Gautier 2015 AFM in Cells Book

CHAPTER
Atomic force microscopy-

based force
measurements on animal
cells and tissues
12
Hélène O.B. Gautier, Amelia J. Thompson, Sarra Achouri, David E. Koser,
Kathrin Holtzmann, Emad Moeendarbary, Kristian Franze1
Department of Physiology, Development and Neuroscience, University of Cambridge, Cambridge,
United Kingdom
1
Corresponding author: E-mail: kf284@cam.ac.uk
CHAPTER OUTLINE
Introduction ............................................................................................................ 212
1. Experimental Setup ............................................................................................ 213
1.1 Setup Design ...................................................................................... 213
1.2 Cantilever Calibration .......................................................................... 214
2. Sample Preparation............................................................................................ 216
2.1 Animal Pretreatment ........................................................................... 216
2.2 Preparation of Measurement Buffers ..................................................... 217
2.3 Sample Immobilization ........................................................................ 217
3. AFM and Optical Imaging .................................................................................... 218
4. Measuring Cell and Tissue Stiffness .................................................................... 220
4.1 Important Parameters for Indentation Measurements.............................. 222
4.2 Analysis of Indentation Experiments ..................................................... 223
5. Measuring Adhesion........................................................................................... 225
5.1 Chemical Force Microscopy.................................................................. 226
5.2 Single-Molecule Force Spectroscopy ..................................................... 227
5.3 Single-Cell Force Spectroscopy............................................................. 228
6. Further Applications ........................................................................................... 228
Conclusions............................................................................................................ 230
Acknowledgments ................................................................................................... 231
References ............................................................................................................. 231
Methods in Cell Biology, Volume 125, ISSN 0091-679X, http://dx.doi.org/10.1016/bs.mcb.2014.10.005 211

© 2015 Elsevier Inc. All rights reserved.
212 CHAPTER 12 AFM force measurements
Abstract
During development, normal functioning, as well as in certain pathological conditions,
cells are influenced not only by biochemical but also by mechanical signals. Over the past
two decades, atomic force microscopy (AFM) has become one of the key tools to
investigate the mechanical properties and interactions of biological samples. AFM studies
have provided important insights into the role of mechanical signaling in different bio-
logical processes. In this chapter, we introduce different applications of AFM-based force
measurements, from experimental setup and sample preparation to data acquisition and
analysis, with a special focus on nervous system mechanics. Combined with other mi-
croscopy techniques, AFM is a powerful tool to reveal novel information about molec-
ular, cell, and tissue mechanics.
INTRODUCTION
The atomic force microscope is a form of scanning probe microscope that was
invented in 1986 (Binnig, Quate, & Gerber, 1986). Over the past two decades,
atomic force microscopy (AFM) has emerged as a versatile tool to study biological
samples (Morris, Kirby, Gunning, & World, 1999; Müller & Dufrene, 2008). Just as
optical microscopy is an extension of our vision, an atomic force microscope can be
thought of as an extension of the sense of touch. It generates and measures physical
interactions between a soft leaf spring, called cantilever, and the sample. When the
cantilever is moved down on the sample, the cantilever deflects while exerting force
on the sample. The cantilever’s deflection is tracked by a laser beam that is reflected
off its surface and is detected by a photodiode, which results in subnanometer spatial
and millisecond temporal resolution (Figure 1(A)).
An atomic force microscope can be used in several modes, including imaging,
conductive measurements, and force spectroscopy. It can image the surface of bio-
logical samples, such as the morphology of living cells (Lamour et al., 2009) or pro-
teins in membranes (Müller & Engel, 2007), in a liquid environment with submicron
resolution. In conduction measurements, current flows through the metal-coated
cantilever tip and the conducting sample, thus assessing the local electrical proper-
ties of the sample. In force spectroscopy mode, AFM can be used to apply and/or
detect forces down to the piconewton range; it has been used extensively to assess
the mechanical properties of biological materials (e.g., their elastic properties)
and mechanical interactions between molecules (e.g., antigeneantibody binding
forces) (Franze, 2011; Goldsbury, Scheuring, & Kreplak, 2009). In this chapter,
we focus on the application of AFM to force measurements of biological samples.
While techniques described in this chapter are applicable to all living systems, forces
specified here are mainly valid for cells that do not possess cell walls (such as animal
cells) and for the tissues comprising these cells (for AFM to probe plant cell me-
chanics, see S.A. Braybrook [Chapter 13 of this volume]).
1. Experimental setup 213
FIGURE 1 Principle and experimental setup of atomic force microscopy (AFM) measurements
on living biological samples.
(A) Schematic drawing of an AFM setup. The cantilever is moved by piezo-elements in the
z-direction with nanometer resolution. A laser beam is reflected by the cantilever and, after
being redirected by a mirror, detected by a photodiode. As the cantilever exerts a force on the
sample it deflects; the magnitude of deflection is proportional to the force. Samples may be
placed in a petri dish on a stage moving in x- and y-directions. Heating and perfusion systems
can be added. (B) Overview of the experimental setup. The AFM is placed on the xey stage,
which holds the sample. The stage is attached to an inverted microscope. The whole setup is
installed on a vibration isolation table.
1. EXPERIMENTAL SETUP
An AFM unit comprises a piezocontroller onto which the cantilever is attached, a
laser that reflects off the cantilever surface, a four-quadrant photodiode, and a mirror
that changes the angle of the laser beam and reflects it onto the photodiode
(Figure 1(A)). A feedback mechanism may adjust the movement of the z-piezo to
maintain a constant force between tip and sample surface.
While this chapter focuses mainly on elastic stiffness and adhesion measure-
ments, several general principles apply to the experimental setup for all types of bio-
logical measurements.
1.1 SETUP DESIGN

First, the specifications of the AFM itself are important to ensure optimally designed
experiments. The movement of the cantilever in the z-direction, for example, is usu-
ally driven by a piezoelectric element (“piezo”). The working range of this z-piezo
determines possible applications. For measurements of proteineprotein interactions,
for example, piezo ranges of a few micrometers are usually sufficient, while for ex-
periments involving living cells the range should be larger than the cell height
(usually 10e30 mm) in order to avoid damage of the cantilever and/or sample when
moving the cantilever across the sample. For work on samples with irregular surfaces,
such as tissue slices, even larger working ranges may be desirable. However, an in-
crease in working range causes an increase in noise; these parameters need to be
balanced. In addition, AFMs can be equipped with piezos for well-controlled motions
in the horizontal plane, which for example is required for surface topography scans.
Second, the cantilevers used need to be appropriate for the sample to optimize
the signal-to-noise ratio. Several shapes of cantilevers exist and both geometry
and dimensions of the cantilever probe need to be well-defined in order for data
to be fitted to a particular model during analysis (see Section 4). It is often difficult
to characterize the sharp, pyramidal-shaped tip geometry of some cantilevers pre-
cisely (Gibson, Watson, & Myhra, 1997), and a sharp tip may also damage fragile
cell and tissue samples as they exert a large stress s (force per area) on the sample,
though this is not always a problem if the tip type and scan parameters are carefully
controlled (Franz & Puech, 2008). Furthermore, indentation measurements taken
with pyramidal-shaped tips often lead to an overestimation of the determined elastic
modulus, likely because of substrate effects due to the high stresses produced by
sharp tips (Carl & Schillers, 2008).
To avoid uncertainties in the tip geometry, spherical, conical and punch-
shaped indenters are commercially available or can be custom-made. For
example, a polystyrene bead can be glued to a tipless cantilever and used as rigid
spherical indenter (Figure 2(B)); different types of beads are commercially avail-
able in defined sizes.
Some AFMs can be combined with optical microscopy, which may allow a better
control of the sample, and also enable measurements that would otherwise not be
possible (see Sections 3e6 for more detail) (Figure 1(B) and (C)). Optical control
is also useful if cantilever probes need to be modified. The entire setup needs to
accommodate the desired sample and eliminate external vibrations that could
decrease the signal-to-noise ratio or, at the worst, prevent measurements. It therefore
needs to be fixed to a vibration isolation system, including all loose cables, which
should be grounded.
The addition of a motorized stage allows automated measurements over a larger
ranges in x- and y-directions than would be accessible with x- and y-piezos only,
which is often required when working with large samples such as tissue slices.
Furthermore, many AFMs that are manufactured for biological applications
(“Bio-AFMs”) also provide sample heaters and flow chambers to enable control
of temperature, pH, and ionic composition of the media used during an experiment.
1.2 CANTILEVER CALIBRATION

Prior to a measurement, the cantilever needs to be calibrated. Although cantilevers
are supplied with approximate values of their spring constant k (a measure of their
stiffness), a precise calibration is required to accurately convert the cantilever deflec-
tion into a force. The calibration of a cantilever proceeds in three steps: (1) adjusting
1. Experimental setup 215
(A) (B) (C)
FIGURE 2 Indentation experiments on tissues and cells.

