Methods in Cell Biology
VOLUME 68
Atomic Force Microscopy in Cell Biology
Series Editors
Leslie Wilson
Department of Biological Sciences
University of California, Santa Barbara
Santa Barbara, California
Paul Matsudaira
Whitehead Institute for Biomedical Research and
Department of Biology
Massachusetts Institute of Technology
Cambridge, Massachusetts
Methods in Cell Biology
Prepared under the Auspices of the American Society for Cell Biology
VOLUME 68
Atomic Force Microscopy in Cell Biology
Edited by
Bhanu P. Jena
Department of Physiology and Pharmacology
Wayne State University School of Medicine
Detroit, Michigan
J. K. Heinrich H ¨orber
Cell Biology and Biophysics Program
European Molecular Biology Laboratory
Heidelberg, Germany
Amsterdam Boston London New York Oxford Paris
San Diego San Francisco Singapore Sydney Tokyo
Paperback edition cover photo credit: The image is the surface topology
of the apical plasma membrane in live pancreatic acinar cell, depicting

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CONTENTS
Contributors xi
Preface xiii
1. Local Probe Techniques
J. K. Heinrich H
¨
orber
I. Introduction 1
II. Scanning Tunneling Microscopy 4
III. Atomic Force Microscopy 7
IV. Force Spectroscopy 13
V. Photonic Force Microscopy 21
References 30
2. The Atomic Force Microscope in the Study of Membrane Fusion
and Exocytosis
Bhanu P. Jena and Sang-Joon Cho
I. Introduction 33
II. Methods 35
III. AFM Studies on Live Cells 37
IV. Identification of New Plasma Membrane Structures Involved in Exocytosis 39
V. Future of AFM in the Study of Live Cells 47

IV. Cell Culture 109
V. Final Remarks 110
References 111
6. Molecular Recognition Studies Using the Atomic Force Microscope
Peter Hinterdorfer
I. Introduction 115
II. Experimental Approach 117
III. Dynamic Force Spectroscopy 124
IV. Recognition Imaging 133
References 137
7. The Biophysics of Sensory Cells of the Inner Ear Examined by Atomic
Force Microscopy and Patch Clamp
Matthias G. Langer and Assen Koitschev
I. Introduction 142
II. Morphology and Function of Cochlear Hair Cells 143
III. AFM Technology 147
IV. Applications 155
V. Discussion 165
VI. Outlook 166
References 167
8. Biotechnological Applications of Atomic Force Microscopy
Guillaume Charras, Petri Lehenkari, and Mike Horton
I. Introduction 172
II. Methods 174
Contents vii
III. Analysis 178
IV. Application Examples 182
V. Future Directions and Improvements 187
References 190
9. Cellular Membranes Studied by Photonic Force Microscopy

References 254
viii Contents
13. Conformations, Flexibility, and Interactions Observed on Individual
Membrane Proteins by Atomic Force Microscopy
Daniel J. M
¨
uller and Andreas Engel
I. Introduction 258
II. High-Resolution AFM Imaging 260
III. Identification of Membrane Proteins 264
IV. Observing the Oligomerization of Membrane Proteins 270
V. Unraveling the Conformational Variability of Membrane Proteins 272
VI. Comparing AFM Topographs to Atomic Models 275
VII. Conformational Changes of Native Membrane Proteins 278
VIII. Observing the Assembly of Membrane Proteins 285
IX. Detecting Intra- and Intermolecular Forces of Proteins 287
X. Conclusions and Perspectives 289
References 292
14. Single-Molecule Force Measurements
Aileen Chen and Vincent T. Moy
I. Introduction 301
II. Experimental Design 302
III. Applications 306
References 308
15. Forced Unfolding of Single Proteins
S. M. Altmann and P F. Lenne
I. Introduction 312
II. The Biological System 313
III. Forced Unfolding 317
IV. Analysis 321

