STM and A F M of biolorganic
molecules and structures
Atsushi Ikai
Laboratory of Biodynamics, Tokyo Institute of Technology, Nagatsuta
Midoriku, Yokohama, 226 Japan
EIS;EVIER
Amsterdam-Lausanne-New York-Oxford Shannon-Tokyo
262
A. lkai/Surface Science Reports 26 (1996) 261 332
Contents
1. Introduction
2. Scanning tunneling microscopy
2.1. Fundamentals
2.1.1. Imaging principle
2.1.2. STM of non-conductive material
2.2. Small molecules
2.2.1. Liquid crystals and fullerenes
2.2.2. Aromatic molecules
2.2.3. Aliphatic hydrocarbons
2.2.4. Fatty alcohols
2.2.5. Fatty acids
2.2.6. More complex molecules
2.2.7. Larger specimens
2.3. Identification of functional groups and individual atoms
3. Atomic force microscopy
3.1. Fundamentals
3.1.1. Imaging principle
3.1.2. New frontiers
3.2. Instrumental
3.2.1. Tips and cantilevers
3.2.2. Substrates
3.2.3. Tip-sample interactions
3.3. Experimental results
3.3.1. Proteins
3.3.2. DNA
3.3.3. Lipid membranes
3.3.4. Viruses and cells
3.3.5. Miscellaneous subjects
3.4. Force measurements
3.4.1. Basic physical interactions
3.4.2. Inter- and intra-molecular forces
3.4.3. Chromosomal manipulations
4. Conclusion
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Acknowledgements
Note added in proof
References
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surface science
reports
ELSEVIER
Surface Science Reports 26 (1996) 261 332
STM and AFM of bio/organic molecules and structures
Atsushi Ikai *
Laboratory of Biodynamics, Tokyo Institute ~[ Technology, Nagatsuta Midoriku, Yokohama, 226 Japan
Manuscript received in final form 2 August 1996
Abstract
Applications of scanning tunneling and atomic force microscopes in bio/organic researches are reviewed with a special
emphasis on the types of researches that are expected to contribute to the creation of a new field of ~single molecule
biochemistry" in the near future. The reviewed articles within the scope as stated above actually include a fairly broad
spectrum of researches. It is, therefore, a hope of the author that this review will be useful to those who are considering
biological applications of the probe microscopy techniques but are not quite familiar with the types of experiments that
have been done in the past. In the section on scanning tunneling microscopy, identification of chemically distinct functional
groups by the difference in their tunneling properties will be discussed as a main focus because it is fundamental for
biochemists to identify molecules by their shapes and properties. In the section on atomic force microscopy, recent
progresses in the imaging techniques of proteins and DNAs are closely reviewed, and rapidly advancing technologies of
single molecule measurements and manipulation of nanometer sized structures are given extensive coverage because the
author considers that such new applications are extremely promising to open an entirely new field in biological sciences.
Keywm'ds: Scanning tunneling microscopy: Atomic force microscopy: Organic molecules: Biological applications:
Chemically functional groups: Single molecule force measurements: Single molecule biochemistry
1. I n t r o d u c t i o n
Scanning tunneling and atomic force microscopes (to be abbreviated as STM and AFM,
respectively) are the two most recent inventions that are making inroads in various disciplines of
biomedical researches. Since almost the beginning of their inventions by Binnig and Rohrer in 1982
for STM [1], and by Binnig et al. in 1986 for AFM [2], applications of these new technologies to
biomedical fields have been actively pursued by devoted researchers. A volume of collected work on
biological applications of STM and AFM [3] and a textbook of more fundamental aspects of STM
[4] and AFM [-5] are available. Many images of DNA, proteins, polysaccharides, membranes, and
even cells taken with STM have been published but the initial dream of imaging biomolecules,
* Tel.: 81-45-924-5828: Fax: 81-45-924-5806: e-mail: aikai/a bio.titech.ac.jp.
0167-5729,/96/$32.00 Copyright c~: 1996 Elsevier Science B.V. All rights reserved
SSDI 0 1 6 7 - 5 7 2 9 ( 9 5 ) 0 0 0 0 8 - 9
264
A. lkai/SmJace Science Reports 26 (1996) 261 332
especially DNAs, with their atomic resolutions as had been done on inorganic materials, quickly
waned as the difficulties of reproducing high quality images began to be recognized. Then came the
age of A F M which does not require the electrical conductivity on the part of the sample. AFM can
produce images of almost anything but so far with not enough resolution to convincingly prove its
usefulness in biomedical fields where various types of electron microscopes have been widely used to
image details of subcellular structures with confidence. At the molecular level, the X-ray crystal
analysis produces ever more astounding images of proteins at the ultimate resolution of single
atoms. A dilemma for the users of STM and AFM in biological researches has been, therefore, that
the former with a good potential resolution requires electrically conductive specimens and the latter
without such a requirement has a lower resolution on soft samples. Working on individual samples
in physiological buffer solutions with AFM should be a distinct advantage over electron microscopes, which are invariably operated in vacuum, or over the X-ray crystallography where the
measurement is restricted to an average over a macroscopic crystal lattice. But, at least at the present
moment, the resolution comes over anything else for the trained eyes of biologists. Currently
researchers are devoted to exploit the potential capabilities of STM and AFM to the fullest extent in
their respective fields. One of the most outstanding advantages of STM and AFM is that probes are
almost in touch with the sample and capable of measuring physical properties of the specimens in
real time, especially when they are soft and deformable, rather than imaging them from a distance. In
the future application of STM and AFM, this advantage will be exploited as their main feature,
particularly in biologically oriented works.
Reviews in this and related fields have been fairly frequently published and I will cite some of the
most informative ones among them for the biologically oriented readers [6-14]. The present review,
when compared with the previous ones, stresses on: (1) in the case of STM, identification of
chemically functional groups and atoms in bio/organic molecules, and (2) in the case of AFM,
measurements of mechanical parameters of soft biological materials. This is based on the author's
own assessment of how the STM and AFM would be accepted in biomedical fields in the near future.
2. Scanning tunneling microscopy
The STM is a precise way of imaging atomic arrangements on the surface of metals, semiconductors and, in selected cases, organic thin layers over a conductive substrate. A metallic tip senses the
local electronic density of states in terms of the intensity of the tunneling current when a certain bias
voltage is applied between the tip and sample. The space between the tip and the specimen could be
vacuum, gas, or liquid. In its application to the study of metal or semiconductor surfaces, STM is
most commonly operated in an ultra-high vacuum of 10 8Pa or below to avoid contamination of
primarily oxygen and organic materials present in the air. Since organic and biomolecules cannot be
cleaned by heating them up to 700 K or above as routinely done for imaging the surface of, say,
silicon crystals, imaging is u.sually done in air or in liquid. The substrate surface is cleaner under
liquid conditions than in air and this situation is more and more widely acknowledged by
researchers. The use of an electrochemical cell is a good example and imaging of molecules at
solid liquid interfaces is now being pursued with promising results. Creation of clean solid liquid
interfaces using an electrochemical cell has been successful but the choice of specimens and matching
substrates for successful imaging is still fairly narrow and the sample layers must be as thin as 0.5 nm
A. lkai/Sutface Scietzce Reports 26 ( 1996 ) 261 332
265
or even less. Examples will be given in the following sections. Another recent development in this
field is the use of a sub-pico ampere (sub-pA) STM to image thick specimens spread over a nonconductive mica surface, for example, 2 nm thick DNA, 20rim thick tobacco mosaic virus, to
mention a few examples (see below). The proposed imaging principle in this case is an electrical flow
through the water layer over the surface of both specimens and the substrate.
2.1. Fundamentals
2.1.I. hna,qin,q principle
Since the imaging principle of STM has been amply reviewed by several authors [6 12], only the
very basics will be given here for those who are not familiar with the technology. When two
conducting metal surfaces are in close proximity of the order of I nm or less and yet not in contact
with each other, and a certain voltage of the order of 1-2 V is applied between the two surfaces,
a tunneling current of the order of 1-2 nA flows between them. Such a current cannot be expected
based on the classical physics because the non-conductive potential barrier between the two surfaces
is much too high to overcome for electrons confined in the two metallic objects. But the quantum
mechanical description of the situation predicts that electrons can in fact flow through the potential
barrier with a non-zero probability. This is called the tunneling probability, and the magnitude of the
tunneling current, i, can be calculated according to the following formula [15]:
i --=0.1R2exp(2R/)+)p(ro; Ev) V,
(1)
where p(r0; E v) = Z,.I tP,.(ro)lZb(E,- Ev); and symbols are: ,i = h/x/2mdp; R the radius of curvature at
the tip of the probe; m the electron rest mass; q5 the work function of the sample; h Planck+s constant
divided by 2?+;p(rt+;E v) the local density of states of the sample at the center of the radius of curvature
of the tip: V the applied bias voltage. Since }~,.(r0)l2 is nearly equal to e x p [ - 2 ( z + R)/2)], where
z represents the distance between the tip apex and the sample surface, the tunneling current i can be
simply approximated as
i--- exp(-2z/]~).