(A) Stiffness map of a cerebellar tissue slice. Left: microscopic image of a cerebellar slice on
which apparent elastic moduli K were measured (right). The light areas in the optical image
correspond to gray matter, dark areas to white matter. The asterisk indicates a region without
tissue. Gray matter is significantly stiffer than white matter. Scale bar: 400 mm. (B)
Photographs of a cantilever with spherical probe before the onset of an approach (top) and
during contact with a cell (images courtesy of D. Larrieu). Scale bar: 50 mm. (C) Typical
atomic force microscopy (AFM) forceedistance curve collected when a sample is indented.
The raw data (dotted black line) is fitted with two independent curves. A linear curve (blue
line (black in print versions)) is fitted to the noncontact portion of the forceedistance curve.
Here, the cantilever approaches the sample (from right to left; the z-piezo position is given at
the x-axis of the plot) and does not deflect. A nonlinear curve (red line (gray in print versions))
is fitted to the indentation (i.e., contact) part of the curve. The cantilever deflection, which is
shown on the y-axis, increases while the z-piezo moves further down. The contact point is
estimated by choosing the point that minimizes the total mean square error of the fitted
curves. ((A) reprinted from (Christ et al., 2010) with permission from Elsevier).
laser and photodiode positions, (2) calibrating the photodiode, and (3) measuring the
cantilever’s spring constant (k).
1. After putting the cantilever in place, the laser is positioned onto the cantilever to
obtain maximum reflection, close to its far enddto optimize the signal-to-noise
ratio. Additional corrections for changes in the refractive index of the medium
may be applied to the system via a tiltable mirror in the laser path. Subsequently,
the photodiode position is adjusted, leading to maximum signal detection.
2. Then the cantilever is approached with a constant approach velocity toward the
sample surface, which for calibration must be infinitely stiff (e.g., glass for soft
cantilevers). Upon contacting the sample surface, the cantilever starts bending.
Once a predefined target force is reached, the movement of the piezoelectric
ceramic is reversed, causing the cantilever to retract. During this procedure, a
voltageedistance is recorded (see Chapter 5). As in this case the sample is not
indented, the deflection of the cantilever equals the relative motion of the z-piezo;
the slope of the voltageedistance curve thus provides a conversion of the voltage
measured on the photodiode into the deflection of the cantilever in nanometers.
3. To determine the cantilever spring constant k, several methods are available:

observing the behavior of the cantilever before and after adding a load of known
mass; using the cantilever to exert a force on a reference sample or second
cantilever of known stiffness; and determining the resonance frequency of the
cantilever at a given temperature (“thermal noise” method) (Cleveland, Manne,
Bocek, & Hansma, 1993; Gates, Reitsma, Kramar, & Pratt, 2011; Gibson et al.,
1997; Hutter & Bechhoefer, 1993). The thermal noise method has the advantage
of not requiring any additional equipment, and unlike the reference sample
method, carries no risk of damaging the cantilever; this method is also often
included in commercially available AFM software.
Once the photodiode is calibrated and the spring constant of the cantilever
known, the voltage measured during cantilever deflection can be converted into a
force. An indentation experiment then yields a forceedistance curve, a plot of the
cantilever deflection (i.e., force) against the height of the piezo in z.
2. SAMPLE PREPARATION
As with any other type of microscopy, a well-prepared sample is a prerequisite for a
good experiment. To guarantee high quality AFM measurements on living biological
samples, specimens must be prepared and maintained in physiological conditions.
Hence, the sample preparation methods and buffers used will influence sample
viability and thus measurement quality. This is particularly important when dealing
with primary cells and tissue samples. We describe here the preparation of acute sli-
ces of rodent central nervous system (CNS) tissue as an example for a highly delicate
sample. The general principles described are applicable to many other tissue and cell
preparation approaches.
2.1 ANIMAL PRETREATMENT

The preservation of CNS slice quality starts before the animal is culled. Anesthetics
at high doses may interfere with brain function, with unknown effects on tissue me-
chanical properties. For example, isoflurane increases the permeability of the
bloodebrain barrier (Tétrault, Chever, Sik, & Amzica, 2008). In addition, culling
with carbon dioxide may alter the health of the slices due to prolonged anoxia, which
modifies the extracellular matrix (ECM) (Jean, Gravelle, Fournie, & Laurent, 2011).
Cervical dislocation causes mechanical stress and potentially hemorrhaging in the
spinal cord, the brain stem, and the cerebellum, as they are close to the area where
the dislocation is done. Thus, the latter method is only suitable if the region of in-
terest is far from these areas (e.g., lumbar spinal cord or cerebrum). Decapitation us-
ing the correct equipment (such as a guillotine) (with and without light
preanesthesia) is a quick and humane method of euthanasia that keeps the brain
in best condition. It is therefore the gold standard for culling rodents (Davie et al.,
2006; Doroshenko & Renaud, 2009) and should be the method of choice.
2. Sample preparation 217
2.2 PREPARATION OF MEASUREMENT BUFFERS

Buffer composition is important to maintain living cells or tissue slices in an environ-
ment resembling their physiological surroundings. The exact buffers used will depend
on species and age, as well as on tissue type and condition (for instance, injured tissues
might be more sensitive to damage from slicing). A standard slicing buffer for CNS
preparations is ice-cold artificial cerebrospinal fluid (aCSF) bubbled with a gas
mixture of 5%CO2 and 95%O2 at a pH of approximately 7.4 containing in mM:
120 NaCl, 26 NaHCO3, 1 NaH2PO4, 2.5 KCl, 2 MgCl2, 2 CaCl2, 10 glucose, and
1 kynurenic acid (a broad-spectrum glutamate receptor antagonist that reduces exci-
totoxicity) (Christ et al., 2010). This buffer can be used for slicing as well as recovery
and maintenance of slices. Several variations exist and have been investigated in
detail: low sodium concentration or sodium replacement (by sucrose, choline, or
(2-Amino-2-(hydroxyméthyl)propane-1,3-diol) TRIS for example) to reduce
neuronal firing and passive sodium influx (which will lead to water movements
and cell swelling); increased osmolarity by adding sucrose to further minimize cell
swelling during slicing; addition of antioxidant or metabolites for energy supply,
as well as adjusted solutions for slicing and recovery (Mitra & Brownstone, 2012;
Moyer & Brown, 1998; Richerson & Messer, 1995; Zhao et al., 2011).
For measuring slices, a (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)
-buffered solution or aCSF with added HEPES increases buffering capacity and re-
duces edema (MacGregor, Chesler, & Rice, 2001). An example of a standard
HEPES buffer at a pH of 7.4 contains in mM: 144 NaCl, 2.5 KCl, 10 HEPES, 1
NaH2PO4, 2.5 CaCl2, 2 MgCl2, 10 glucose. HEPES-only solutions must be bubbled
with 100% 02, whereas HEPESeaCSF should be bubbled with 5%CO2 and 95%O2
to achieve the right pH. A perfusion system should be in place, ensuring constant
renewal of the solution to keep the pH value stable. HEPES buffers are suitable
for experiments at room temperature, while aCSF can be used up to w37 C.
Cells cultured in CO2-dependent media can be kept in culture media without
perfusion, but the experiment should not last longer than 20e30 min (depending
on the surface to volume ratio of the medium) due to the lack of an enriched CO2
atmosphere. Alternatively, a CO2-independent medium can be used, or the sample
chamber can be perfused with HEPES or HEPESeaCSF.
If investigating mechanosensitivity of cells or cell mechanics, special care should
be taken to avoid components that could interfere with mechanosensitive ion channels
(MSC) or deplete calcium, an essential second messenger for different mechanotrans-
duction pathways. For instance, aminoglycoside antibiotics such as streptomycin and
gentamicin are known MSC blockers (Hamill & McBride, 1996), and buffer solu-
tions such as phosphate buffered saline do not contain calcium ions.
2.3 SAMPLE IMMOBILIZATION