Volumes in Series 409
This Page Intentionally Left Blank
CONTRIBUTORS
Numbers in parentheses indicate the pages on which authors’ contributions begin.
S. M. Altmann (311), Cell Biology and Biophysics Program, European Molecular
Biology Laboratory, D-69117 Heidelberg, Germany
Nathan Becker (213), Department of Physics, University of California, Santa Barbara,
Santa Barbara, California 93106
Martin Benoit (91), Center for Nanoscience, Ludwig-Maximilians-Universität
München, D-80799 Munchen, Germany
Guillaume Charras (171), Bone and Mineral Center, Department of Medicine, The
Rayne Institute, University College London, London WC1E 6JJ, United Kingdom
Aileen Chen (301), Department of Physiology and Biophysics, University of Miami
School of Medicine, Miami, Florida 33136
Sang-Joon Cho (33), Department of Physiology and Pharmacology, Wayne State Uni-
versity School of Medicine, Detroit, Michigan 48201
Daniel M. Czajkowsky (231), Department of Molecular Physiology and Biological
Physics, University of Virginia School of Medicine, Charlottesville, Virginia 22908
Andreas Engel (257), M. E. Müller Institute, Biocenter, University of Basel, CH-4056
Basel, Switzerland
Ernst-Ludwig Florin (193), Cell Biology and Biophysics Program, European Molecular
Biology Laboratory, D-69117 Heidelberg, Germany
Marie-C´ecile Giocondi (51), Center of Structural Biochemistry, French National
Institute for Health and Medical Research U414, 34090 Montpellier Cedex, France
Christian Le Grimellec (51), Center of Structural Biochemistry, French National
Institute for Health and Medical Research U414, 34090 Montpellier Cedex, France
Helen G. Hansma (213), Department of Physics, University of California, Santa
Barbara, Santa Barbara, California 93106
Peter Hinterdorfer (115), Institute for Biophysics, University of Linz, A-4040 Linz,
Austria

D-01097 Dresden, Germany
Emin Oroudjev (213), Department of Physics, University of California, Santa Barbara,
Santa Barbara, California 93106
Lia I. Pietrasanta (213), Department of Physics, University of California, Santa Barbara,
Santa Barbara, California 93106
Arnd Pralle (193), Cell Biology and Biophysics Program, European Molecular Biology
Laboratory, D-69117 Heidelberg, Germany
†
Manfred Radmacher (67), Drittes Physics Institute, Georg-August Universität, 37073
Göttingen, Germany
‡
Bruno Samor
`
ı (357), Department of Biochemistry, University of Bologna, 40126
Bologna, Italy
Zhifeng Shao (231, 243), Department of Molecular Physiology and Biological Physics,
University of Virginia School of Medicine, Charlottesville, Virg inia 22908
Sitong Sheng (243), Department of Molecular Physiology and Biological Physics, Uni-
versity of Virginia School of Medicine, Charlottesville, Virg inia 22908
John C. Sitko (213), Department of Physics, University of California, Santa Barbara,
Santa Barbara, California 93106
Mario B. Viani (213), Depar tment of Physics, University of California, Santa Barbara,
Santa Barbara, California 93106
Giampaolo Zuccheri (357), Department of Biochemistry, University of Bologna, 40126
Bologna, Italy
*
Present address: HNO-Klinik, D-72076 T¨ubingen, Germany
†
Present address: Department of Molecular Cell Biology, University of California, Berkeley, Berkeley,
California 94720