(2)
Thus the tunneling current is sensitively dependent on the distance z between the two conducting
surfaces, and therefore one can calculate the distance by measuring the magnitude of tunneling
current assuming everything else is equal. One surface in STM measurement is that of an atomically
sharp metal tip and the other is the surface of electrically conductive samples. The first workable
STM was introduced by Binnig et al. in 1982 [1] and it was an enormous success in the field of
surface science. One of the earliest successes of the technique was the demonstration of the atomic
arrangement on the so-called reconstructed surface of silicon crystal as was proposed by Takayanagi
et al. [ 16].
2.1.2. S TM of non-conductive material
Following a remarkable success of imaging DNA with STM by Driscoll et al. [17] many reports
have been published on the imaging of biological macromolecules using STM, including nucleic
acids, proteins, polysaccharides, lipid membranes, and even cells, though the imaging principle
remained obscure from a physical point of view. Earlier data on non-conductive macromolecules
turned out to be not quite reproducible, and, in some cases, could be attributed to artifacts on a not
266
A. Ikai/Sur[ace Science Reports 26 (1996) 261 332
well-characterized surface of graphite (HOPG). In fact, Clemmer and Beebe [18] showed that
a freshly cleaved graphite surface often presented various nanometer sized structures in the absence
of intentionally placed samples, and some of such structures looked like a double helical DNA. After
artifactual images on the graphite surface became known, STM images of non-conductive materials
on H O P G have become to be regarded with reservations. Although thick biological macromolecules are now recognized as difficult specimens for STM measurements, smaller molecules such
as hydrocarbons, fatty alcohols, fatty acids, aromatic molecules, liquid crystal molecules, etc. are
now routinely imaged with STM in vacuum or at solid liquid interfaces.
2.2. Small molecules
2.2.1. Liquid crystals and fullerenes
As an introduction to the STM of organic molecules, results on liquid crystalline materials and
fullerenes will be given as examples of adsorbed layers of carbon compounds on conducting solid
surfaces. Smith et al. [19] demonstrated that they could image 4-n-octyl-4'-cyanobiphenyl (8CB)
molecules on H O P G in 1990. The liquid crystal molecule is not electrically conductive in the bulk
state. Crystalline cyanobiphenyl was placed on H O P G and warmed above the transition temperature to the liquid crystalline state and imaged with STM. A regular array of long chain molecules was
recognized and molecules were individually distinguished with two intramolecular domains, one
with a higher (brighter in image) and the other with a lower (dimmer in image) tunneling probabilities.
The former was assigned as corresponding to a series of aromatic rings and the latter to the aliphatic
group. Such a pattern has since been repeatedly observed for several different liquid crystals and
organic molecules. Nejoh [20] tried to explain why molecules with a band gap of 11 eV or so between
H O M O (for the highest occupied molecular orbital) and L U M O (for the lowest unoccupied
molecular orbital) could be imaged with a bias voltage as low as 0.8 V and suggested that the
interaction between the substrate and the liquid crystalline molecule might have caused a shift in
energy levels, or the layered molecules formed new energy bands. Molecular orbital calculations were
done on various arbitrary fragments of 8CB molecule. The resulting tendency was that the presence
of graphite carbons at 0.154 nm (a very short distance) from the aromatic rings of cyanobiphenyl
group would have caused some noticeable narrowing of the gap between its H O M O and LU MO.
H6rber et al. [21] studied the dependence of STM image of 10-alkylcyanobiphenyl on the
magnitude of bias voltage and found that the aromatic and aliphatic parts of the molecule alternated
in their tunneling characteristics as follows. When the bias voltage was less than 0.5 V, the tunneling
current of the aromatic part of the molecule had an exponential dependence on the applied voltage
and the aliphatic part had more or less a linear dependence. When the applied voltage was raised
higher than 0.5 V, the reverse situation resulted but the aromatic part remained topographically higher.
They also eliminated the possibility that the images of non-conducting organic liquid crystals was due
to a lateral conductivity over the molecule by checking the absence of a current between the two tips.
Hara and his colleagues [22,23] studied n-alkylcyanobiphenyls on another popular substrate,
MoS2, and studied the alignment of the molecules in a 2D array. Their study was aimed at an elucidation
of the basis for an exact arrangement of molecules with respect to each other and to the crystal lattice
of the substrate. The STM images obtained for the alkylcyanobiphenyls were interpreted in terms of
the differential tunneling properties of the aromatic and aliphatic parts of the molecule.
,4. lkai/SmJ~we Science Reports 26 (1996) 261 332
267
An exceptionally clear image of an organic molecule called MDW 74 was obtained by Walba et al.
[24] as reproduced in Fig. 1 and an interpretation of the image based on the molecular orbitals of an
isolated molecule was undertaken. A conclusion obtained from the comparison of the molecular
orbital calculation and the STM image was that no single molecular orbital, be it H O M O or
LUMO. could explain the observed image. Superposition of several molecular orbitals in the
Fig. 1. Thc superposition of the lowest four unoccupied orbitals of MDW 74 (top) and the raw STM image of arrayed
structure of MDW 74 on HOPG (bottom). The molecular structure and two possible arrangements of the molecules are
given at the bottom (after Walba et al. [24]; reproduced with permission of authors and publisher, copyright [1995]
American Association for the Advancement of Science).
268
A. lkai/Sur[~tce Science Reports 26 (1996) 261 332
Bright Column
] Orientation A i (_
..,h,.
o
Row-
"~
-- °'~--/7~ o
........£>.~o/~_~ o~
o_0_o
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°
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. Row
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Bright Column
Fig. 1 (continued).
neighborhood of H O M O and L U M O was suggested to represent the STM image of the molecule on
H O P G . A comprehensive calculation of the molecular orbitals of an adsorbed molecule on H O P G
is awaited for a quantitative explanation of the imaging principle of non-conductive molecules on
conductive surfaces.
A successful explanation of STM images based on the molecular orbital calculation for a combined sample-substrate system was carried out for fullerene molecules adsorbed on a crystalline
silicon surface [-25,26]. A strong interaction between the substrate and adsorbate had been
experimentally indicated when the STM image of fullerene molecule was shown to have a two-fold
symmetry characteristic to the dimer rows of the substrate rather than of its own.
2.2.2. Aromatic molecules
The benzene molecule was the first among aromatic molecules to be imaged at a submolecular
resolution. It was evaporated in vacuum and adsorbed on a clean metal surface, and imaged with
STM under high vacuum. Fig. 2 shows an example of such a study [27].
Benzene is composed of six equivalent atoms but in the STM images it is a collection of three
conductive spots. Such images were interpreted based on the known molecular orbital calculation of
benzene molecules.
Among the most popular molecules for imaging with STM are various forms of phthalocyanine
and porphyrin. A clear image of four-lobed copper phthalocyanine was reported by Gimzewski et al.
[28] in 1987 and Lippel et al. [29] in 1989. Both metal free and metal associated porphyrins have
been clearly imaged with STM in liquid. Imaging is especially clean and clear under electrochemical
,4. lkai/Surl~lce Science Rem~rts 26 ( 1996 ) 261 332
269
Fig. 2. STM image of benzene molecules emphasizing the ring appearance (after Ohtani et al. [27]: reproduced with
permission of authors and publisher).
STM conditions with porphyrin molecules adsorbed on a clean gold surface [30]. ltaya pioneered in
an electrochemical STM and showed that it was possible to image an atomically flat, clean gold
(1 1 1) surface with STM in electrolyte solutions. The STM image of porphyrin from this group has
clearly distinguishable four lobes presumably corresponding to four pyrrole rings (Fig. 3).
An advantage of using electrochemical STM seems to be that the adsorption condition can be
controlled by changing the voltage applied to the substrate, and its surface can be kept as clean as in
high vacuum. Imaging of biologically important molecules that readily dissolve in aqueous
electrolyte solutions should be tried with an electrochemical STM for the elucidation of their
electronic states under conditions with varying biological activities.
2.2.3. Aliphatic hydrocarhons
It was a surprise that long chain aliphatic hydrocarbons, which are non-conductive to electricity
and less likely to be imaged with STM than aromatic molecules, were imaged with STM at a quasiatomic resolution both in vacuum [31] and in liquid [32]. In ultra-high vacuum, C>H,,z was heat
evaporated and deposited on H O P G [31]. The subsequent imaging with STM revealed a highly
regular 2D array of the molecules which is reproduced in Fig. 4.
