or a much
lower number, can be detected.

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the proteins are highly fluorescent. In highly fluorescentcells (top right of figure 3), specific PA-GPI events can bedetected within a range of 100 pN (see representative
retraction curve in yellow). In non-fluorescent cells,
some GPI-anchored proteins are still present inside theplasma membrane, but as they are not over-expressed,
the number of specific unbinding events is decreased byapproximately a factor of five. This type of work highlights
how straightforward it is to correlate the number of specific
events and the fluorescence intensity of the targeted cells.

AFM Probe-Induced Mechanical Stimulation of
Living Neurons and Simultaneous Fluorescence
Monitoring of Transduction Signals
In vivo pain-evoking stimuli are detected by the primarysensory neurons through their endings. Signals aretransmitted by a series of action potentials along theneurons until the spinal chord, and then to the brain where
they are interpreted as pain. Those specific neurons thatenable the transmission of painful noxious stimuli are called
nocireceptors.16 Although it’s accepted that mechanicallyinduced pain plays a clear role in trauma- and inflammation-

mediated death, the detailed process (particularly thesignaling pathways following the stimulation) remains
poorly understood. A first step to enhanced understanding
is to develop a new device that allows both application ofcarefully controlled mechanical stimuli and the monitoring of
the consequences at the biochemical level.
A tipless cantilever modified with a 10 μm polystyrene
bead at its end can be used to apply a controlled nominal
force on living dorsal root ganglions. Simultaneously, theintracellular calcium concentration can be measured bymonitoring the intensity of the fluorescence signal on
the optical image. The somas can easily be targeted byusing 20x objectives and navigating the AFM probe on the(bright field or DIC) optical image, but the stimulation of theneurite endings requires a much more accurate localizationand the use of high-magnification objectives. Single force
stimulations were applied with the tip at different locationsof the neurites, and each time a fluorescence change wassystematically observed at the contact point or far from it(sometimes several hundreds of micrometers away) provingthat the signals are transmitted along the cell and between

Figure 4: Mechanical stimulation of living dorsal root ganglions by a modified AFM probe. As soon as the tip touches the surface, someprimary and secondary flows of action potentials can be observed, indicating a mechano-sensitive activation and transduction of the signal,
through calcium and/or sodium receptors. In force volume mode, regular indentations can be applied onto the endings, which are perfectly
synchronized with the fluorescence pulses seen on the cell core.

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neighbor cells.17 The fluorescence intensity could bequantified and clearly distinguished from the basal activity,
but one interesting unanswered question was whether or
not the cells can be stimulated several times by the AFM
tip. To address that question, the modified tip was engaged
in force volume mode with a surface delay of 0 ms, aretraction delay of 500 ms and a scan size of 0 nm (seefigure 4). The results indicate that the living neurites can be
stimulated 4 or 5 times by the tip before being completely
depolarized. These types of discoveries demonstrate thepotential of such an approach to detect signal propagationand transduction in living neurons and to obtain better
insight into the mechanisms of mechanotransduction in
those cells.

Combining Confocal Laser Scanning Microscopy
and AFM Data
Fluorescence microscopy techniques have been becoming
more and more important in biology over the past decadeor so, and they have enabled the development of moreadvanced applications, such as confocal18 or total internalreflection microscopies.

Voluminous samples, even when properly stained, are hard
to image in standard fluorescence microscopy, and generally
result in blurry images. To overcome this problem, confocal
microscopy is often used since most of the light from thesample that is not from the microscope’s focal plane is
excluded. As a result, the corresponding image has muchbetter contrast and represents a thin section of the sample
at a certain height. By stacking several of those images
taken at different heights, it is possible to reconstruct a
three-dimensional image of the specimen.
Figure 5 shows confocal and atomic force microscopy
images of Hela cells labeled with DAPI and α-phalloidin.
A, B, C and D images were captured on a dense cluster ofcells having more than 15 μm in thickness. A, B and C are
three thin-sections of a z-stack movie recorded on a few
cells protruding from the rest of the cluster, at different
heights along the vertical axis. In A, the nuclei are clearlyvisible because they are very close to the bottom layer ofunderlying cells (whereas the actin cytoskeleton exhibits
an average illumination density). In B, the actin fibers areeven more visible, whereas the nuclei tend to disappear.
In C, only the very top part of the sample is visible, here,