and introduce a force-measuring technique, which is the main theme of the third section.
The last section focuses on actual instrumental developments and new methods. The con-
tribution by Hinterdorfer describes how, at the AFM tip, ligands can be used to measure
specific interactions even on cell surfaces. Humphris and Miles developed a new type of
AFM which is able to measure forces in a dynamic way, whereas Chen and Moy describe
static force measurements with a conventional AFM. Altmann and Lenne invented a new
type of active stabilization for the AFM making force-clamp measurements, used for
protein unfolding studies, more accessible.
Recently, the photonic force microscope (PFM) was developed (see first chapter by
H¨orber) by combining the principles of AFM, confocal microscopes, and optical twee-
zers into a new nanotechnological tool. The advantage of the PFM is its capability
xiii
xiv Preface
of entering the force range from 50 pN down 1/10 pN. This allows imaging of very
soft membrane structures. Furthermore, the instrument provided new methods to study
molecular structures with the observation of the thermal movement of the small particles
used, e.g., the tip in an AFM. This became possible by using a new optical technique
to detect the three-dimensional position of the particle with respect to the trapping laser
focus, which allows imaging of three-dimensional networks as formed by the cytoskele-
ton with the position resolution determined by the instrument, which is actually about
1 nm. Thermal fluctuations of a particle also reflect all the influences of its environment.
In this way, the technique can be used to map surface potentials, to study mechanical
properties at the molecular level, and to measure viscosity. Pralle and Florin demonstrate
in the last chapter how the PFM can be used to examine the biophysical properties of
the plasma membrane in live cells.
In general, the book is designed to provide a working knowledge of the AFM and
its potential for use in cell biology studies. The strengths and limitations of the AFM
technique are discussed from a practical perspective. The book provides a wide range of
applications in cell biology, which by no means are exhaustive. The examples described
in the book will enable the reader to appreciate the power and scope of the AFM to study

velopments. Local probe techniques extend our sense of touching into the micro- and
nanoworld and in this way provide complementary new insight into these worlds with mi-
croscopic techniques. Furthermore, touching things is an essential prerequisite to manip-
ulating things, and the ability to feel and to manipulate single molecules and atoms
certainly marks another of these revolutionizing steps in our relation to the world we
live in.
Local probes are small objects, e.g., the very end of sharp tips, whose interactions
with a sample, or better, the surface of a sample, can be sensed at selected positions.
METHODS IN CELL BIOLOGY, VOL. 68
Copyright 2002, Elsevier Science (USA). All rights reserved.
0091-679X/02 $35.00
1
2 J. K. Heinrich H¨orber
Proximity to or contact with the sample is required for high spatial resolution. This, in
principle, is an old idea that appeared in literature from time to time, in context with
bringing a source of electromagnetic radiation into close contact with a sample (Synge,
1928; O’Keefe, 1956; Ash and Nicolls, 1972), yet found no resonance and therefore
was not pursued until recently. Nanoscale local probes require atomically stable tips
and high-precision manipulation devices. The latter, based on mechanical deformations
of spring-like structures by given forces—piezoelectric, mechanical, electrostatic, or
magnetic—to ensure continuous and reproducible displacements with precision down
to the picometer level, also require very good vibration isolation. The resolution that
can be achieved with local probes is mainly determined by the effective probe size, its
distance from the sample, and the distance dependence on the interaction between the
probes and the samples measured. The latter can be considered to create an effective
aperture by selecting a small feature of the overall geometry of the probe tip, which then
corresponds to the effective probe.
The first of these local probe instruments was the scanning tunneling microscope
(STM), which emerged during the early 1980s as a response to an issue in semicon-
ductor technology (Binnig et al., 1982). Inhomogeneities on the nanometer scale had

