Hawkes P.W., Spence J.C.H. (Eds.) Science of Microscopy. V.1 and 2
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1020 P. Sutter
Acknowledgments. The compilation of this chapter was supported
by the U.S. Department of Energy under Contract No. DE-AC02-
98CH10886.
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1025
16
Atomic Force Microscopy in the
Life Sciences
Matthias Amrein
He sees the face and the moving hands, even hears it ticking. If he is ingenious he may
form some picture of a mechanism, which could be responsible for all the things he
observes, but he may never be quite sure his picture is the only one that could explain
his observations. He will never be able to compare his picture with the real mechanism
and he cannot even imagine the possibility of the meaning of such a comparison.
Albert Einstein, describing a man trying to understand a closed watch.
1 Introduction
Cells are highly organized in compartments and functional units down
to the macromolecular level. The frameworks of these functional and
structural units are either protein complexes or complexes of proteins
with nucleic acids or with lipids. The recently completed map of the
human genome and the systematic mapping of the proteins expressed
in tissues and cells (proteomics) are part of a concerted effort to rapidly
advance the understanding of the functions of macromolecular units
and the cell. But proteomics reveals only the basic inventory of a cell and
the inventory is insuffi cient to explain the function of each element and
the orchestration of the components. As with Einstein’s image of the
closed watch, understanding life is inconceivable without observing the
structures behind function. Microscopy plays an important role by
placing the molecular elements into a structural context. Because the
pace of discovery of these elements has been increased substantially by
proteomics, the need for more sophisticated microscopy has also sub-
stantially increased in recent years. Atomic force microscopy (AFM)
plays a specifi c role in life science microscopy by allowing imaging to be
combined with locally probing functions of macromolecular elements.
Most microscopes depend on radiation, emitted and recorded at a dis-
tance from the sample, using lenses. The resolution power of these micro-
scopes in the life sciences is limited by diffraction and/or damage to the
sample by the illuminating beam. In contrast, the scanning tunneling
microscope (STM) has been the fi rst microscope to rely on an effect only
present in the immediate vicinity of a physical probe and the sample. In
an STM, an electrically conductive needle approaches the sample until
1026 M. Amrein
current fl ows. The current is based on a quantum-mechanical tunneling
effect and fl ows before the tip and sample physically touch. During scan-
ning, the tip–sample distance remains constant by keeping the tunnel-
ing current constant. The movement of the tip relative to the sample
refl ects the sample topography and is recorded. Not only has the atomic
surface lattice structure been revealed for many crystalline samples, but
also single atom defects imaged directly. The STM immediately created
great interest among biologists because of its outstanding resolution
power and the absence of radiation damage (Amrein et al., 1989, 1993;
(Baró et al., 1985; Voelker et al., 1988; Guckenberger et al., 1989;
Stemmer et al., 1989; Welland et al., 1989; Miles et al., 1990; Arscott and
Bloomfi eld, 1993; Lindsay and Tao, 1993; Miles and McMaster, 1993). But
the application of STM in the life sciences suffered from the poor electri-
cal conductivity of most biological matter, and reproducible images were
obtained only after metal coating the specimens (Travaglini et al., 1987;
Zasadzinski et al., 1988; Blackford and Jericho, 1991; Amrein et al., 1988,
1989b, 1993). Nevertheless, early applications demonstrated the resolu-
tion power and high signal-to-noise ratio at the macromolecular level
offered by the new microscopy. Although unintentional at fi rst, it also
became clear that the probe could be used to manipulate macromolecu-
lar structures individually (Figure 16–1).
The basic principles of the STM were soon extended to form a suite
of new devices, the scanning probe microscopes (SPM). For all SPM, a
physical probe is scanned over the sample in nanometer distance at
most. Sample properties are mapped from a very small interaction
volume in the near fi eld of the sample and the probe. The meaning of
the term of near fi eld is different for the various types of SPM. In an
AFM, short-range forces between a tip and the sample take the role of
the tunneling current in the STM, thus making the new paradigm
applicable to electrically nonconductive samples. This is how AFM
gained ground in the life sciences.
The direct measurement of the position of the surface means that the
achieveable resolution is not limited by the environment—the AFM
lends itself well to imaging molecular and cellular specimens under
physiological conditions, in buffer. In many cases, the sample prepara-
Figure 16–1. STM topographies of Rec-DNA complex (left) and an aberrant bacteriophage capside
(right). In the upper right corner, a hole has been created applying a voltage pulse between the tip and
the sample. Both images immediately reveal the molecular arrangement of the respective complex
structure. (Left, from Amrein et al., 1988, reproduced with permission. Right, from Amrein et al., 1998b,
reproduced with permission.) (See color plate.)
Chapter 16 Atomic Force Microscopy in the Life Sciences 1027
tion is very straightforward, and at a basic level requires only the
immobilization of the sample to be imaged. In the case of adherent
cells, for instance, the sample can be imaged directly without any
special preparation. The local measurement at the sample surface also
allows direct quantitative measurement of dimensions and forces. In
addition to the topographic image that is built up by scanning the tip
over the surface, AFM can simultaneously probe the mechanical prop-
erties of the surface, by dynamically oscillating the cantilever that
supports the tip. Tip–sample adhesion can also be probed, and maps
of surface (visco)elasticity or adhesion built up. One application is to
coat the tip with appropriate molecules, such as antibodies, other pro-
teins, or sugars to generate maps of specifi c ligand–receptor binding
over the surface. The AFM is also well suited to combination with other
techniques—a combination with optical microscopy is often particu-
larly worthwhile for life science research. Other techniques, such as
electrochemistry or electrophysiology, can also be used simultane-
ously, and in situ probing of cell response to electrical, chemical, or
mechanical stimulation is possible.