Taki ct al. collected an assortment of STM images from various fields of a given sample and
interpreted them as representing different stages of the molecular arrangement that preceded the
formation of the regular array as given in Fig. 4. Hydrocarbon molecules are aligned side by side and
formed wide bands of 4.3 nm in width, exactly matching the length of a C>H,,, molecule. One can
270
A. lkai/Surface Science Reports 26 (1996) 261 332
-12.5
10.0
?.5
-5,0
"2,5
-0
0
2.5
5.0
7.5
10.0
12.5
rtM
(a)
-12.5
-10.0
-?,5
-5,0
o
u~_
2,5
o -]
0
(b)
FO
2 .5
~
5.'0
?.'5
10'.0
12'.5
nM
Fig. 3. Electrocheanically adsorbed porphyrin molecules in liquid condition (a) and its molecular structure (b) (afte~
Kunitake et al. [30]; reproduced with permission of authors and publisher).
A. lkai/Su@we Science Reports 26 (1996) 261 332
271
Iil
J
m
2rim
Fig. 4. STM image of C30H62 as interpreted in various stages of 2D array formation in vacuum. Numbers on each panel in
the figure are from the original article. Panel 11 represents a perfect 2D array (after Taki et al. [31]: reproduced with
permission of authors and publisher).
A. Ikai/SuJfate Science Reports 26 (1996) 261 332
272
:
.....
0.246
nm
:
i
Fig. 5. Top view of the surface lattice of HOPG with atomic distances. A and B site atoms are in different environments
with respect to the atoms in the underlying layer.
trace individual molecules from one end of the band to the other, and the molecules were nearly in
right angle to the edge of the band. Bands are separated by a narrow dim space of an approximate
width of 0.4 nm. It is likely that the hydrocarbon chains were epitaxially adsorbed on the H O P G
surface because the distance between every other methylene group (0.24 nm) is approximately equal
to one of the lattice distances (0.246 nm) of H O P G as depicted in Fig. 5. It is clear from the image that
the methylene groups that were precisely overlapped on the lattice structure of H O P G are brightly
imaged. The epitaxial interaction with the substrate is apparent since the methylene groups that are
brightly imaged in the figure form continuous linear patterns between the molecules in side by side
alignment to each other.
Very recently there appeared a paper by Venkataraman et al. [33] on triacontane (C30H62)/
triacontanol molecules dissolved in phenyloctane and adsorbed on HOPG. They changed the
mixing ratio of the two chemicals and observed phase separation phenomena on HOPG, and STM
images were obtained with a bias voltage in the range of 1.2-1.5 V and the tunneling current of
60-120pA. Both alcohol and alkane molecules formed band patterns on the graphite but the
alignment of individual molecules in such bands was different. Alcohol molecules had an angle of
about 60 'Dagainst the edge of the band whereas the alkane molecules were at right angles against the
edge (Fig. 6). The STM image of triacontanol was brighter on its two molecular ends but no
interpretation was given in terms of the differential tunneling properties of functional groups. When
the mixing ratio of the two compounds was changed, alcohol was preferentially adsorbed on the
H O P G surface over the pure alkane.
2.2.4. Fatty alcohols
One of the early examples of investigation of fatty alcohols was reported for dodecanol on H O P G
[34]. Unlike pure hydrocarbons, long chain alcohols, acids and other hydrocarbon derivatives are
difficult to evaporate in vacuum without causing heat degradation; therefore an adsorption method
to the liquid-solid interface at an ambient temperature was cultivated. When crystalline dodecanol
was placed on H O P G and heated from 6 ° to 36 ~' above the bulk melting temperature of 297 K,
A. 1kai/Sutface Science Reports 26 (1996) 261 332
273
a presumably monolayer of straight chain molecules was formed where molecules were arranged
side by side, and one could identify individual methylene groups forming a zigzag backbone of the
molecule. It is not possible to identify hydroxyl groups in the image as a distinct chemical group.
Octadecanol, tetracosanol, triacontanol were imaged in adsorbed states on H O P G from
phenyloctane solutions showing well-ordered arrangements of elongated molecules forming rows of
bands with width comparable to the length of the molecules [35]. The hydrocarbon parts of the
molecule were imaged as conductive to tunneling current but identification of hydroxyl group as
a differentially conducting part of the molecule was not possible. It is most likely that the hydroxyl
group was less readily conducting toward the tunneling current and thus immersed in the interband
gap in the image.
2.2.5. Fatty acids
Long chain fatty acids were taken up for STM imaging by Rabe and Buchholz [35]~ Kishi et al.
[36], Hibino et al. [37], and Yoshimura et al. [38]. Results were similar to those for fatty alcohols in
Fig. 6. Alignment of triacontanol (a) and triacontane (b) on H O P G (after Venkataraman et al. [33]: reproduced with
permission of authors and publisher).
274
A. lkai/SmT[ace Science Reports 26 (1996 ) 261 332
Fig. 6 (continued).
the sense that only the hydrocarbon backbones were visible and no indication of the carboxyl group
as a differentially conducting part in the molecule. Rabe recognized a dynamic change of molecular
arrangement within a very short time scale. Hibino et al. [37] reported that when they imaged
arachidic acid and elaidic acid both dissolved in phenyloctane and placed on H O P G , they clearly
identified a lone double bond in the central part of elaidic acid as the most readily tunneling part of
the molecule (Fig. 7 and [37]). Although the interest of the authors was more focused on the
molecular arrangement and the mechanisms that made such neat arrays possible, the result was
encouraging from the chemical point of view that convincingly a way was shown to use the STM in
the identification of chemically functional groups in organic molecules.
In the study by Hibino et al. [37], long chain fatty acids from myristic acid to behenic acid were
dissolved separately in phenyloctane and dropped on the surface of HOPG. The STM images of long
chain fatty acids were composed of more readily conducting bands with a width corresponding to the
length of the hydrocarbon chain of the molecule and much narrower interband gaps less conductive to
the current. It is also an interesting subject to investigate the dynamics and equilibrium alignment of
these rod-like molecules on the surface of HOPG, but details are out of the scope of this article.
,4. lkai/SuJ;[~lce Science Reports 26 (1996) 261 332
275
(a)
(b)
Fig. 7. STM image of arachidic acid (a), and elaidic acid (b) in 2D arrays on HOPG, with a bright spot in the middle of the
latter molecule corresponding to a lone double bond in the molecule {after Hibino et al. [37]: reproduced with permission
of authors and publisher).
276
A. lkai/Surface Science Reports 26 (1996) 261 332
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278
A. lkai/Surface Science Reports 26 (1996) 261 332
2.2.6. More complex molecules
Yoshimura et al. [38] studied the STM images of stearic acid and its derivatives, i.e. stearoyl
alcohol, amide and anilide, together with cholesteryl stearate. They found that, whereas the acid and
alcohol were imaged with a more or less uniform tunneling probability along the molecular axis, the
amide and anilide derivatives showed distinct bright spots against the aliphatic backbones,
respectively, corresponding to the size ofamide and anilide groups. Cholesteryl stearate is more than
twice as thick as stearic acid and the obtained STM image was not clear but represented a rough
shape of the molecule in geometrically interesting relation to each other. Takeuchi et al. [39]
confirmed and extended the results of Yoshimura et al. [38] on the amide and anilide showing very
clear inv,~ges of the functional groups in the molecule as shown in Fig. 8.
K ~l\~li and his colleagues [40,41] have been successful in imaging adenine, guanine, cytosine, and
thymine, the four bases in DNA using vacuum deposition on a silicon surface and scanning with an
ultra-high vacuum STM. Bases were observed to form clusters on a crystalline surface of silicon.
Identification of purines and pyrimidines was claimed to be possible but further differentiation
between adenine and guanine, or thymine and cytosine was difficult. Simple molecular orbital
calculations were carried out for an identification of bases adsorbed on the surface.
Katsumata and Ide [42] imaged bases and their sulfur containing analogs in the adsorbed state to
a gold surface under aqueous conditions. Bases were found to form regular arrays on gold but there
was a difficulty in defining the territory corresponding to a single molecule.
Arrangements of organic molecules on crystalline surfaces were discussed by Frommer [43] in
terms of hydrogen bond formation, Van der Waals interactions, etc.
2.2.7. Larger specimens
Since STM images of biological macromolecules and cells that were reported in the past have
already been reviewed on various occasions and many of the results turned out to be not
reproducible enough to be used as practical and reliable experimental techniques, I will limit myself
to cite the most recent and new development of the use of STM on large scale specimens.
An interesting and promisi~g method of imaging thick organic materials such as tobacco mosaic
virus and DNA with sub-pA STM has been proposed by Guckenberger and his colleagues [44,45].
They started to study the dependence of the quality of DNA images taken with STM on the relative
humidity (RH) of the experimental conditions and found that a small tunneling current of less than
0.01 to 10pA could be detected on a normally non-conductive substrate such as mica. When
biological samples such as tobacco mosaic virus and DNA were placed on mica, dried in air and
scanned with STM under a relative humidity of about 65%, clear images such as the ones given in
Fig. 9 were obtained.