piezo-tube scanner is widely used to produce movements in all three directions easily and
consists of a thin-walled hard piezo-electric ceramic that is radially polarized. Electrodes
are attached to the internal and external faces of the tube. The external electrode is split
into quarters parallel to the axis as shown in Fig. 2. By applying a voltage between
the inner and all the outer electrodes, the tube expands or contracts and in this way
either moves a tip closer to a surface or retracts it from a surface, respectively. If the
voltage is applied just between the inner and one outer electrode, the tube will bend,
i.e., moving the tip along the surface, with a precision determined by both the noise of
the voltage source used and the overall mechanical stability. The disadvantage of these
piezo-tubes is that the tip is not scanned exactly parallel to the surface but is moved
on an arc, leading to an effect known as “eyeballing” when large scans are carried out.
Another problem of piezo-materials is the hysteresis, which like the arc motion must
be corrected by the electronic equipment controlling the movement by providing the
necessary voltage.
In the meantime, many other types of scanning probe microscopes using various
types of interactions have been developed and are too numerous to mention in this short
Fig. 2 Piezo-electric effect of quartz and the piezo-ceramic tube scanner with inner and segmented outer
electrodes used in scanning probe microscopes.
4 J. K. Heinrich H¨orber
introduction. I prefer, therefore, to name only one other: the scanning nearfield optical
microscope (SNOM), developed by Pohl et al. (1988), which is, as the name implies,
the near-field equivalent to the conventional optical microscope working in the farfield
of the radiation. The STM, on the other hand, can be seen as the nearfield equivalent
to the electron microscope. The optical microscope, like other types of microscopes
using radiation in the farfield range, is limited in its resolution by the wavelength of the
radiation. This limit, reported by Abbe in 1873, restricts the optimal resolution to several
hundred nanometers for using visible light. The only way of overcoming this limit is by
using nearfield effects observed within a wavelength from a radiation source. In high
resolution, the very small tip can be used again. The tip of a SNOM is, at least in many
instruments, a specially prepared end of an optical fiber, which acts as a light source. The

1. Local Probe Techniques 5
formation in the STM for biological samples. For example, the electron microscope can
produce a three-dimensional image of a helical structure, e.g., a bacteriophage tail. For
such experiments performed by my group, T5 tails were purified and adsorbed to glow
discharged indium tin oxide (ITO) surfaces in solution (Gu´enebaut et al., 1997). The
surface was washed with distilled water, which was removed partially by blotting, leaving
only a thin layer of aqueous solution. The STM used was a noncommercial “pocket-size”
type (Smith and Binnig, 1986), equipped not only with tungsten tips etched in KOH
by alternating currents but also with a patch-clamp amplifier allowing measurements
down to 0.5 pA with an equivalent noise current of 200 fA. Importantly, all the current
measurements were carried out in the picoampere range. The feedback circuit controlling
the movement of the tip in the z direction, which is the distance to the sample, was
equipped with a logarithmic amplifier to correct for the exponential behavior of the
current. However, the direct measurements of the current variation giving the constant
height images usually are not corrected for this exponential behavior. Therefore, the
logarithms of these images were extracted, before combining the left-to-right and right-
to-left scans, to produce a real-space representation of the specimen. Both scans can be
normalized by histogram equalization and, after combining the different scan directions,
they could be compared to the transmission electron microscope (TEM) reconstruction
of the phage tail structure. The time constant of the feedback used was the limiting factor
in the tip movement, and adding both right- and left-scan images significantly suppressed
the z feedback effect. With this setup, recording the feedback signal simultaneously with
the current signal is alsopossible. Inprinciple, thiscombined constantheight andconstant
current imaging mode increases the height resolution of the instrument, showing the fine
structure on the top of the phage tails as a constant height image (Fig. 3).
Fig. 3 STM image of the tail of the bacteriophage T5 prepared on an ITO surface. The scan size is
18 × 18 nm
2
. The picture was taken with a 30-pA current at a 120-mV tip voltage within a thin layer of water.
6 J. K. Heinrich H¨orber