Figure 5: Full compatibility of AFM and confocal laser scanning microscopy. In confocal microscopy, several thin sections of the specimen
can be captured along the z-axis (A, B, C and D) to reconstruct a 3D model. The green dashed circle spots cells in mitosis, both visible on thethin section (C) at high resolution and on a higher plane (D). A key requirement to overlay atomic force microscopy and fluorescence images
is to make sure the AFM tip can easily be located on the optical images. Most of the commonly used AFM probes like MCLT (D), DNP (E andF), OBL, OTESPA (not shown here) were successfully tested. We also made sure that none of the confocal parameters (like the wavelength,
the size of the pinhole or the rotation scanfield) impact the quality of the AFM operation and vice versa. An example of overlay with an atomic
force microscopy height image is shown in G.
5highlighting the two cells under mitosis. D is a zoom out of
the same area showing the AFM cantilever tip in contact
with the sample. As in epifluorescence, overlaying AFM and
optical images is possible but requires precise localizationof the AFM tip. Images D, E and F show different confocal
images with different types of AFM tips clearly visibleon each. The yellow square in F indicates where thetopography AFM image (represented in 3D in G) has been
performed. This example is focusing on the combinationof fluorescence data with atomic force microscopy height
information. However, with these two techniques it would
also be possible to combine confocal images with local
mechanical properties of the sample to provide even more
relevant information.19

Combining TIRF and Atomic Force Microscopy Data
In regular epifluorescence, the intrinsic fluorescent signalof the fluorophores bound to the feature of interest is overimposed with the background fluorescence of the unboundmolecules, which causes the resulting image to exhibit
a certain part of non-specific fluorescence and be blurry.
TIRF was developed to selectively illuminate and excite
fluorophores at the very neighborhood of the glass-waterinterface by using the evanescent wave that is generated
only when the incident light is totally and internally reflected
at this interface. The evanescent field decays very quickly
from the interface and penetrates into the sample bya depth inferior to 200 nm. TIRF can hence be used to

Figure 6: Combination of TIRF and atomic force volume measurements for biomolecular detection. The TIRF principle (left) is base on theevanescence wave. Unlike in confocal where thick features can be analyzed, in TIRF, only the first 100 nm close to the surface can beanalyzed. The PEG-ylated nano-rings are mounted on a glass slide so that they are 1.3 μm apart from each other and have an inner diameterof less than 30 nm. As soon as different antibodies bind to the PEG layer, the TIRF signal is modified and can be monitored (right, backgroundimage), simultaneously with atomic force volume measurements (right; inset), which enable tracking the local changes in topography andmechanical properties.

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visualize very thin structures like basal plasma membraneor small features like proteins or DNA, with a remarkable
sharpness. The atomic force microscopy/TIRF combination
has proven to be extremely powerful for investigating theproperties of biological fibrils, such as myosin filaments.20
The future of TIRF/atomic force microscopy combination
might also dwell in biomimetic devices. Biomaterials
capable of biological recognition with a high specificity areof considerable importance in the field of biosensors andseveral key medical applications.21,22 By taking advantageof the intrinsic affinity between Poly(ethylene-glycol) PEG
and PEG-binding antibodies, some researchers23 haverecently proposed an original device (figure 6) based on
a combination of TIRF and AFM that perfectly mimics
the structure and function of biomolecular adaptors
that transport molecules from a very complex biologicalenvironment to site-specific targets. PEG is known for
its unique properties of biocompatibility and protein
resistance.24 In this setup, gold nano-rings are functionalizedwith various types of thiol-based PEG derivatives in regularmicro-patterns. Several types of anti-PEG antibodies areplaced in the buffer and exhibit various affinity toward
the PEG molecules. Such specific bindings result in a
conformational change of the so-called “protein-like”
structure of the PEG and can be monitored by using TIRF.
On the other hand, force volume can be simultaneously
used to probe the different changes in height andmechanical properties versus the different antibodies
bound to the polymer brush. Figure 6 represents the TIRF/
AFM setup (left) and the type of data that can be collected(right). As the antibodies bind to the PEG-coated ring, the
TIRF signal can be detected (background image) correlatedwith any corresponding changes in height and mechanicalproperties via force volume measurements, and comparedwith AFM images (inset). This type of device is rather
unique since it allows the detection of both nanomechanicaland biochemical information, and opens the way to many
promising outcomes in nano-biomimetism and biosensing.