The bias voltage was from + 1 to _+8 V and the tunneling current level was between 0.1 and 0.5 pA.
They attributed their success in imaging to the presence of lateral conductivity through a thin layer
of water that was supposed to continuously cover the surface of the sample and substrate. According
to their calculation, the surface water layer had a more than five orders higher electrical conductivity
than that of the bulk water, which is a very interesting finding. DNA has also been shown to be
imaged with STM in water under electrochemically deposited state on a gold surface [46,47].
STM can be useful to image metal coated specimens prepared for the shadowing experiment of
transmission electron microscopy (TEM). For example, DNA and protein complexes were imaged
with STM after metal coating and the result is reproduced in Fig. 10 from [48]. The advantage of
Fig. 9. Sub-pA STM imaging applied to DNA molecules on mica (after Guckenberger et al. [45]:. reproduced witl~
permission of authors and publisher, copyright [1994] American Association for the Adwmcemcnt of Sciencel.
Fig. 10. DNA protein complex coated with metal and imaged with STM (after Mfiller et al. [48]~ reproduced witt
permission of authors and publisher).
A. lkai/Sm;liwe Science Reports 26 (1996) 261 332
281
using STM in this case is in the measurement of relative height of the various parts of the specimen
with respect to the open surface, since the height measurement in TEM is difficult and unreliable.
An interesting example of the application of STM was made on the structural study of petroleum
asphaltenes in combination with an N M R study [49]. The multi-ring aromatic nature of asphaltene
substance was imaged but resolution of individual rings was not exactly attained. Applications of
STM and AFM in such areas where molecular level researches have been difficult due to a lack of
suitable methods to characterize individually different components will be strongly encouraged if
cautions are taken to validate the data from multidisciplinary points of view.
2.3. htentilication olilimctional groups and imtividual atoms
From the above examples where aromatic, methylene, ethylenic, amide, anilide groups were
imaged with STM as distinct substructures within larger molecules, it has become clear that there is
a definite possibility of identifying chemically functional groups within a single organic molecule
based on their differential tunneling properties and indeed such attempts have recently been
discussed by Venkataraman et al. [33]. First, they observed in their STM images of fatty alcohol, thiol,
disulfide, and chloro compounds that SH and S S were imaged as readily conducting parts in the
molecule whereas hydroxyl and chloro substitutions were less conductive than the aliphatic backbones. The authors attributed the observed differences among chemically functional groups to the
relative magnitude of atomic polarizabilities of carbon, oxygen, sulfur, chlorine, and hydrogen. The
rationale behind invoking polarizability is that the surface work function which is one of the main
factors to determine the tunneling probability is influenced by the polarizability of adsorbates [50].
Tentative conclusions that can be deduced from the cited works above on the possibility of
differentiating chemically functional groups in organic molecules with STM are as follows:
(1) Saturated hydrocarbon backbones of many organic molecules can be imaged on HO PG as well
as on MoS2, but the imaging principle is not clear yet.
(2) Carbon-~carbon double bonds, aromatic rings, amide, thiol, disulfide groups are more readily
conductive to tunneling current and imaged brighter than saturated aliphatic backbones.
(3t Carboxyl, hydroxyl chloro groups are less conductive to tunneling current compared with the
saturated aliphatic backbone.
(4) The contents of the tentative conclusion given above are likely to be modified when more details
become known on the dependence of molecular images on the sign and magnitude of applied
bias voltage.
(5) Molecular orbital calculations for a combined system of the sample and substrate should be
carried out and a guiding principle for the interpretation of STM images of organic compounds
in terms of a limited number of molecular orbitals must be established. For molecules with
clearly different shapes of H O M O and L U M O , a sample negative bias is expected to give an
image resembling H O M O , and a positive bias likely to give one similar to LUMO.
(6) Difficulties in reliable imaging of organic compounds and limitations on the choice of suitable
samples either in vacuum or in liquid state STM are still enormous~ and consequently no precise
measurements of STS (for scanning tunneling spectroscopy) have been done on organic
compounds, These difficulties should be overcome by devising reliable and less tedious methods
to image organic molecules with STM.
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I did not cite many excellent reports on the behavior of simple organic molecules adsorbed on
solid catalysts, simply because they are beyond the scope of this article. In some of the work, the
carboxyl group of formic acid is brightly imaged in contrast with the result on H O P G cited above. It
will therefore be interesting to see how the work done in different disciplines and with different
purposes can be understood in a broader perspective. STM will undoubtedly be a very versatile
method that will open a new field in biologically oriented researches.
3. Atomic force microscopy
The atomic force microscopy applied to biological molecules and systems has been reviewed by
many authors with usually special emphasis on the principle, instrumental innovation, and the
imaging accomplishments within the previous few years [6-12].
3.1. Fundamentals
3.1.1. Imagin9 principle
The A F M as mentioned briefly in Section 1 does not require the sample to be electrically
conductive, thus enabling the atomic and molecular level of studies on biological structures with
a hitherto unprecedented clarity. A F M has an either triangular or rectangular cantilever equipped
with a small stylus, more often called a tip, extending perpendicularly from the free end of the
cantilever. The tip apex has a radius of curvature in the range of 10-100 nm. In a modern commercial
instrument, the tip cantilever unit is securely attached to the cantilever holder of the AFM
and stays stationary during the AFM operation. Samples are usually adsorbed on the atomically
flat surfaces of freshly cleaved mica, glass, H O P G , silicon wafer, gold-coated mica, etc. and securely
held on a small piece of magnetized disk with adhesive tape or removable glue. After the disk is
magnetically secured on the metallic top of the tube piezo motor/scanner, the motor raises the
sample holder until the sample surface comes into contact with the tip apex. A continued upward
movement of the sample stage will bend the cantilever slightly upward. The upward deflection
of the cantilever exerts a spring force on the sample surface. The back of the cantilever is made
mirror flat and a laser beam that shines down on the cantilever is reflected in the direction of
a split photodiode detector with either two or four faces. The two-face detector detects up and
down deflections of the reflected laser beam and sends a current proportional to the light intensity
to the upper and the lower diode to the feedback circuit. The feedback circuit tries to keep the
difference between the currents of the upper and lower diodes to a certain preset value keeping
a constant small force on the surface of the sample. This is the so-called, "repulsive" or "contact"
mode of A F M and the force maintained during the scanning is usually 1 10 nN depending on the
circumstances. This scanning force is necessary to trace the topography of the sample surfaces. One
of the problems in biological samples is that this force is still strong enough to cause their
deformations.
A recent development of AFM instrumentation has achieved a truly molecular resolution when
applied on hard crystalline surfaces [51-53] but the resolution on soft and deformable biological
samples remained at a similar stage as it was five years ago, i.e. 2 3 nm. It is true that the uncoated
DNA or tail fibers of T4 phage with known diameters of 2-3 nm can be routinely imaged but with
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a width substantially greater than the real width due to the tip-sample convolution effect. Other
factors such as attractive interactions between the tip and sample contribute to lower the resolution
as well.
It is now almost imperative to operate AFM in water not only because the natural environment
for biological samples is in aqueous media but also that various types of tip sample or tip substrate
interactions are reduced to insignificant levels compared with those in the air. Not only can
electrostatic interactions be made much smaller in aqueous salt solutions but also do the Van der
Waals interactions get smaller by a factor of 10 50, and the capillary force is practically non-existent
(see below). In some special cases of DNA imaging, propanol has been the choice of investigators.
3.1.2. Ne~v lJ'ontiers
When compared with the two most frequently used means of resolving the molecular structures of
biochemical interest, i.e. electron microscopy and X-ray crystallography, the lateral resolution of
present-day AFM, as mentioned above, is not very remarkable yet. Its vertical resolution, though
once claimed to be a definite advantage over other microscopic methods, is now seriously questioned
because of possible deformations of the sample under scanning force and other unclarified reasons
such as flattening of adsorbed samples on the substrate. Improvement in resolution is being actively
pursued and we hope that it will reach a sub-nanometer level on isolated molecules in the near future.
At the same time, we should be constantly trying to expand the application of the probe microscopy
technique with a wider perspective. What, then, would be the most interesting and profitable ways of
using AFM in biological applications in the future? In my opinion, there are emerging new frontiers
in several aspects of the AFM application in biosciences involving force measurements and I will try
to give a fair amount of coverage to them even if some of them are still in their preliminary stages.