Complete sample immobilization is essential for accurate AFM measurements. This
can be achieved in different ways depending on the type of sample and experiment.
In most indentation experiments, the sample needs to be attached to a surface; in
adhesion measurements the sample needs to attach to the substrate, other cells, or the
cantilever. For nonadherent cells and small tissue slices (e.g., spinal cord slices) a
nonspecific cell and tissue adhesive such as polylysine or Cell-Tak will be sufficient.
Larger slices, however, can be more challenging to immobilize and a chemical ad-
hesive might not be sufficient. Here a harp grid will provide adequate fixation, or, if
the sample is sufficiently large, it can also be pinned down with insect pins on a
sylgard-coated dish.
3. AFM AND OPTICAL IMAGING

Optical imaging techniques are an invaluable addition to the Bio-AFM toolbox. The
laser position on the photodiode already provides a precise readout of the cantile-
ver’s deflection. However, observing the cantilever’s position in the sample plane,
morphological changes of the sample, or changes of its fluorescence signal in
response to an applied load may also provide crucial information.
To date, many different optical techniques have been combined with AFM (for a
current review see Kainz, Oprzeska-Zingrebe, & Herrera, 2014): e.g., epifluores-
cence microscopy (Mangold, Harneit, Rohwerder, Claus, & Sand, 2008), confocal
microscopy (Harris & Charras, 2011), infrared spectroscopy (Dazzi et al., 2012)
and super-resolution microscopy (Chacko, Zanacchi, & Diaspro, 2013). Often, the
optical component is used to track dynamics of cellular structures; for example,
Kidoaki and Matsuda (2007) used epifluorescence to image the cytoskeleton in cells
with artificially imposed geometries and correlated actin structures with changes in
elastic stiffness. Until recently, the slow speed of AFM image acquisition compared
to optical approaches has been a barrier to real-time imaging, but this issue is now
being overcome by the development of high-speed AFM heads, such as the tip-
scanning system in Suzuki et al. (2013).
When planning a combined optical/AFM setup, the first technical consider-
ation is whether to use an inverted or upright microscope (Figure 3). Inverted
setups are comparatively easy to implement, regardless of the desired optical
approach. A typical setup might include the AFM head above an xey motorized
stage, mounted on an inverted microscope with epifluorescence and bright-field
(Figure 3(A)). This approach is ideal for optically transparent samples such as bio-
films, gels, or single cells in culture, and many currently available optical/AFM
combinations use inverted imaging (Kainz et al., 2014). Upright imaging
(Figure 3(B) and (C)) is more challenging: either the presence of the AFM head
becomes a spatial constraint for optical components, or imaging must be done
through the AFM apparatus. However, upright imaging is essential to characterize
optical properties of the sample surface, and for thick, opaque samplesdi.e., most
tissue and in vivo measurements.
The basic requirements for an upright optical/AFM setup are summarized in
Table 1.
3. AFM and optical imaging 219
FIGURE 3 Combination of atomic force microscopy (AFM) and optical imaging.

Schematics of typical combined AFM/fluorescence imaging setups: (A) standard
epifluorescence using an inverted microscope, (B) upright epifluorescence setup, in which
both the excitation light source and the camera are located above the sample (note: the
working distance here usually needs to be quite large to provide space for the AFM, limiting
NA of the objective), and (C) transfluorescence setup, in which the excitation light reaches
the sample from beneath and fluorescence emission is collected by a camera mounted
above the AFM (suitable for transparent samples). (D) Schematic of sample preparation
when using a side-view cantilever holder. The sample needs to be elevated; the elevating
“unit” can consist of a portion of microscope slide. Blue denotes excitation wavelength, green
fluorescence emission, and yellow transmitted light. (See color plate)
The AFM apparatus itself can also be modified to suit optical imaging. For
example, cantilevers can be mounted on glass blocks. These glass blocks can
additionally be fabricated with a mirror attached, allowing “side-view” imaging
to visualize the physical deformation of the sample (Figure 3(D)). The precision
of side-view imaging may be increased by using fluorescent beads glued to
the cantilever. Thus, the position of both indenter and sample may be tracked
simultaneously when (for instance) using AFM with confocal imaging of
Table 1 Basic Requirements for an Upright Optical/AFM Setup

Essential
Components Technical Considerations Examples
Light source • Illumination must be on For low-magnification
same side as AFM bright field:
cantilever for thick/opaque • Ring LED
samples • Swan neck lamp
(transfluorescence may be For fluorescence:
ineffective) • Mercury lamp
• Metal halide source
Optical apparatus and • AFM head must be • Wide-field camera with
detector optically transparent telescope lens, mounted
around cantilever mount above AFM head
• Lenses must have long • Fluorescence
working distance (limiting stereomicroscope
NA)
• Detector and optical
elements must be
perpendicular to AFM
apparatus to ensure good
image quality
• Detector mount must be
stable and drift free
x–y stage • Micron-level control of x–y • Manual stage
movement (if needed) • Motorized stage
• Must be stable • Shuttle stage (moves
• Must be compatible with between AFM and
correct sample mounts separate optical
microscope)
AFM, atomic force microscopy.
fluorescently labeled cells (Moeendarbary et al., 2013). An alternative is to add a

fluorescent dye to the surrounding medium, which will make the cantilever visible
as a dark shape (Harris, Daeden, & Charras, 2014). However, the use of this holder
requires the sample to be elevated from the bottom of the dish to allow room for the
mirror, and therefore also requires microscope objectives with long working
distances.
Although combining AFM with optical imaging is still technically challenging,
much progress has been made. Further development will lead to an even wider scope
of possible AFM experiments in biology.
4. MEASURING CELL AND TISSUE STIFFNESS

There are a number of different ways to measure the mechanical properties of cells
and tissues with an AFM (for cell mechanics measurements with another method,
4. Measuring cell and tissue stiffness 221
parallel plate technique, see Bufi et al. [Chapter 11 of this volume]). Most commonly
used are indentation experiments, in which the sample is indented by the cantilever,
which is usually moved at several micrometers per second, and its elastic stiffness is
determined. Classical creep and stress relaxation experiments can also be per-
formed, in which either a constant stress (force/contact area) or constant strain (rela-
tive deformation) is applied to the sample, and the accompanying change in strain or
stress is recorded. However, these approaches are more complicated because when
the force applied to a viscoelastic medium (such as cells or tissues) is kept constant,
the cantilever will continue indenting the sample, and the contact area between
probe and sample will increase over time for most probe geometries. Thus, with
standard probes it is very difficult to maintain a constant stress. However, this lim-
itation may be overcome using custom-built probes such as wedged-AFM cantile-
vers (Stewart et al., 2013). See (Miri, Heris, Mongeau, & Javid, 2014) for further
information on creep or stress relaxation experiments. Therefore, we here focus
on indentation experiments (for a summary of important considerations to optimize
indentation experiments see Table 2).
AFM indentation experiments can be performed on single cells (Figure 2(B))
(Pagliara et al., 2014) and even on isolated cell compartments such as the nucleus
(Krause, Te Riet, & Wolf, 2013; Lu et al., 2006). Furthermore, bulk mechanics
was measured at the tissue level, for instance in mammalian brain slices and retinal
tissue (Figure 2(A)) (Christ et al., 2010; Elkin, Azeloglu, Costa, & Morrison, 2007;
Franze et al., 2011). AFM indentation measurements can either be performed on re-
gions of interest of a sample or programmed to collect data in a raster scan covering
Table 2 Important Considerations and Common Problems When Using AFM in

Force Spectroscopy Mode
Important Considerations Common Problems and Solutions
Cantilever stiffness appropriate for the Sample moving: Cell/tissue adhesive,
sample harp grid, or insect pins (see Section 2.3)
Calibrate each cantilever in air and the Baseline shifted: Reduce approach speed
photodiode on uncoated glass in a drop of or change the cantilever. If the problem
buffer/medium persists, sample integrity is likely to be an
Adjust parameters (force, approach issue
speed) for each sample type
Once optimal parameters found, keep Acquisition time of indentation
them constant through each set of measurements too long: increase
experiments retraction speed
Do not use water as liquid when Poor visualization of the sample: Ensure
characterizing materials as it will generate optical path is clean, verify alignment of
electrostatic interactions light source and AFM and/or add an extra
light source
AFM, atomic force microscopy.
a larger area (the maximum area is determined by the survival time of the tissue and
the desired resolution). Local stiffness distributions can then be visualized (e.g., as a
color map) or used to perform region of interest analysis (Figure 2(A)) (Christ et al.,
2010).
4.1 IMPORTANT PARAMETERS FOR INDENTATION MEASUREMENTS