tails freshly adsorbed on ITO-coated glass retained a thin (50- to 100-nm) film of water.
While imaging, the tip was immersed several tens of nanometers into this film; at these
distances, currents of 5–50 pA were observed. In this situation (Fig. 4), the electrons had
to cross a water layer of up to several tens of nanometers in addition to the molecular
structure, but still could provide a resolution of 3 nm. The exponential distance depen-
dence of the measured current decays faster in water than through the macromolecules,
leading to a positive contrast. Without hypothesizing on the nature of the electron transfer
mechanism across biological material and through water, the observation that the protein
structure has less resistance to the current than to the surrounding aqueous solution is
very interesting. This produces a positive image of the specimen, while cryo-TEM, based
on high-energy electron scattering by the specimen, produces a negative image. A pos-
sible explanation might be that as denser protein structures are more ordered low-energy
electrons do not scatter as frequently.
1. Local Probe Techniques 7
Fig. 4 Scaled schematic drawing of the imaging situation as determined by current/distance measurements.
The diameter of the tail is 11 nm and the tip surface distance while imaging is about 60–70 nm above the
surface. The water is kept by cooling the sample as a thin layer of 100–200 nm on top of the sample.
If the physical basis for the use of the STM on biological structures can be identified,
then the STM can become an important complement to TEM in structural studies, as
completely different preparation methods are used and the samples remain hydrated
under close to physiological conditions.
III. Atomic Force Microscopy
A. Combination with Optical Microscopy
It has been shown in many experiments that the AFM can be used to study biological
structures under physiological conditions. It is even possible for the AFM to both image
living cells (H¨aberle et al., 1991) and study dynamic processes at the plasma membrane,
although such experiments are quite difficult, as the AFM cantilever is by far much
more rigid than cellular membrane structures (Schneider et al., 1997; Jena and Cho
in this book). The preparation of cells and the parallel optical observation, which are
necessary for having standard biological controls for cell activities available, present

the endocytosis of the virus might have been due to a shadowing by the lever and the
imaging tip, which prevented the penetration of viruses into this area. On the other hand,
at about the time when the virus would be expected to enter the cell (a few minutes after
adding viruses according to estimates of diffusion times in the surrounding liquid) we
noticed a strong softening of the cells, which was always accompanied by the danger
of the tip easily penetrating the membrane and the images losing considerable contrast.
One might imagine that a virus only locally modifies the membrane to enable its entry
into the cell. However, from the fact that the dramatic softening of the cell membrane
is always observed when viruses are added, we conclude that the cell membrane as a
whole is affected by the penetration or adhesion of the viruses. It is known that 4 to
1. Local Probe Techniques 9
Fig. 6 Exocytotic process imaged by AFM 3 h after monkey kidney cells were infected by pox viruses. The
size of the structure seen is about 200 nm and similar to the size of viral particles.
6 h after infection the first viruses reproduced inside the cell and emerged from the cell
through the cell membrane. However, approximately 2.5 h after infection we observed
a series of processes occurring in our SFM images. Single clear protrusions became
visible and grew in size. The objects quickly disappeared and the original structures on
the cell surface were more or less restored. Such processes can occur several times in
the same area and last about 90 s for a small protrusion (about 20-nm lateral extent) and
up to 10 min for a larger one (cross section of about 100 nm). Each process proceeds
distinctly, apparently independently of the others, and is never observed with uninfected
cells and never prior to 2 h after infection.
The fact that the growing protrusions abruptly disappeared after a certain time led us
to believe that we observed an exocytodic process but not the virus release. First-progeny
viruses are known to appear 5–8 h after infection and they are clearly bigger than the
structures observed. It is alsoknown, however, that after2–3 h only the early stageof virus
reproduction is finished and the final virus assembly has just begun. Since the protrusions
are observed after this characteristic time span, we believe that they are related to the
exocytotic processes connected to the virus assembly. Significantly more than 6 h after
infection even more dramatic changes are seen in the cell membrane (Fig. 6). Large

mation is provided regarding cell membranes and their dynamics in various situations
during the life of the cell. To separate the elastic and topographic properties, additional
information is needed, which can be provided either by topographic data from electron
microscopy or by the use of AFM modulation techniques. The pipette–AFM concept
is very well suited for such modulation measurements, because, as mentioned earlier,
perturbation by the excitation of convection or waves in the solution are extremely small
compared to the normal situation in AFM measurements. Furthermore, the cells held
by a pipette are supposedly in a state much more comparable to the natural situation
than a cell adhering to a substrate. For a thorough analysis of a cell membrane elasticity
map, one would have to record pixel by pixel a complete frequency spectrum of the
cantilever response and derive image data from various frequency regimes. This would
require too much time for a highly dynamic system like a living cell. Nevertheless, we