3.2. Instrumental
In addition to the basic "repulsive mode" AFM, two other modes of AFM have been developed:
the tapping mode AFM in air [54], in gas [55], and finally in liquid [56]; and an alternative current
(AC) or non-contact mode AFM [57,58]. Both the tapping mode and AC mode AFM try to reduce
the contact time between the tip and sample to a minimum. A commercialized version of the tapping
mode AFM (registered name by Digital Instruments, Santa Barbara, CA, USA) has been a big
success in its application to weakly adsorbed samples on a substrate. In many studies of biological
materials, a weak binding between the sample and substrate is desirable to preserve the activity and
function of the samples. The use of tapping mode AFM, therefore, has become virtually a standard
for imaging soft samples.
In tapping mode AFM, a stiffcantilever is forced to oscillate at a certain distance from the sample
surface while it is scanning laterally. When the tip-sample distance becomes shorter due to the
presence of protruding structure on the sample surface, the frequency of cantilever oscillation is
forced to change and its amplitude at a given frequency is lowered. The feedback system then lowers
the position of the sample stage so that the cantilever resumes its original amplitude of oscillation.
Thus the topography of the sample surface is recorded as changes in the input voltage to the z-piezo
motor. Operating A F M in the tapping mode, a force or forces acting laterally to the sample can be
virtually eliminated. A lateral force, together with a vertically applied scanning force, not only
damages soft samples but also often displaces them.
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The tapping mode A F M in liquid operates in a slightly different way. In this case, the sample stage
rather than the cantilever is oscillated and the deflection of the cantilever when the sample touches
the tip is monitored by a feedback circuit. The sample stage is either raised or lowered to keep the
cantilever deflection at a constant level. Compared with the tapping mode in air, a much softer
cantilever is used. Both tapping mode AFMs have proved to be extremely useful in the imaging of
lightly adsorbed soft biological samples in air as well as in aqueous environments.
The AC mode operation A F M in liquid described by Wong and Descourts [58] is not a true
non-contact mode AFM. It is similar to the tapping mode in liquid developed by Hansma and his
colleagues [56]. They compared the performance of AC mode AFM at three different frequency
ranges, a very low, intermediate (acoustic), and high frequency (second overtone) region, and
obtained the best resolution in the intermediate range of 5 20 kHz using a cantilever of 0.37 N/m
with a natural frequency of 60 kHz in air.
The non-contact mode A F M or AC mode A F M works at a distance from the surface of the
sample. The true non-contact mode AFM working in the attractive region of the Van der Waals
force with the capability of resolving subnanometer atomic lattices has recently been completed
[53]. The major advantage of the non-contact mode AFM is its truly non-invasive character. It is not
altogether clear, though, if the non-contact mode AFM can operate without ever touching the
samples higher than 100 nm and as high as a few ktm such as live cells and chromosomes.
In biological applications of AFM, especially when one is working on cells or subcellular
organelles such as chromosomes, it is desirable that one can locate the samples on the substrate with
an optical microscope and subsequently start imaging with A F M or start taking force-distance
curves on selected areas of the sample. Stemmer described a hybrid AFM with interference optics for
3D optical sectioning [59]. According to Stemmer, "scanning force microscopy and light microscopy are mutually complementary techniques, the former being primarily surface sensitive while the
latter rather volume oriented, their combination into a single hybrid instrument is expected to
provide unique and unprecedented experimental access to problems such as how composition and
biophysical properties of the cell surface determine or are affected by the 3D dynamic organization
inside the living cell". His instrument is an A F M constructed on the sample stage of an inverted
optical microscope with a wide numerical aperture so that the full capability of differential
interference contrast (DIC) and that of the polarization microscope can be pursued. The aim of
combining the DIC capability of optical microscopy was to get 3D sectioning of a living cell and
correlate the intracellular events with the events taking place on the surface of the cell. A cockroach
blood cell was immersed in saline solution and imaged as a test specimen using the light reflected
from the gold-coated cantilever as an illuminating light. The result showed the presence of granular
structures inside the cell which could also be imaged from the outside using AFM. An important
point was not in the preliminary result presented in the paper, but realization of the idea of
combining an optical microscope with an AFM, which has been recognized as early as 1988 by
Guckenberger et al. [60] and Stemmer et al. [61].
As long as A F M was being used as an instrument for molecular imaging, the need for an auxiliary
optical microscope was not felt so strongly for the obvious reason that molecules cannot be seen by
the latter. In the application of A F M to biology, however, the initial location of the target cells with
an optical or fluorescence microscope is a very convenient one and, in some applications of AFM, it
is a vital requirement. For example, in the force-distance curve mode of AFM, it is not desirable to
spend much time repeating scans looking for a sample. Unnecessary scans would contaminate the
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285
tip or even damage it. Especially when one is working on a sample like chromosome, there is much
unidentifiable cellular debris around the target chromosome and much of it sticks to the scanning
tip. In such cases, it is most desirable to have an auxiliary optical microscope to help targeting
a chromosome without the need for scanning around it. In this respect, Mariani et al. [62] developed
an AFM equipped with optical microscope and took images of human chromosomes. Putman et al.
reported construction of a confocal laser scanning microscope with AFM [63].
3.2.1. Tips and cantilevers
There are several kinds of AFM tips available for biological research. The most popular kind is
prefabricated silicon nitride cantilevers with an integrated tip. The tip has a pyramidal shape with an
opening angle at the apex of about 70 ° which is in fact not very sharp. Sharper tips can be
manufactured, and etched silicon tips and electron beam deposited (EBD) tips have been introduced.
The former was microfabricated from crystalline silicon and the latter was found to be grown at the
apex of a commercial pyramidal tip when placed in the scanning electron microscope and exposed to
a focused electron beam. Both are commercially available under labels of either ultra tips or super
tips. In their comparison of the performance of the three kinds of tips, Schwarz et al. [64]
interestingly rated an overall performance of ordinary pyramidal tips rather high, primarily on the
basis of their robustness under conditions where they often make inadvertent crashes with protruded
features of the sample. Especially when a sample is hard and with convoluted topographies, the
robustness of a tip comes first to maintain stable scans. According to the authors, when flat surfaces
atomically are scanned, the resolution is not so sensitive to the type or the sharpness of the tip as
suspected.
Double tip artifacts are a more serious problem for biologically oriented researchers as pointed
out by the authors. A double tip is one with twin or multiple peaks at its apex. Since both the twin
peaks produce images, the resulting superimposed images could often be misinterpreted as
structurally interesting. The problem of tip shape was also taken up by Siedle et al. [65]
Sheiko et al. [66] proposed the use of heat treated SrTiO 3 as a gauge or a standard sample for the
evaluation of tip quality. A mechanically and chemically polished crystal of SrTiO 3 after heat
treatment at 750°C in vacuum and annealing has a regular saw tooth profile with approximately
50 nm repeats and an edge angle of 90 °. They imaged the surface of this crystal with commercially
available pyramidal tips and an EBD super tip. According to them, only one out of 20 commercial
tips from the same lot could be judged as good, the rest having various defects such as a truncated
(flattened) pyramid, double peaked, knife-edged, truncated pyramid with several sharp protrusions,
etc. Some of the defective tips did still image the crystalline NaCl surface almost perfectly. The
selected tip according to their criteria imaged various hard samples rather nicely including the
gel-drawn ultra-high molecular weight polyethylene film at the atomic or molecular level. Some of
the images obtained with defective tips are very educational for the routine users of AFM because
they sometimes give falsely detailed images of the sample surface.
Siedle et al. [65] tried to image the tip shape by scanning over the edges of small crystals of NaC1
and MgO of sizes up to 2 gm. They imaged the crystals and assuming that the crystals had sharp
edges, reconstructed the tip image and determined its equivalent radius of curvature. The result was
compared with the radius determined from the TEM image of the tips. They emphasized that their
method of checking AFM tips did not require any instrument other than AFM, once the reliability is
established between the results from AFM and TEM.
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(a)
(b)
(c)
(d)
a~
Fig. 11. Collection of tip artifacts. Panels from (a) to (d) are the results of imaging of spherical gold particles (10 20 nm in
diameter) with four different tips. Panels from (e) to [j) are: (e) and (f) gold (1 1 1) surface with a monolayer of a H6berg
compound (0.7 0.8 nm) adsorbed via thiol groups taken with different Si3N,~tips. Protrusions are imaged with pyramidal
in (e) and columnar (f) structures; (g) and (h) gold sputtered surface of stainless steel. The round elevations with small
holes in the center in (g) are looking interesting but are artifactual; (i) and (j) gel drawn ultrahigh molecular weight
polyethylene films. The image of fibrillar structure is wider in (i) which was taken with an ordinary pyramidal tip, compared
with the result in [j) taken with a super tip (after Xu et al. [67]; reproduced with permission of authors and publisher).
(e)
(f)
(g)
(h)
fi)
(j)
Fig. 11 Icontinued).