The main technical considerations that facilitate successful indentation experiments
concern the types of cantilevers used and the measurement parameters.
Cantilevers are leaf springs, they follow Hooke’s law: the force F that is applied
to a cantilever is proportional to its deflection Dx:
F ¼ kDx;
where k represents its spring constant. In general, spring constants of cantilevers
need to be matched to the sample stiffness. For instance, if a stiff cantilever is
used with a sample that is orders of magnitude softer, large sample deformations
would barely lead to any detectable cantilever deflection. However, there is also
a lower limit for cantilever spring constants, as noise increases with decreasing k,
and the drag force of liquid media may cause significant cantilever deflections
with increasing cantilever velocities at low k-values, which may limit force measure-
ments. Some examples of suitable magnitudes of k are 0.1e1 N/m for stiffer
animal tissue samples (with an elastic modulus E in the kilopascal range), or
0.01e0.05 N/m for measuring softer animal tissue mechanics (E ranging from
100 to 500 Pa) (Table 3) (Christ et al., 2010; Park, Costa, Ateshian, & Hong,
2009). Stiffer cantilevers are required for stiffer samples such as plant cells (see
S. Braybrook [Chapter 13 of this volume]).
Cantilevers come in different shapes, are made of different materials, and may or
may not have a reflective coating. Also, there is a wide variety of probe geometries
commercially available. Every application requires optimally chosen cantilevers,
Table 3 Stiffness of Various Animal Tissues

Tissue Elastic Modulus (E) (Pa) References
Braindwhite matter (w2.25) 10 2
Christ et al. (2010)
Braindgray matter (w3.4) 102 Christ et al. (2010)
Breastdnormal (1–4) x 103 Samani, Zubovits, and Plewes
(2007)
Breastdcancerous (3–50) x 103 Samani et al. (2007)
Muscle tissue 12 x 103 Engler et al. (2004)
Articular cartilage (0.4–200) x 106 Nemir and West (2010)
Bone (0.008–40) x 109 Nemir and West (2010)
4. Measuring cell and tissue stiffness 223
and the choice may sometimes be difficult. For biological measurements, uncoated
(as coating not only increases reflectivity but also cantilever drift) beam-shaped or
triangular silicon or silicon-nitride cantilevers with spherical probes are often
appropriate.
The second important parameter, which needs to be carefully controlled, is the
speed at which the cantilever approaches the sample. Since cells and tissues must
be maintained in medium, Bio-AFM measurements are usually performed in liquid.
If the approach speed is too fast when the cantilever moves through liquid, the
viscous drag may lead to a deflection of the cantilever before it reaches the sample,
which will distort data analysis (see Table 2). In practice, this can be seen by a pro-
gressive tilt in the baseline (larger tilt with increasing approach speed). This is espe-
cially problematic for soft cantilevers (k < 0.05 N/m) used for biological
applications; however, setting an appropriate approach speed of w5e15 mm/s
will usually overcome this problem. Once a suitable approach speed is selected, it
must be kept constant for experiments to be comparable, as most biological mate-
rials are viscoelastic, and their response to an applied force depends on the frequency
at which they are probed.
Finally, all structures in the sample within the area that is deformed by the load
during AFM indentation measurements will contribute to some degree to the overall
elastic modulus that is measured. Therefore, indentation depth is a crucial parameter.
Small indentations of animal cells (w100 s of nm) will mostly measure actin cortex
mechanics, while at larger indentations (wmm) contributions of cell organelles such
as the nucleus are increasingly visible. Furthermore, large indentations of a thin
sample will lead to an increasing contribution of the underlying substrate to the
measured elastic modulus (Kuo, Xian, Brenton, Franze, & Sivaniah, 2012). There-
fore, this parameter needs to be tightly controlled, which can be done by choosing an
appropriate force for a measurement and/or by restricting data analysis to an appro-
priate range of the forceedistance curve (i.e., by discarding data of large indenta-
tions). To avoid substrate effects, indentations should be smaller than w a tenth
of the sample height if using the Hertz model for data analysis, or correction terms
for thin samples should be applied (Mahaffy, Park, Gerde, Kas, & Shih, 2004).
4.2 ANALYSIS OF INDENTATION EXPERIMENTS

An indentation experiment produces forceedistance curves. These are plots of the
applied force versus distance between cantilever and sample during the cantilever’s
approach to the sample (Figure 2(D)). During indentation the force F applied to the
cantilever is related to the deflection of the cantilever Dd and its spring constant k via
Hooke’s law
F ¼ kDd ¼ kðd d0 Þ (1)
The indentation depth d is calculated by subtracting the cantilever deflection Dd
from the piezo translation Dz
d ¼ Dz Dd ¼ ðz z0 Þ ðd d0 Þ ¼ ðz dÞ ðz0 d0 Þ ¼ w wo (2)
where z0 is the translation of the piezo at the contact point and w ¼ (z d) and
w0 ¼ z0 d0 are the transformed variables.
By fitting an appropriate mechanical model to the recorded force-indentation
curves (red line in Figure 2(C)) the elastic modulus of the sample is obtained, which
is a measure of its elastic stiffness. The mechanical models must be chosen carefully,
as they rely on various preconditions of the setup to deliver valid results. The most
widely used model to determine an elastic modulus of a wide range of biological
materials is the Hertz model (Hertz, 1881). It describes the indentation of an elastic
half-space (i.e., the contact area between indenter and sample is much smaller than
the characteristic radius of the sample) with a rigid spherical indenter for small
strains, when the deformation is elastic. In practice, those conditions can be met
to a sufficient degree in a typical AFM setup; both indenter and sample must be large
in comparison to the area indented, friction and adhesion between sample and
indenter must be negligible (this can be confirmed with some preliminary force dis-
tance curves and adhesion analysis) and the sample can only be indented up to
10e15% of its thickness to avoid artefacts from the underlying substrate. Under
these conditions, the relationship between the applied force F and the indentation
depth d is
4 E pffiffiffi 3=2
F¼ Rd (3)
3 1 n2
with n being the Poisson’s ratio which is the measure of material’s compressibility, and
R the radius of the spherical indenter. For most biological samples, the Poisson’s ratio is
unknown, and an apparent reduced elastic modulus K is often given as K ¼ E/(1 n2).
The large number of forceedistance curves that can be recorded during a typical
experiment requires designing an automated analysis routine for fitting the model to
the raw data. The critical step in the analysis process is determining the contact
point, where the indenter touches the sample without applying any force
(Figure 2(C)). For most biological samples, the contact point is not well defined
and must be inferred from fitting a model to the raw data, using for example methods
described by (Lin, Dimitriadis, & Horkay, 2007). If the Hertz model is applicable,
most analysis routines employ a sequential search algorithm, where after exclusion
of end parts of the curve, each point is treated as a potential contact point and both
the noncontact and the indentation part of the curve are fitted with a linear or
Hertzian fit, respectively. The point with the best least-squares fit of the force dis-
tance curve is then chosen as contact point (Figure 2(C)). This approach requires
forceedistance curves to be continuous with concave curvature.
Models applied to extract elastic moduli of biological materials from indenta-
tion or, more generally, rheological measurements have to make assumptions with
respect to the mechanical properties of the samples. These assumptions are often
required to simplify the problem and thus to actually enable fitting the data. In
this regard, even the widely used Hertz model is limited in its scope; it does
5. Measuring adhesion 225
not take viscous effects or adhesion into account. However, viscosity can be
determined by introducing dynamic (e.g., oscillatory) measurements (Mahaffy,
Shih, MacKintosh, & Kas, 2000). The effects of adhesion are described by
Johnson-Kendall-Roberts (Chu, Dufour, Thiery, Perez, & Pincet, 2005) and
Derjaguin-Muller-Toporov (Derjaguin, Muller, & Toporov, 1975), and implemen-
tations of those theories can be found in (Lin et al., 2007). The model is also only
valid for indentations that are small compared to the sample height. Correction
terms have been introduced, extending the application of the Hertz model for
example to thin regions of cells (Dimitriadis, Horkay, Maresca, Kachar, & Chad-
wick, 2002; Mahaffy et al., 2004). Without such correction terms, the apparent
elastic modulus increases with indentation depth if a soft sample is adhered to a
much stiffer substrate (Kuo et al., 2012). Furthermore, the Hertz model assumes
the sample to be homogenous and isotropic, which biological samples rarely
are. However, if the right experimental conditions are chosen, the model is actually
very useful. For small and fast indentations, for example, elastic moduli obtained
by fitting the Hertz model to AFM raw data are remarkably accurate and
reproducible.
5. MEASURING ADHESION
In indentation measurements, as described above, an elastic modulus is inferred
from the forceedistance curve generated during cantilever approach. During the
indentation, the surfaces of probe and sample physically interact. This interaction
can result in the establishment of bonds resulting from physical and chemical inter-
actions, which contribute to adhesion. Such adhesive interactions will have already
occurred by the time the cantilever starts to retract, and can be promoted by
increasing the contact time and/or contact area (by exerting a larger force) prior
to retraction. The information about these adhesive interactions that can be extracted
from retraction curves depend on the nature of the probe and the sample, and also on
the level of control the experimenter holds over these. Depending on the type of
experiment, three main AFM techniques can provide insight into adhesion based
on the analysis of retraction curves:
• Chemical force microscopy (CFM), when the whole probe, which is designed to
have a rather large contact area, is chemically coated.
• Single-molecule force spectroscopy (SMFS), when the sharp tip of a probe is
coated with biomolecules.
• Single-cell force spectroscopy (SCFS), when the probe consists of a cell.
These techniques have several applications; here we will focus on their use for
adhesion measurements. It is important to note that since these measurements are
obtained from retraction curves, what is measured is effectively an unbinding/
detachment force, which is not necessarily the adhesion force (as not only cell adhe-
sion bonds may break), although highly correlated with it.
5.1 CHEMICAL FORCE MICROSCOPY