The foundation of AFM in the life sciences has now been laid by a
profound understanding of the interactions between the tip and the
sample and the possibility to subtly control the interactions by choos-
ing an appropriate sensor and by adjusting the imaging environment
(Weisenhorn et al., 1992; Butt et al., 1995; Heuberger et al., 1996).
To appreciate the contribution of AFM to the life sciences, it is neces-
sary to consider it in relation to other microscopy techniques currently
in use. In the realm of single molecules, the motivation for using AFM
comes mainly from the high resolution achievable and the direct (real-
space) measurement of samples without coating. Most of the knowledge
so far gained by AFM in the life sciences comes from studying single
macromolecules or macromolecular complex structures. Biological
membranes, for example, can be imaged in their native state at a lateral
resolution of 0.5–1 nm and a vertical resolution of 0.1–0.2 nm. Conforma-
tional changes that are related to functions can be resolved to a similar
resolution. In the study of macromolecular structure, AFM competes
with transmission electron microscopy. In many cases, the outcome is
comparable and either microscope can be used with equal success. For
example, measuring the contour length of a plasmid DNA or determin-
ing the binding site of a protein to a DNA may be performed by either
microscope. However, solving the three-dimensional structure of mac-
romolecules or macromolecular complexes that cannot be solved by X-
ray crystallography or by nuclear magnetic resonance (NMR) is better
accomplished by (cryo)electron microscopy, because AFM reveals only
a topographical view of the structures. On the other hand, when a
molecular assembly is not highly defi ned structurally and individual
units differ from each other substantially, AFM may allow a specifi c
question to be answered in a straightforward manner, because of its
uniquely high signal-to-noise ratio at molecular dimensions.
The strength of AFM does not lie in imaging alone, but in the possi-
bility of combining microscopy with an experiment at the molecular
level. In a number of cases, molecular images have been obtained with
1028 M. Amrein
suffi cient resolution to individually recognize single macromolecules
and then address the molecules individually with the stylus of the
AFM (Figure 16–2).
Most prominently, single molecule force spectroscopy combined
with single molecule imaging has provided unprecedented possibili-
ties for analyzing intramolecular and intermolecular forces including
the study of the folding pathway of proteins. Probing the self-assembly
of macromolecular complexes and measuring the mechanical proper-
ties of macromolecular “springs” are other examples in which AFM
has made substantial contributions in the life sciences.
Imaging living cells with AFM is also performed in conjunction with
local probing of the sample. The images by themselves usually are used
only to obtain proper experimental control. The extreme fl exibilty of
the cell membrane means that the images often show a combination of
the cell topography with the mechanical stiffness of cytoskeletal fi bers
and vesicles below the cell surface. An important example for a mean-
ingful application of AFM in living cells is the measurement of the
constrained diffusion of elements of the plasma membrane. These mea-
surements have substantially contributed to establishing the nature of
lipid rafts. In other applications, the cellular response on defi ned
mechanical stress has been studied in conjunction with hearing or with
the myogenic effect (Figure 16–3).
Future applications and future instrument developments in life sci-
ences AFM are certain to capitalize even more strongly on performing
local spectroscopy or manipulation of samples at the nanoscopic scale
in addition to imaging. Such experiments will certainly extend the
possibilities of probing function directly in many ways. But future
AFM technology might also contribute to revealing the spatial relation-
ships of the macromolecules of cells and tissues. Today, immunohisto-
chemistry and immunocytochemistry are mostly in use to establish
these structural relationships in cells and tissues. This implies that
macromolecular identity is revealed only after labeling. However, the
number of different proteins and other macromolecules at work at any
time in a cell makes it inconceivable to extend this approach to deter-
mine even a substantial subset of all of its macromolecular relation-
Figure 16–2. The ATP synthase is a proton-driven molecular motor with a
stator (seen here) and a rotor. Simply counting the number of individual pro-
teins inside the ring structure has not been possible from electron micrographs.
(From Seelert et al., 2000, reprinted with permission.)
Chapter 16 Atomic Force Microscopy in the Life Sciences 1029
ships by current techniques. AFM might offer a means to overcome the
need for labeling. It has recently been demonstrated that thin sections
of fi xed and embedded tissue and cell samples make the cell’s interior
accessible to AFM. High-resolution cell images give a clear picture of
the cell’s ultrastructure, sometimes down to the molecular level.
Although AFM will not likely allow identifying macromolecules by
their shape, it could be combined with powerful local spectroscopy. It
is tempting to speculate that spectroscopy might then enable micro-
scopy to identify macromolecular specimens under the tip directly
(Figure 16–4).
Figure 16–3. Neuronal cell, cultured on an electronic chip. The chip is
designed such as to pick up an action potential of the cell. The image demon-
strates proper tracing of the cell surface. In a future application, an appropri-
ately designed stylus might be used as an additional electrode to excite or
record an action potential at any location of the cell body or a process of the
neuronal cell. (See color plate.)
Figure 16–4. AFM image of the block face surface (left) and TEM image of an
ultrathin section (right) from the nematode Caenorhabditis elegans embedded
in epoxy resin. Scale bars equal 500 nm. Arrows point to actin fi laments,
m, mitochondria; G, gut. (From Matsko and Mueller, 2004, reprinted with
permission.)