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Xu and Arnsdorf [67] recommended the use of colloidal gold particles of known diameters of 10
and 2 0 n m supplied by Sigma (St. Louis, MO, USA) as a standard sample for quickly selecting
"good" tips for macromolecular imaging using the contact mode AFM. Double tips can easily be
recognized because images of colloidal gold particles look dumbbell shaped as reproduced in
Fig. 11. Tips that are not axisymmetric but knife-edged, for example, at its apex will produce
non-spherical images of colloidal gold particles.
According to the authors, 80% of the silicon nitride tips supplied by a tip manufacturer were
defective in one way or another and requirement for a quick and reliable method for testing the tips
before imaging was started. They also presented a method to reconstruct the shape of the tip from the
scanned image of gold particles assuming a perfect sphericity for the gold particles and a spherical
shape of the tip apex. The tip was characterized by two parameters, namely the angle of the conical
part and the radius of curvature at the apex. Both parameters were shown obtainable from the
scanned image of the gold particles. From the images taken with a "good" tip, one can reconstruct
the tip image and finally a corrected image of the sample for tip sample convolution effects. It is
important to note that obtaining an atomically resolved image of mica surface did not serve as
a guide for the search of good tips, because many bad tips discovered using gold particles actually
produced atomically resolved images of mica surface. A small scan area and a regular array of atoms
tend to obscure the presence of doublets in such images.
The optimal scan speed of an A F M tip to obtain distortion free images under various conditions
was treated by Butt et al. [68]. Their analysis is based on the constant height mode of the A F M which
is less popular than the constant force mode in biological applications. Their reason that the
constant height mode allows much faster scan speed and therefore more important in the analysis of
scan speed limitation to the image seems to emphasize the potential use of A F M as a data storage
apparatus. The scan speed in the constant force mode is limited by the piezo electric scanner
response rather than the dynamics of the cantileverl They also presented a way to measure the force
constant of a cantilever by measuring its direct deflection when it was brought into contact with
a pendulum weight. They also measured the resonance frequency in the heat noise movement of the
cantilever and confirmed that the force constant obtained in the two different ways agreed well.
Theoretical calculation of the force constant based on the geometry and material constant of the
cantilever was different from the measured value by a factor of 2. Their calculation was based on
a parallel rectangular cantilever approximation as well as an exact calculation of the triangular
cantilever. Although the authors did not refer to possible sources for the observed difference between
the calculated value and that determined from their experiments, one possibility may be in the value
of Young's modulus of silicon nitride. Young's modulus of silicon nitride is E = 15 × 10 I° N / m 2 while
that of silicon is 10.5 × 101°N/m 2, and the composition of a silicon nitride tip is known not to be
constant and variations in the composition may influence Young's modulus.
From the examination of dynamics of the cantilever over the surface with sinusoidally changing height,
they concluded on a practical limit of scan speed for atomic resolution operation in vacuum of 0.1 ~tm/s.
What was again found from their careful experimentation was a wide variation in the quality of
commercial cantilevers. They reported a variation of cantilever thickness from 0.40 to 0.66 lam. Since
the cantilever force constant is proportional to the cube of its thickness, the force constant can easily
vary by a factor of 4 among the cantilevers from the same batch.
Cleveland et al. developed a reliable method of force constant determination [69]. They measured
the natural frequency v of a cantilever with an attached mass (tungsten balls) of varying weight (M).
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Since 27n, is proportional to x/k/(mc, + M), where k and reeff stand for the force constant and the
effective mass of the cantilever, 1/(2rrv) 2 was plotted against M and the slope of the straight line gave
an accurate value ofk. After the values ofk for several cantilevers were determined and plotted vs. v3,
a straight line was obtained which enabled an estimation of k (of a cantilever whose k is not known)
from the determination of its resonant frequency only, provided other material constants were
identical.
It is also possible to measure the mean square amplitude of thermal oscillation of a cantilever,
(Ax2), and equate the spring energy, (1/2)k(Ax 2) to the thermal energy, (1/2)k~T, where k~ is
Boltzmann constant and T is temperature in kelvin. This method does not require additional
weights to be fixed on a test cantilever. (Ax-') can be obtained as the Fourier transform of the
average of the autocorrelation function of Ax(t), i.e. (x(t)x(t + v))~,,.~.
Mitsui et al. [-70] constructed a macroscopic cantilever from a thin gold wire of 0.025 mm in
diameter and pressed this cantilever with an A F M tip. The spring constant of a cylindrical cantilever
of length L a n d diameter D is given by k = (37rED4/64L~), where E is Young's modulus of gold. It is
always necessary to calibrate a macroscopic cantilever beforehand by measuring its deflection
against a known applied force. One may then record the force curve on an A F M apparatus by
pressing a test A F M cantilever against the calibrated one. The slope of resulting force curve is used to
calculate the force constant of the test cantilever.
Since in the future, A F M will be increasingly used for the measurement of the force involved in
various biochemical processes, calibration of the cantilever force constant will be a very important
routine procedure in every A F M laboratory and therefore a simple and reliable method of
calibration must be developed.
3.2.2. Substrates
Another important factor in A F M measurement is that samples must be kept in a stationary state
and, at present, it means that they should be securely attached to a flat substrate. This is more
important in the case of contact m o d e A F M where laterally acting forces are strong and often
remove or scrape off lightly attached ("physisorbed") samples on the substrate. Tapping mode and
non-contact mode A F M s have been put into use with a primary concern to reduce such deleterious
effects observed in the contact mode operation of AFM. In the case of measurement in liquid,
samples often come into solution if they are not strongly attached or chemically cross-linked to the
substrate. This poses a dilemma that if samples are strongly held by the substrate, they are likely to
be deformed or even denatured and inactivated due to the binding energy, and if they are only lightly
adsorbed to the substrate, they are easily removed with the tip movement. In future, these difficult
situations may be alleviated by the use of laser tweezers which can keep cells and molecules in space
or in liquid totally free of any substrate and in a desired orientation. At present, we will investigate
various substrates that have been tested by m a n y devoted workers.
I1) H O P G , mica, and glass are three most popular substrates used in biological applications of
AFM. Silicon, zeolite, MoS 2 are also used. The criteria here are the flatness for at least several
hundreds of nanometers. For proteins and DNA, mica is a preferred substrate and the surface of
mica is often made hydrophilic by the treatment with MgC1 z solution. A mica surface can be
made particularly adsorptive to D N A by the treatment with NiC12 presumably due to the
d-electron orbitals on Ni ion adsorbed on the mica surface [71,72].
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(2) Mica and glass surfaces are often coated with an epitaxially grown crystal surface of gold (1 1 1).
It is most often employed to create chemically reactive surfaces toward thiol-bearing reagents
which can either cover the entire gold surface or act as a cross-linker for a sample to be examined
with AFM (or STM) [73]. Glass surfaces were shown to be activated with silane reagents [74]
and made functional toward reactive groups of samples.
(3) Denaturation of proteins, DNA, and other biological structures in the adsorbed state on the
solid surface is another problem to be considered carefully. It has been known that cytochrome
c when adsorbed to the surface of gold does not have activity (Hara, private communication).
The protein was then anchored to a gold surface covered with alkane thiol reagents to preserve
its electron transport activity. Alkane thiol reagents are considered to protect proteins from
being too strongly adsorbed to the surface.
(4) Contamination is another problem when working with biological samples. In a solid state
physics experiment, substrates are routinely cleaned by heating them in vacuum to a high
temperature and any possible organic contamination is decomposed and/or evaporated. If this
ultimate way of surface cleaning should be applied for a biological study, sample application
must be done either in vacuum or in an ultra-clean gas chamber after the substrate is cleaned. The
problem is that the air is highly contaminated and a clean gold surface, once exposed to air, gets
highly contaminated with particles of various sizes, including those of sizes similar to protein
molecules. Gold-coated mica substrates can be stored under ultra-clean water in a relatively
contamination free state because contaminants from air are adsorbed preferentially on the liquid
surface. So if experiments are to be done in aqueous environments, gold-coated mica substrates
should be kept in water until immediately before use, and exposure to air should be kept minimal
during sample application. Particles that adhere to a gold surface from air were shown to be
easily scraped during scanning with a contact mode AFM resulting in a deceptively clean image
of the surface and at the same time contaminating the tip apex.
(5) Since many kinds of animal cells adhere to glass or polystyrene surfaces, AFM scanning of such
cells is easy but resolution is often compromised. Plant cells, however, do not usually have an
affinity to a glass surface. For example, spherical yeast cells are difficult to image on any of the
substrates mentioned above. In such cases, special methods to harness them alive must be
devised. From this point of view, two methods devised by Kasas and Ikai [75] and Gad and Ikai
[76] are useful.
3.2.3. Tip sample interactions
Basically there are the following tip sample interactions to be considered in relation to the image
quality. If there is any specific interaction between the tip and surface other than the fundamental
repulsive and Van der Waals attractive force, AFM images will be distorted. An ideal situation for
a contact as well as non-contact mode A F M is that any force other than the L o n d o n - V a n der Waals
type repulsive-attractive force is not operating during the scanning.