In CFM, the probe is a chemically coated cantilever probe, which usually is either a
tip or a sphere (Figure 4(A)). The sample surface can be chemically coated as well,
or it can simply be the surface of a biological specimen (e.g., a cell). The two sur-
faces are brought in contact with a set force for a fixed period of time and separated
again at a predefined retraction velocity. The force required to unbind probe and
sample is extracted from retraction curves (Figure 4(B)). CFM can probe forces
FIGURE 4 Adhesion measurements.

(A) Schematic of a chemical force microscopy (CFM) sample: The probe is a cantilever tip
coated with CH3 and the sample is a polymer bead coated with COOH. (B) Typical CFM
retraction curve: the detachment force of the interaction between CH3 and COOH is extracted
from the retraction curve as the difference between the minimal force value and the curve’s
baseline. (C) Schematic of a single-molecule force spectroscopy (SMFS) sample: the sample
is a cell possessing membrane receptors; the probe is a pyramidal-shaped tip coated with a
ligand binding to a specific receptor. (D) The detachment force of the interaction between the
ligand and its receptor is extracted from the retraction curve as the difference between the
minimal force value and the curve’s baseline. (E) and (F) Schematics of a single-cell force
spectroscopy (SCFS) sample with a protein-coated substrate (E) or cell-seeded substrate (F).
(G) Example of SCFS retraction curve: the detachment force of the interaction between the
cell-probe and the substrate is extracted from the retraction curve as the difference between
the curve’s minimum and its baseline. The work necessary to fully detach the cell from the
substrate is calculated as the area underneath the baseline. (H) Schematic of how to coat
cantilevers. CFM, chemical force microscopy; SMFS, single-molecule force spectroscopy;
SCFS, single-cell force spectroscopy.
5. Measuring adhesion 227
based on van der Waals interactions, hydrogen bonds (Noy, Vezenov, & Lieber,
1997), or covalent bonds (Grandbois et al., 1999).
In biological applications, CFM can be used to detect, quantify, and map cell sur-
face hydrophobicity (Dague et al., 2007). Although CFM can quantify a variety of
chemical interactions, it can only probe nonspecific interactions.
5.2 SINGLE-MOLECULE FORCE SPECTROSCOPY

In SMFS, the probe (which usually is a sharp tip) is coated with biomolecules
(Figure 4(C)) and the sample surface is most commonly a surface coated with biomol-
ecules, the surface of a cell, a tissue, or a polymer gel. This technique is ideal for the
study of proteineligand interactions. SMFS was used for the first time to characterize
the strength of the biotineavidin bond (Moy, Florin, & Gaub, 1994) and its use to
functionalize AFM cantilever tips with additional molecules such as antibodies.
Specific coatings of the probe and sample surface allow the selection of pro-
teineligand pairs for detachment force measurements. This way, SMFS provides
a powerful tool to map the distribution of specific membrane proteins on cell sur-
faces (Lee, Mandic, & Van Vliet, 2007). Here, cantilever probes are coated with
antibodies that specifically bind to certain proteins, and a raster scan of the cell sur-
face reveals locations at which adhesive forces are significantly larger due to pro-
teineantibody interactions.
Using SMFS, it has furthermore been discovered that during detachment/separa-
tion experiments involving globular proteins, the retraction curves yield information
on their mechanical architecture, as it records the unfolding of the proteins before
rupture of proteineprotein bonds (Puchner & Gaub, 2009). For instance, reversible
unfolding of individual titin immunoglobulin domains has been detected using AFM
(Rief, Gautel, Oesterhelt, Fernandez, & Gaub, 1997). Hence, it is crucial to keep in
mind the variety of behaviors that are recorded in the retraction curves (e.g., stretch-
ing of cross linker, protein unfolding, step-wise dislocation of membrane proteins,
etc.) (Figure 4(D)).
While SMFS may provide important information on the ultrastructure, interac-
tions, and distribution of proteins and other macromolecules, it is technically chal-
lenging. Sample preparation is critical in this type of experiment. It is key to
adsorb biomolecules properly onto the probe/sample surface, which is a tedious
process, and to immobilize the sample (El Kirat, Burton, Dupres, & Dufrene,
2005; Hinterdorfer & Dufrêne, 2006; Müller & Engel, 2007). Due to the molecular
complexity of cell surfaces, the specificity of the interaction being measured must be
verified with appropriate controls (e.g., use of blocking antibodies). When probing
cell surfaces, particular care must be given to sample preparation and maintenance
during measurements (see Section 2). Furthermore, during successive detachment
measurements, the probe’s coating is progressively lost, limiting the number of mea-
surements that can be performed using the same probe. It is therefore very important
to design and perform experiments depending on what is to be measured, and to
know how to extract relevant data (Noy, 2011).
5.3 SINGLE-CELL FORCE SPECTROSCOPY

In order to measure cell adhesion, SCFS uses a cell as a probe. In this approach,
a cell is strongly attached to the cantilever and used to probe coated surfaces
(Helenius, Heisenberg, Gaub, & Muller, 2008) or other cells strongly attached to
the surface of the sample holder (Puech, Poole, Knebel, & Muller, 2006).
To attach cells to the cantilever, tipless cantilevers are coated with extracellular
matrix compounds, such as laminin, collagen, fibronectin, or Cell-Tak, or with un-
specific compounds, such as polylysine, which promote cell adhesion mainly by
electrostatic interactions. Cells are seeded on a nonadherent substrate, and treated
cantilevers are then approached onto such cells for a defined amount of time (usually
10 s of seconds) with a defined force (wnN). After retraction of the cantilever, the
cell sticks to it and can be used for a number of measurements (often for up to half an
hour, before the cell starts degrading). The compliant probe cell, which is now firmly
attached to the cantilever, is brought in contact with the sample for a fixed period of
time before it is pulled away. In this configuration, the cell-probe deforms when
brought in contact with the surface of the sample (and in case of cellecell experi-
ments the sample cell as well), resulting in a relatively large contact area between
the cell and the sample if compared to CFM or SMFS. The increased contact area
facilitates the generation of multiple bonds and prevents single moleculeemolecule
interaction measurements.
When probing coated surfaces (Figure 4(E)), specific interactions can be moni-
tored; however, the proteins at the surface might not be in their native states. This
technique allows a compartmentalized investigation of cell adhesion by measuring
adhesive properties between cells and isolated ligands or receptors. Such an
approach is ideal when investigating adhesive interactions of cells with extracellular
matrix proteins. In cellecell adhesion measurements, sample cells are strongly
attached to the surface of the sample holder, which may be coated with above
mentioned proteins or polypeptides (Figure 4(F)).
Independently of the sample’s nature, in SCFS measurements multiple adhesion
bonds are established within the contact area. When retracting the cantilever, these
bonds are successively broken one or more at a time until complete probe-sample sep-
aration occurs. These rupture events are recorded in the retraction curve, which reaches
a maximum amplitude before a step-wise return to the extension curve’s baseline, cor-
responding to complete detachment of the cell (Figure 4(G)). The detachment work
can be calculated as the area between the baseline and the retraction curve.
Table 4 summarizes the three key steps to perform adhesion measurements: canti-
lever coating, making a cell-cantilever probe and performing the experiment itself.
6. FURTHER APPLICATIONS
In addition to force-indentation and pulling experiments to measure the mechanical
resistance of a sample to a controlled load, Bio-AFMs can be used in many other
ways to assess different aspects of the mechanical interaction of the sample with
6. Further applications 229
Table 4 Key Steps for Adhesion Measurements Using AFM