Figure 7: 100x100 μm scans of fixed glioblastoma cells recorded on a FLIM/AFM setup. The fluorescence intensity and FLIM signals are
expressed in arbitrary units but all AFM channels are directly quantitative. The z-scales are (from bottom to top of the color bar): topography =0–5 μm; PeakForce error = 0–400 pN; Elasticity = 0–700 kPa; Adhesion = 0–150 pN = Deformation = 0–150 nm; and Dissipatio = 0–3 keV.

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Combining AFM with Fluorescence Lifetime
Imaging Microscopy Measurements
Every fluorescent dye has a specific lifetime at the excitedstate. By monitoring differences in lifetime, one candistinguish between different molecular species, even if
they have the same fluorescence color. The lifetime of amolecule is influenced by such factors as the intermolecularbinding or the ionic strength, but is not dependent of
the fluorophores concentration, the light intensity, or thephotobleaching. One of the major benefits of using FLIM
versus other fluorescence techniques is that it is possible toobserve specific molecules while imaging an entire cell.
As mentioned above, atomic force microscopy is asurface investigation technique that can be combined withFLIM in one single tool. An ideal example of this is the
BioScope Catalyst, which is a sample-scanning systemwith a powerful NanoScope® V controller that allows scansynchronization signals and very accurate positioning of the
AFM tip in x,y,z relative to the confocal beam. With respect
to the FLIM integration, the Catalyst can be combinedwith a MicroTime 200 (PicoQuant, Berlin), which is a highperformance time-resolved fluorescence microscope withsingle-molecule sensitivity.25 The synchronization of thetwo techniques is possible via the so-called time-tagged
time-resolved mode, which allows the branching of external“marker” synchronization signals within the continuously
monitored flow of photon arrival times. For each AFM
scan, the markers are given at the beginning and at the endof each scan line, where the recorded photon signals areultimately converted into pixels. The “line start” and “linestop” synchronization signals are readily available from the
NanoScope V controller. Figure 7 gives a representative
example of simultaneously acquired FLIM, fluorescenceintensity and PeakForce QNM® AFM signals on fixed
glioblastoma cells. PeakForce QNM is the only AFM modethat provides directly quantitative mechanical informationof the sample at a pixel resolution, making it the mostinformative AFM mode that can be combined with the
FLIM technique. After data acquisition, the images can bepost-processed in 3D so that atomic force microscopy andfluorescence data are overlaid, allowing a straightforward
identification of specific features and immediate
correlation with their intrinsic mechanical, chemical or
fluorescence properties.

Figure 8: 3D representation of a 100x100 μm AFM height image of
fixed glioblastoma cells with a fluorescence intensity skin. The center
part of the cell (nucleus) is highly fluorescent. The intensity tends todecrease in the rest of the cell, to eventually be weak on the edges.
Such a rendering allows a direct identification of the cell parts andimmediate correlation with the fluorescence lifetime of fluorophores.

Conclusions
This application note provides a non-exhaustive list of
combinations of atomic force microscopy and various
fluorescence microscopy techniques, as well as the typicalapplications that can be addressed by using such setups.
The fact that all of these combinations are made possible by
the use of a single fully integrated tool makes the operator’stask easy and user-friendly. The development of newly
released AFM modes (such as PeakForce QNM) opens theway to even more promising applications.

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Authors
Alexandre Berquand, Bruker Nano Surfaces(alexandre.berquand@bruker-nano.com)
Frank Lafont (Institut Pasteur Lille, France)
Mathias Hafner (Institute of Molecular Cell Biology, Universityof Applied Sciences Mannheim, Germany) and Marlen Petersen(University of Heidelberg, Germany)
Jan Schroeder, Stefanie Landwher, and Ulf Schwarz(Leica Microsystems, Mannheim, Germany)
Janne Hyotyla and Roderick Lim (Biozentrum and the Swiss
Nanoscience Institute, Basel, Switzerland)

Bruker Nano Surfaces Divison
Santa Barbara, CA · USA+1.805.967.1400/800.873.9750
productinfo@bruker-nano.com

www.bruker .com

trademarks or registered trademarks of Bruker Corporation. All other trademarks are the property of their respective companies. AN138, Rev. A0©2012 Bruker Corporation. All rights reserved. BioScope Catalyst, MIRO, NanoScope, PeakForce QNM, Peak Force Tapping, and TappingMode are

Marcelle Koenig (PicoQuant, Berlin, Germany)