(1) Electrostatic interactions. Electrostatic distortions of the image become serious problems
especially when the image of non-conducting sample is taken in air because both the surface of the
sample and that of the tip can be statically charged in dry air. In aqueous electrolyte solutions, such
electrostatic charges are effectively shielded by an ionic atmosphere. It is a good idea to choose a pH
of the solution near the isoelectric point of the sample. In aqueous electrolyte solutions, the
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291
characteristic length of electrostatic interaction is determined by the Debye length which is
dependent on the inverse square root of the ionic strength. According to Israelachvilli et al. [77] the
electrical double layer interaction is apparently more complicated than a simple charge-charge
interaction and can be attractive or repulsive depending on the distance between two such surfaces.
(2) Van der Waals interactions. Van der Waals attractive force is significantly weaker in water due
to a higher refractive index of water than that of air, according to Israelachvilli [77].
(3) Capillary force due to the surface layer of water. As both tip and sample surfaces are covered
with thin layers of water when the RH is higher than 50% [78], the tip is strongly attracted to the
surface due to the capillary force between the two water layers. The force is often damaging to soft
samples especially during an initial contact of the two surfaces.
(4) Contaminant mediated interactions. While a tip scans over a substrate surface with lightly
adsorbed molecules, it is conceivable that some of the molecules become attached to the tip and the
subsequent interaction between the tip and the sample is mediated by the adsorbed molecules. This
situation can lead to either attractive or repulsive interactions depending on the nature of the
molecules on the tip and those on the substrate.
Thus the tip-sample interaction is complicated when working on biological samples and one
must routinely check the status of the tip by periodically scanning standard samples.
3.3. Experimental results
I will cite experimental results from the selected work published between 1991 and 1995. The
choice of cited literature is based on the following criteria:
( 1) those of immediate biological interest;
(2) those with helpful suggestions and useful data for biologically oriented AFM users;
(3) those predicting interesting future applications of A F M in bio-medical fields.
3.3.1. Proteins
Expectation is naturally high for successful imaging of isolated protein molecules with sufficient
atomic details to allow biochemically meaningful discussions of the structure function relationship.
That task, however, still remains to be fulfilled. Imaging of two-dimensionally aligned molecules in
quasi-crystalline states has been more successful than for isolated molecules, and examples of such
studies will be given below. If a protein can be crystallized, there are better suited methods for
structural analysis than AFM such as electron diffraction and X-ray crystallography. Nonetheless, it
is worth to know, if carefully applied, AFM can produce the surface topography of 2D protein arrays
with resolution of less than 1 nm.
Schabert et al. [79] presented an AFM image of 2D crystalline E.coli O m p F porin protein at
a very high resolution. The O m p F porin protein is a membrane channel protein known to mediate
an uptake of extracellular molecules. The protein was purified and embedded in lipid bilayers in
a crystalline state and adsorbed on a mica surface without covalent fixation. By using a contact mode
AFM (Nanoscope III) with oxide-sharpened silicon nitride tips (Olympus, Tokyo), they achieved
a resolution of 0.8 nm on a periplasmic (facing the plasma side of the cell} side and 1.3 nm on an
extracellular surface. Since naturally grown crystals exposed only the periplasmic side, they
mechanically scraped a part off the top lipid- protein layer of the crystal and imaged an exposed
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Fig. 12. High resolution images of OmpF porin from the periplasmic side (a) and from the extracellular side (b). The left
figures are tip views and the right figures are topographic views (after Schabert et al. [79]; reproduced with permission of
authors and publisher, copyright [1995] American Association for the Advancement of Science).
face of the extracellular side. Fig. 12 reproduces the original high resolution images from the listed
paper.
This is undoubtedly one of the best resolved data with AFM for 2D crystalline proteins. Since the
X-ray crystal analysis of the trimeric structure of the protein has been known [80], the authors
reconstructed a model of the membrane bound state of the protein and discussed the important
interactions between the trimeric units in the membrane. They also noticed two structural states for
the extracellular face of the protein which are likely to be correlated to an expected conformational
change of functional importance. To attain such a high resolution with AFM, a contact mode
scanning with a sharp tip (oxide sharpened) at a low contact force of 0.1-0.3 nN was an essential
requirement. According to the authors, the periplasmic side was more resistant to the scanning force
and could be scanned for at least 10 times with a force of 0.3 nN. The extracellular side was more
flexible and less resistant to the scanning force.
A. Ikai/SurJace Science Reports 26 (1996) 261 332
293
Lal and Yu [81] chose the nicotinic acetylcholine receptor, another membrane protein of
pentameric structure and expressed it in Xenopus oocytes using in vitro transcribed mRNAs which
were mixed at a ratio of2:1 : 1 : 1 for four subunits of the protein. This artificial expression system was
chosen on the basis that: (1) there is no need for the purification and crystallization of the membrane
proteins to be studied; (2) the extracellular surface can always be identified, and (3) structural and
functional studies can be done on the same cell, among other reasons. An oocyte specimen for AFM
imaging was prepared either as a whole cell or by cutting it into halves. The sample was then dried in
air and scanned with a contact mode AFM. Expression of the pentameric receptor on the oocyte
surface was confirmed by an electrophysiological measurement of the generation of action potentials
by the addition of acetylcholine, and the surface density of the expressed protein was checked by
using a specific binding of c~-bungarotoxin, an inhibitor of the acetylcholine mediated neural
transmission. The expressed receptor was found to be clustered in patches on the oocyte surface in
disagreement with the previous macroscopic studies. The molecular image of the receptor as
a pentameric protrusion from the surface was obtained although only in limited cases according to
the authors. The result is reproduced in Fig. 13.
The gap junction that forms tight connections between two cells is another popular 2D crystalline
sample for AFM imaging. The junction is formed by a tight coupling of hexameric proteins called
connexons {each subunit is called connexin with a molecular weight (MW) = 28000) embedded as
hexameric arrays in the membranes between two cells. Hoh et al. [82] isolated gap junctions from
a rat liver and prepared patches of double layered membrane pairs containing connexons. They are
sometimes called plaques and observed to attach spontaneously and tightly to a freshly cleaved mica
surface. The top layer of the paired membranes was a periplasmic lipid layer and should be removed
so that hexagonal arrays of connexons were to be exposed. Removal of the lipid layer was done by
scanning with an AFM tip with an increased force or increased scan rate. Fortunately, the force that
is holding the gap junction plaques to the substrate was strong enough to withstand this removal
procedure, and the hexagonal array of connexon proteins facing the outside of the cell was exposed
and imaged with AFM. A contact mode AFM was operated in a liquid cell filled with phosphate
buffered saline at pH 7.2. Not only the hexagonal array of hexameric proteins but also the internal
structure of each protein was imaged with a large pore in the center. Many of the pores were imaged
with a depth extending to 0.8 nm but the authors observed closed pores as well. In some cases the
connexon molecules looked C-shaped possibly indicating the loss of one subunit during the removal
procedure of the top layer. This paper showed that the AFM could change the physical state of the
sample to expose an embedded layer to be studied, by controlling the force during the micro-surgery
of the sample and that of scanning.
The dissection of double layered membrane pairs with an AFM tip had been observed by Hob et
al. [83] prior to the above cited paper. It was noted that lateral dissection of a membrane pair with
a thickness of 14.4 nm reduced that of the remaining membrane to 6.4 nm. The apparent asymmetry
of the membrane thickness was attributed to a strong adhesion of the bottom layer to a glass and/or
mica surface. One advantage of using the force method to dissect the membrane pair was that when
other methods were used, it was sometimes difficult to tell the extracellular side from the periplasmic
side, and the dissection methods are often chemically harsh so that there was little guarantee of the
resulting structure being an intact one. The thickness of the gap junction preparation was measured
under several different conditions including trypsinization. The average thickness of hepatic gap
junction (14.4nm) was not reduced upon trypsin treatment (15.5 nm) which is known to remove
294
A. Ikai/Surface Science Reports 26 (1996) 261 332
!
Fig. 13. Molecular image of acetylcholine receptor expressed on the Xenopus oocyte showing a pentameric subunit
structure [after Lal and Yu [81 ]; reproduced with permission of authors and publisher).
60 70 amino acid residues from individual connexins. Apparently the removed part ofconnexin was
not contributing to the original thickness of the preparation. Glutaraldehyde fixation changed
neither the thickness nor the easiness of microdissection of the plaque.