Coating cantilevers with unspecific adhesive (all types)
• Prepare a microscope slide or petri dish by sticking two superposed pieces of double
sided tape aligned along their edge (Figure 4(H)).
• Place calibrated cantilevers on the edge of the tape and flip them over so that the ends
protrude as shown in Figure 4(H). Be careful not to press the cantilever down onto the
tape as this will make them stick and make later handling difficult, as well as attaching
glue residues to the cantilever’s surface which could affect later measurements.
• Using a pipette, place a small drop of coating solution (e.g., Cell-Tak) as close as
possible to the cantilever’s tip side without touching it with the pipette tip. Leave to
incubate following instructions of the according protocol. If the coating solution dries out
completely in this process, rehydrate the cantilevers before trying to move them.
Making a cell-cantilever probe
• Coat a calibrated tipless cantilever with the cell adherent chemical or protein of choice
(e.g., Cell-Tak or Concanavalin A) following instructions of the according protocol and the
ones listed above.
• Seed cells sparsely in an AFM-compatible petri dish. For best results, coat the surface of
the dish with a passivating molecule suitable for the cell type used (e.g., bovine serum
albumine (BSA), Polyethyleneglycol). This will reduce the cells’ adhesion affinity for the
dish’s surface.
• Mount the dish and cantilever on the AFM and recalibrate the photodiode.
• Select a cell and approach it with the cantilever at a speed of 10 mm/s or less and a force
of approximately 1 nN. A contact time of 1s can be sufficient; if not, increase this time until
the cell is attached to the cantilever when the latter is retracted.
• Allow the cell to further attach to the retracted cantilever for several minutes before
starting measurements. This cell-probe can be used to perform adhesion measurements
on coated surfaces or adhering cells.
• If the cell is not attached to the cantilever despite several attempts and a long contact
time (up to 30 s), select another cell and repeat the procedure. If no cell attaches to the
cantilever despite several attempts, check that the coating solution is efficient, and that
the cells are kept in good conditions (use buffered CO2-independent media where
possible, and limit the use of cells to 30 min on the AFM).
Performing a cell–cell detachment measurement
• Prepare an AFM-compatible petri dish by coating one area with the cell adherent
chemical or protein of choice, and one area with a passivating molecule (to facilitate cell-
cantilever probe making). In parallel to this, coat a cantilever with a cell adherent chemical
or protein.
• Seed cells sparsely on both areas (to avoid cells touching each other on the substrate).
Mount the dish and the coated cantilever on the AFM and calibrate for sensitivity.
• Attach a cell from the passivated area to the cantilever as described above and allow to
adhere on the retracted cantilever for several minutes.
• Move the cell-cantilever probe across the adherent area of the dish and select a cell
(avoid cells that look too spread or too lightly attacheddlooking at the cells while
knocking on the microscope’s side reveals the ones that are not attached). To avoid
excessive spreading of the cells, aim to start measurements within 10 min of seeding.
• Align the probe cell with the measured cell and perform an “adhesion measurement”
using a 1 nN force and a 10 mm/s approach. Set contact time duration according to the
needs of the experiment.
AFM, atomic force microscopy; BSA
the cantilever. For example, a sample may be exposed to a short-term mechanical

stimulus and its response to the applied force optically measured. For example,
z-stacks of a fluorescently labeled sample can be recorded during force application
using confocal microscopy, and a 3D-reconstruction of the images yields the defor-
mation of the sample along the load direction (Moeendarbary et al., 2013). The
deformation of the sample can also directly be visualized with a side-view optical
setup (Chaudhuri, Parekh, Lam, & Fletcher, 2009), or a side-view cantilever holder
equipped with a side mirror.
Vice versa, AFM can be used to control the deformation of the sample and
observe its response. Using this approach it was discovered that stem cells on their
way from pluripotency to fate commitment undergo a transition during which their
nuclei become auxetic, i.e., they assume a negative Poisson’s ratio (their cross-
sectional area decreases during compression and their apparent elastic modulus in-
creases with indentation depth) (Pagliara et al., 2014). Here, the cantilever is first
brought into contact with the sample by approaching it with a low set-point force
(w100 pN), and then the z-piezo is lowered by a predefined distance, while (fluores-
cence) pictures of the sample are simultaneously acquired.
In combination with contrast enhancing microscopy methods (e.g., phase
contrast or differential interference contrast microscopy), active cellular responses
to an applied load, such as retraction of neuronal processes after exposure to a me-
chanical stress, can be detected (Franze et al., 2009). Furthermore, cellular activity
in response to a mechanical signal can be monitored, either simultaneously to pre-
viously mentioned measurements or independently, for example, by combining cal-
cium imaging (Franze et al., 2009) or patch clamp (Mosbacher, Langer, Hörber, &
Sachs, 1998) with AFM.
AFMs possess feedback loops, which allow maintaining a preset force at a
given position irrespective of a change in height. This way, cantilevers can be
“parked” (i.e., approached on the surface with a predefined force) in front of a
migrating cell to measure the force required to stop the cell, i.e., the stall force
(Brunner et al., 2006; Prass, Jacobson, Mogilner, & Radmacher, 2006). Similarly,
a cantilever can also be placed on top of a sample to detect changes in sample height
with high spatial and temporal resolution (Domke, Parak, George, Gaub, & Rad-
macher, 1999; Hardie & Franze, 2012). When the sample contracts, cantilever
deflection initially decreases, until a feedback loop quickly lowers the cantilever
to maintain the deflection (i.e., force) constant. The cantilever height signal thus pro-
vides a readout of the sample height, with much better resolution than would be
possible with any type of conventional light microscopy.
CONCLUSIONS
In this chapter, we described the use of AFM in force spectroscopy mode to measure
elastic stiffness of cells and tissues as well as adhesion forces between cells and mol-
ecules. Indentation experiments are applicable over a wide range of biologically
References 231
relevant scales, from individual cellular compartments through to tissue samples,

providing information on the mechanical properties of both cells and their physical
environment. In addition, adhesion measurements allow detailed characterization of
cells’ interactions with their neighbors and surroundings. Such data provide valuable
understanding of the role of mechanical factors in development, tissue maintenance,
and regeneration.
The range of techniques, such as biological imaging, that can be combined with
AFM is also increasing. This is widening the versatility and potential scope of Bio-
AFM experiments, and is now allowing us to draw new links between biomechanics
and cellular responses. Importantly, during physiological processes, mechanical
cues may act together with biochemical signals, such as hormones, guidance cues,
or neurotransmitters. It is likely that cells need to integrate both types of signals
to produce an appropriate response. Therefore, when the role of mechanics in tissue
processes is better characterized (e.g., through the experimental approaches
described above), the next step will be to integrate mainstream biochemical ap-
proaches into the design of AFM experiments. Ultimately, this will strengthen the
powerful combination of physical and biological techniques, which will contribute
key insights into development, health, and disease.
ACKNOWLEDGMENTS
The authors would like to thank the Wellcome Trust (Fellowships to AJT and SA), Cambridge
Commonwealth, European & International Trust (Fellowship to AJT), NanoDTC (Student-
ship to KH), Köln Fortune Program/Faculty of Medicine, University of Cologne (Fellowship
to DEK), German National Academic Foundation (Scholarship to DEK), Herchel Smith
Foundation (Fellowship to EM), UK Medical Research Council (Career Development Award
to KF), and the Human Frontier Science Program (Young Investigator Grant to KF) for finan-
cial support.
REFERENCES
Binnig, G., Quate, C. F., & Gerber, C. (1986). Atomic force microscope. Physical Review
Letters, 56(9), 930e933.
Brunner, C. A., Ehrlicher, A., Kohlstrunk, B., Knebel, D., Kas, J. A., & Goegler, M. (2006).
Cell migration through small gaps. European Biophysics Journal, 35(8), 713e719.
Carl, P., & Schillers, H. (2008). Elasticity measurement of living cells with an atomic force
microscope: data acquisition and processing. Pflugers Archiv, 457(2), 551e559.
Chacko, J. V., Zanacchi, F. C., & Diaspro, A. (2013). Probing cytoskeletal structures by
coupling optical superresolution and AFM techniques for a correlative approach. Cyto-
skeleton, 70(11), 729e740.
Chaudhuri, O., Parekh, S. H., Lam, W. A., & Fletcher, D. A. (2009). Combined atomic force
microscopy and side-view optical imaging for mechanical studies of cells. Nature
Methods, 6(5), 383e387.
Christ, A. F., Franze, K., Gautier, H., Moshayedi, P., Fawcett, J., Franklin, R. J., et al. (2010).
Mechanical difference between white and gray matter in the rat cerebellum measured by
scanning force microscopy. Journal of Biomechanics, 43(15), 2986e2992.
Chu, Y. S., Dufour, S., Thiery, J. P., Perez, E., & Pincet, F. (2005). Johnson-Kendall-Roberts
theory applied to living cells. Physical Review Letters, 94(2), 028102.
Cleveland, J. P., Manne, S., Bocek, D., & Hansma, P. K. (1993). A nondestructive method for
determining the spring constant of cantilevers for scanning force microscopy. Review of
Scientific Instruments, 64, 403e405.
Dague, E., Alsteens, D., Latgé, J.-P., Verbelen, C., Raze, D., Baulard, A. R., et al. (2007).
Chemical force microscopy of single live cells. Nano Letters, 7(10), 3026e3030.
Davie, J. T., Kole, M. H. P., Letzkus, J. J., Rancz, E. A., Spruston, N., Stuart, G. J., et al.
(2006). Dendritic patch-clamp recording. Nature Protocols, 1(3), 1235e1247.
Dazzi, A., Prater, C. B., Hu, Q., Chase, D. B., Rabolt, J. F., & Marcott, C. (2012). AFM-IR:
combining atomic force microscopy and infrared spectroscopy for nanoscale chemical
characterization. Applied Spectroscopy, 66(12), 1365e1384.
Derjaguin, B. V., Muller, V. M., & Toporov, Y. P. (1975). Effect of contact deformations on the
adhesion of particles. Journal of Colloid and Interface Science, 53(2), 314e326.
Dimitriadis, E. K., Horkay, F., Maresca, J., Kachar, B., & Chadwick, R. S. (2002). Determi-
nation of elastic moduli of thin layers of soft material using the atomic force microscope.
Biophysical Journal, 82(5), 2798e2810.
Domke, J., Parak, W. J., George, M., Gaub, H. E., & Radmacher, M. (1999). Mapping the me-
chanical pulse of single cardiomyocytes with the atomic force microscope. European
Biophysics Journal, 28(3), 179e186.
Doroshenko, P., & Renaud, L. P. (2009). Acid-sensitive TASK-like Kþ conductances
contribute to resting membrane potential and to orexin-induced membrane depolarization
in rat thalamic paraventricular nucleus neurons. Neuroscience, 158(4), 1560e1570.
El Kirat, K., Burton, I., Dupres, V., & Dufrene, Y. F. (2005). Sample preparation procedures
for biological atomic force microscopy. Journal of Microscopy, 218(Pt 3), 199e207.
Elkin, B. S., Azeloglu, E. U., Costa, K. D., & Morrison, B., 3rd (2007). Mechanical hetero-
geneity of the rat hippocampus measured by atomic force microscope indentation. Journal
of Neurotrauma, 24(5), 812e822.
Engler, A. J., Griffin, M. A., Sen, S., Bonnemann, C. G., Sweeney, H. L., & Discher, D. E.
(2004). Myotubes differentiate optimally on substrates with tissue-like stiffness: patholog-
ical implications for soft or stiff microenvironments. Journal of Cell Biology, 166(6),
877e887.
Franze, K. (2011). Atomic force microscopy and its contribution to understanding the develop-
ment of the nervous system. Current Opinion in Genetics and Development, 21(5), 530e537.
Franze, K., Francke, M., Günter, K., Christ, A. F., Körber, N., Reichenbach, A., et al. (2011).
Spatial mapping of the mechanical properties of the living retina using scanning force
microscopy. Soft Matter, 7(7), 3147e3154.
Franze, K., Gerdelmann, J., Weick, M., Betz, T., Pawlizak, S., Lakadamyali, M., et al. (2009).
Neurite branch retraction is caused by a threshold-dependent mechanical impact. Biophys-
ical Journal, 97(7), 1883e1890.
Franz, C. M., & Puech, P. H. (2008). Atomic force microscopy: a versatile tool for studying
cell morphology, adhesion and mechanics. Cellular and Molecular Bioengineering, 1(4),
289e300.
References 233
Gates, R. S., Reitsma, M. G., Kramar, J. A., & Pratt, J. R. (2011). Atomic force microscope
cantilever flexural stiffness calibration: toward a standard traceable method. Journal of
Research of the National Institute of Standards and Technology, 116, 703e727.
Gibson, C. T., Watson, G. S., & Myhra, S. (1997). Scanning force microscopyecalibrative
procedures for “best practice”. Scanning, 19, 564e581.
Goldsbury, C. S., Scheuring, S., & Kreplak, L. (2009). Introduction to atomic force micro-
scopy (AFM) in biology. Current Protocols in Protein Science (Chapter 17), Unit
17.7.1e17.7.19.
Grandbois, M., Beyer, M., Rief, M., Clausen-Schaumann, H., & Gaub, H. E. (1999). How
strong is a covalent bond? Science, 283, 1727e1730.
Hamill, O. P., & McBride, D. W., Jr. (1996). The pharmacology of mechanogated membrane
ion channels. Pharmacological Reviews, 48(2), 231e252.
Hardie, R. C., & Franze, K. (2012). Photomechanical responses in Drosophila photoreceptors.
Science, 338(6104), 260e263.
Harris, A. R., & Charras, G. T. (2011). Experimental validation of atomic force microscopy-
based cell elasticity measurements. Nanotechnology, 22(34), 345102.
Harris, A. R., Daeden, A., & Charras, G. T. (2014). Formation of adherens junctions leads to
the emergence of a tissue-level tension in epithelial monolayers. Journal of Cell Science,
127, 2507e2517.
Helenius, J., Heisenberg, C.-P., Gaub, H. E., & Muller, D. J. (2008). Single-cell force
spectroscopy. Journal of Cell Science, 121(Pt 11), 1785e1791.
Hertz, H. (1881). Über die Berührung fester elastischer Körper. Journal für die reine und
angewandte Mathematik, 92, 156e171.
Hinterdorfer, P., & Dufrêne, Y. F. (2006). Detection and localization of single molecular
recognition events using atomic force microscopy. Nature Methods, 3(5), 347e355.
Hutter, J. L., & Bechhoefer, J. (1993). Calibration of atomic-force microscope tips. Review of
Scientific Instruments, 64(7), 1868e1873.
Jean, C., Gravelle, P., Fournie, J.-J., & Laurent, G. (2011). Influence of stress on extracellular
matrix and integrin biology. Oncogene, 30(24), 2697e2706.
Kainz, B., Oprzeska-Zingrebe, E. A., & Herrera, J. L. (2014). Biomaterial and cellular prop-
erties as examined through atomic force microscopy, fluorescence optical microscopies
and spectroscopic techniques. Biotechnology Journal, 9(1), 51e60.
Kidoaki, S., & Matsuda, T. (2007). Shape-engineered fibroblasts: cell elasticity and actin
cytoskeletal features characterized by fluorescence and atomic force microscopy. Journal
of Biomedical Materials Research Part A, 81A(4), 803e810.
Krause, M., Te Riet, J., & Wolf, K. (2013). Probing the compressibility of tumor cell nuclei by
combined atomic force-confocal microscopy. Physical Biology, 10(6), 065002.
Kuo, C. H., Xian, J., Brenton, J. D., Franze, K., & Sivaniah, E. (2012). Complex stiffness
gradient substrates for studying mechanotactic cell migration. Advanced Materials,
24(45), 6059e6064.
Lamour, G., Journiac, N., Souès, S., Bonneau, S., Nassoy, P., & Hamraoui, A. (2009). Influ-
ence of surface energy distribution on neuritogenesis. Colloids and Surfaces B Bio-
interfaces, 72(2), 208e218.
Lee, S., Mandic, J., & Van Vliet, K. J. (2007). Chemomechanical mapping of ligand-receptor
binding kinetics on cells. Proceedings of the National Academy of Sciences of the United
States of America, 104(23), 9609e9614.
Lin, D. C., Dimitriadis, E. K., & Horkay, F. (2007). Robust strategies for automated AFM
force curve analysisdI. Non-adhesive indentation of soft, inhomogeneous materials.
Journal of Biomechanical Engineering, 129, 430.
Lu, Y. B., Franze, K., Seifert, G., Steinhauser, C., Kirchhoff, F., Wolburg, H., et al. (2006).
Viscoelastic properties of individual glial cells and neurons in the CNS. Proceedings of
the National Academy of Sciences of the United States of America, 103(47), 17759e17764.
MacGregor, D. G., Chesler, M., & Rice, M. E. (2001). HEPES prevents edema in rat brain
slices. Neuroscience Letters, 303(3), 141e144.
Mahaffy, R. E., Park, S., Gerde, E., Kas, J., & Shih, C. K. (2004). Quantitative analysis of the
viscoelastic properties of thin regions of fibroblasts using atomic force microscopy. Bio-
physical Journal, 86(3), 1777e1793.
Mahaffy, R. E., Shih, C. K., MacKintosh, F. C., & Kas, J. (2000). Scanning probe-based fre-
quency-dependent microrheology of polymer gels and biological cells. Physical Review
Letters, 85(4), 880e883.
Mangold, S., Harneit, K., Rohwerder, T., Claus, G., & Sand, W. (2008). Novel combination of
atomic force microscopy and epifluorescence microscopy for visualization of leaching
bacteria on pyrite. Applied and Environmental Microbiology, 74(2), 410e415.
Miri, A. K., Heris, H. K., Mongeau, L., & Javid, F. (2014). Nanoscale viscoelasticity of extra-
cellular matrix proteins in soft tissues: a multiscale approach. Journal of the Mechanical
Behavior of Biomedical Materials, 30, 196e204.
Mitra, P., & Brownstone, R. M. (2012). An in vitro spinal cord slice preparation for recording
from lumbar motoneurons of the adult mouse. Journal of Neurophysiology, 107(2),
728e741.
Moeendarbary, E., Valon, L., Fritzsche, M., Harris, A. R., Moulding, D. A., Thrasher, A. J.,
et al. (2013). The cytoplasm of living cells behaves as a poroelastic material. Nature Ma-
terials, 12(3), 253e261.
Morris, V. J., Kirby, A. R., Gunning, A. P., & World, S. (1999). Atomic force microscopy for
biologists. London: Imperial College Press.
Mosbacher, J., Langer, M., Hörber, J. K., & Sachs, F. (1998). Voltage-dependent membrane
displacements measured by atomic force microscopy. Journal of General Physiology,
111(1), 65e74.
Moyer, J. R., Jr., & Brown, T. H. (1998). Methods for whole-cell recording from visually pre-
selected neurons of perirhinal cortex in brain slices from young and aging rats. Journal of
Neuroscience Methods, 86(1), 35e54.
Moy, V. T., Florin, E. L., & Gaub, H. E. (1994). Intermolecular forces and energies between
ligands and receptors. Science, 266(5183), 257e259.
Müller, D. J., & Dufrene, Y. F. (2008). Atomic force microscopy as a multifunctional molec-
ular toolbox in nanobiotechnology. Nature Nanotechnology, 3(5), 261e269.
Müller, D. J., & Engel, A. (2007). Atomic force microscopy and spectroscopy of native mem-
brane proteins. Nature Protocols, 2(9), 2191e2197.
Nemir, S., & West, J. L. (2010). Synthetic materials in the study of cell response to substrate
rigidity. Annals of Biomedical Engineering, 38(1), 2e20.
Noy, A. (2011). Force spectroscopy 101: how to design, perform, and analyze an AFM-based
single molecule force spectroscopy experiment. Current Opinion in Chemical Biology,
15(5), 710e718.
Noy, A., Vezenov, D. V., & Lieber, C. M. (1997). Chemical force microscopy. Annual Review
of Materials Science, 27(1), 381e421.
References 235
Pagliara, S., Franze, K., McClain, C. R., Wylde, G. W., Fisher, C. L., Franklin, R. J., et al.
(2014). Auxetic nuclei in embryonic stem cells exiting pluripotency. Nature Materials,
13(6), 638e644.
Park, S., Costa, K. D., Ateshian, G. A., & Hong, K.-S. (2009). Mechanical properties of
bovine articular cartilage under microscale indentation loading from atomic force
microscopy. Proceedings of the Institution of Mechanical Engineers H, 223(3), 339e347.
Prass, M., Jacobson, K., Mogilner, A., & Radmacher, M. (2006). Direct measurement of the
lamellipodial protrusive force in a migrating cell. Journal of Cell Biology, 174(6), 767e772.
Puchner, E. M., & Gaub, H. E. (2009). Force and function: probing proteins with AFM-based
force spectroscopy. Current Opinion in Structural Biology, 19(5), 605e614.
Puech, P.-H., Poole, K., Knebel, D., & Muller, D. J. (2006). A new technical approach to quan-
tify cell-cell adhesion forces by AFM. Ultramicroscopy, 106(8e9), 637e644.
Richerson, G. B., & Messer, C. (1995). Effect of composition of experimental solutions on
neuronal survival during rat brain slicing. Experimental Neurology, 131(1), 133e143.
Rief, M., Gautel, M., Oesterhelt, F., Fernandez, J. M., & Gaub, H. E. (1997). Reversible
unfolding of individual titin immunoglobulin domains by AFM. Science, 276(5315),
1109e1112.
Samani, A., Zubovits, J., & Plewes, D. (2007). Elastic moduli of normal and pathological hu-
man breast tissues: an inversion-technique-based investigation of 169 samples. Physics in
Medicine and Biology, 52(6), 1565e1576.
Stewart, M. P., Hodel, A. W., Spielhofer, A., Cattin, C. J., Müller, D. J., & Helenius, J. (2013).
Wedged AFM-cantilevers for parallel plate cell mechanics. Methods, 60(2), 186e194.
Suzuki, Y., Sakai, N., Yoshida, A., Uekusa, Y., Yagi, A., Imaoka, Y., et al. (2013). High-speed
atomic force microscopy combined with inverted optical microscopy for studying cellular
events. Scientific Reports, 3, 2131.
Tétrault, S., Chever, O., Sik, A., & Amzica, F. (2008). Opening of the bloodebrain barrier
during isoflurane anaesthesia. European Journal of Neuroscience, 28(7), 1330e1341.
Zhao, S., Ting, J. T., Atallah, H. E., Qiu, L., Tan, J., Gloss, B., et al. (2011). Cell type-specific
channelrhodopsin-2 transgenic mice for optogenetic dissection of neural circuitry
function. Nature Methods, 8(9), 745e752.

Gautier 2015 AFM in Cells Book

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Gautier 2015 AFM in Cells Book

Uploaded by

Copyright:

Available Formats

CHAPTER

Atomic force microscopy-

Methods in Cell Biology, Volume 125, ISSN 0091-679X, http://dx.doi.org/10.1016/bs.mcb.2014.10.005 211

1.1 SETUP DESIGN

1.2 CANTILEVER CALIBRATION

(A) (B) (C)

FIGURE 2 Indentation experiments on tissues and cells.

3. To determine the cantilever spring constant k, several methods are available:

2.1 ANIMAL PRETREATMENT

2.2 PREPARATION OF MEASUREMENT BUFFERS

2.3 SAMPLE IMMOBILIZATION

3. AFM AND OPTICAL IMAGING

FIGURE 3 Combination of atomic force microscopy (AFM) and optical imaging.

Table 1 Basic Requirements for an Upright Optical/AFM Setup

fluorescently labeled cells (Moeendarbary et al., 2013). An alternative is to add a

4. MEASURING CELL AND TISSUE STIFFNESS

Table 2 Important Considerations and Common Problems When Using AFM in

4.1 IMPORTANT PARAMETERS FOR INDENTATION MEASUREMENTS

Table 3 Stiffness of Various Animal Tissues

4.2 ANALYSIS OF INDENTATION EXPERIMENTS

5.1 CHEMICAL FORCE MICROSCOPY

FIGURE 4 Adhesion measurements.

5.2 SINGLE-MOLECULE FORCE SPECTROSCOPY

5.3 SINGLE-CELL FORCE SPECTROSCOPY

Table 4 Key Steps for Adhesion Measurements Using AFM

the cantilever. For example, a sample may be exposed to a short-term mechanical

relevant scales, from individual cellular compartments through to tissue samples,

You might also like