The gap junction from a heart muscle was prepared and studied by Lal et al. [84] before and after
trypsin treatment. The molecular weight of connexin of the cardiac gap junction is 43 000 and
substantially larger than that of hepatic connexin. Consequently the thickness of the plaque was
about 25 nm under liquid and 27 nm when dried, and in this case trypsin treatment was effective to
reduce the thickness to 17nm under liquid and 20rim in dry state. In addition to the intact gap
junction plaques as described above, the authors found in their samples, plaques with a thickness of
9 nm in PBS and 11 nm when dried. The plaques had a morphology similar to the trypsinized half
junctions and were considered to represent the native half junctions called hemi-junctions that had
A. Ikai/Surface Science Reports 26 (1996) 261 332
295
been proposed from indirect electrophysiological data. The quality of the images representing
hexagonal arrays of connexons was not very high compared to that of hepatic gap junctions.
The nuclear pore complex is a huge unit of estimated MW of 1.2 x 108 encompassing the double
membraned nuclear envelope. The complex has a cylindrical doughnut structure with a diameter of
120 nm and a height of 70 nm as revealed by electron microscopic studies (references are cited in
[-85]). It is supposed to act as a conduit for nucleocytoplasmic transport of transcription enhancer
molecules such as intracellular receptor complexes for steroid hormones and the ribonucleoprotein
complexes produced in the nucleus. Goldie et al. [85] prepared nuclear pore complexes by rupturing
Xenopus oocyte nuclei on a chemically modified glass surface. Chemical modification of the glass
surface consisted of either poly-lysine coating or carbon coating. The nuclear envelope was adsorbed
to the modified glass surface with its cytoplasmic side, and the nuclear face was exposed to the
solvent for the study with AFM. The Xenopus nucleus has a diameter of about 0.1 mm and could be
located by the eye against a dark background if it was cross-linked with glutaraldehyde. The carbon
coated glass surface was superior to either poly-lysine coated glass or freshly cleaved mica as
a substrate for the immobilization of this sample. Glutaraldehyde fixation of the nuclear envelope
helped rendering the sample more resistant to mechanical damages inflicted on them by a scanning
tip. The paper gives dimensions of the observed structure on the exposed surface of the nuclear
envelope but the lateral resolution of given images was not high.
The images of the nuclear pore complex given by Oberleithner et al. [86] show a doughnut-like
structure with an improved clarity. They attempted to study the structure function relationship of
an aldosterone sensitive clone of M a d i n - D a r b y canine kidney cells and prepared the nuclei from the
cultured cells. They counted the number density of nuclear pore complexes in 1 gm 2 with AFM and,
by multiplying the number with the calculated total area of the nucleus, estimated the total number
of the complex. They measured the electrical conductivity of the nuclear envelope under physiological conditions and divided the result by the number of pore complexes per nucleus. The single
nuclear pore conductance thus obtained was compared between the control cells and the aldosterone stimulated ones. The number of pores was greater for the latter cells but the conductance per
single pore complex was approximately constant between two types of the cells leading them to
a conclusion that the aldosterone stimulated increase of the conductance was solely due to an
increase in the number of conductive pores. The calculation was based on the assumption that
nuclear pore complexes were the only ion channels on the nuclear envelope. Individual complexes
were imaged as doughnuts with a central pore with a diameter of 30-40 nm but a calculation based
on the electrophysiological data taking the concentrations and transfer numbers of ionic species into
consideration gave an estimate for the pore size of 11 nm. The difference between the two results led
them to assume an existence of electrically blocking material within the cylinder-like structure of the
complex. The blocking material with a pseudo three-fold symmetry was sometimes imaged to be
sitting inside the cylindrical pore complex as shown in Fig. 14.Such a plugging substance in the
nuclear pore complex has been imaged by electron microscopy as well.
The purple membrane of Halobacterium halobium is another very popular subject in high
resolution imaging of protein crystals. The purple membrane contains a retinal containing protein
known as bacteriorhodopsin which acts as a light driven proton pump to produce a finite difference
in the proton concentration between the inside and outside of the cell membrane. The combined
chemical and electrical potential difference thus created on the two sides of a membrane is utilized as
a chemical energy to regenerate ATP. When the density of the protein in lipid membrane is high,
296
A. lkai/Sur['ace Science Reports 26 (1996) 261 332
Fig. 14. The plug substance imaged in the pore of nuclear pore complex. (a) and (b) are open channels whereas (c) and (d)
are channels plugged with a body having a distorted three-fold symmetry (after Oberleithner et al. [-86]; reproduced with
permission of authors and publisher).
bacteriorhodopsin molecules form 2D crystalline patches which can be readily extracted from the
membrane as flat sheets of several micrometers in diameter. The purple membrane has been used in
the A F M study since very early stages of its application in biochemical field 1-87-91] and one of the
most recent one by MiJller et al. [92] achieved a lateral resolution of 0.7 nm on the cytoplasmic side
of the membrane. They studied the adsorption property of purple membrane on a mica surface at
different pH's and found that a high pH of 8.2-9.4 and the presence of KC1 in the buffer solution were
important factors for the stable imaging with AFM. For imaging of the cytoplasmic side of the
membrane, samples were adsorbed on a freshly cleaved mica surface and submerged in liquid.
The result of height measurement of purple membranes at different pH's was reported by Mfiller
et al. [92]. The change in the membrane thickness around pH 5.2 which corresponded to the
isoelectric point of bacteriorhodopsin was tentatively interpreted as due to altered electrostatic
interactions between different protein segments or between the protein and mica surface. The
corresponding change in the interlammelar distance has been noted in diffraction experiments at
around the same pH value. The height value of 5.6nm in the neutral pH range was in good
agreement with the reported value of 5.3 nm by Henderson et al. [93].
A. lkai/Surface Science Reports 26 (1996) 261 332
297
The image of bacteriorhodopsin in 2D arrays is reproduced in Fig. 15. The hexagonal array of
protein molecules is clear and the filtered images give a trimeric pattern of bacteriorhodopsin as
a ring with a large hole in the center. This is the tentatively assigned cytoplasmic side of the sample
and the protrusion on this side of the membrane was three times higher than on the other side of the
same membrane.
The remarkable success of Miiller et al. in obtaining a high resolution image of the purple
membrane probably depended, among other factors, on: (1) their careful study of adsorption
properties of the sample to the substrate including the choice of most effective pH and KCI
concentrations; (2) scanning with a low force of less than 1 nN: and (3) minimization of thermal drift
by waiting for several hours before taking scanning images.
Mou et al. [94], working on AF'M imaging of cholera toxin proteins, devised a way of artiticially
incorporating non-membranous proteins on a lipid bilayer prepared on a mica surface. Cholera
toxin B is a pentameric protein of small subunits of MW = 12 000. They first used a gel phase bilayer
prepared from 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (transition temperature from gel to
fluid phase bilayer is 41°C) containing 10% of a ganglioside called GM1 which, being a natural
membrane receptor for cholera toxin B, served as anchor to hold the toxin molecules on the bilayer.
The gel phase membrane was stable on a mica surface so that an artificially created defect with the
A FM tip was preserved for a long enough time to be imaged in subsequent scans. When the substrate
was submerged under a buffer solution in a liquid cell and cholera toxin B was added, the protein was
adsorbed to the bilayer and could be imaged at a high resolution revealing a ring-like structure with
an outer and inner diameter of, respectively, about 5 and 1 nm, and a height of 1 nm above the lipid
layer. The authors claimed that they were able to discern a pentameric substructure but it was not
clear from the published images. They then tried several methods of their own device to use bilayers
in a fluid phase. By using a fluid phase lipid layer prepared from egg-phosphatidyl choline
containing 10% GM1, they showed that crystalline 2D arrays of protein molecules were spontaneously formed (see Fig. 16) presumably due to an increased mobility of the lipid molecules allowing
relocation of protein molecules from original adsorption sites.
The paper by M o u et al. briefly described various ways to prepare fluid phase lipid layers over
a mica surface [94]. The method developed by Mou et al. will be useful in future not only for the
membrane binding proteins but also for any soluble proteins if a suitable anchoring molecule is
embedded in lipid layers formed on an atomically flat surface of, for example, mica.
Other soluble proteins were also prepared on chemically and/or physically treated substrate and
imaged with AFM. Droz et al. [95] used H O P G that was glow discharged to increase the
hydrophilicity. Immunoglobulin were molecules adsorbed better to a glow discharged H O P G
surface but adsorption as well as the imaging with AFM seemed to be better when the protein was
adsorbed on a bare mica surface. They treated the protein with phenylglyoxal which eliminated
some of the positive charges due to arginine residues with an intention to increase the hydrophobicity of the protein surface and as a consequence adsorption on a hydrophilic mica surface was
reduced.
Ohnishi et al. [96] irradiated a silicon wafer with UV light and placed the wafer in a desiccator
filled with hexamethyldisilazane vapor. The wafer was then heated to 60°C for covalent binding of
the reagent. They then transferred a double layer of ferritin (an iron containing protein of
MW = 5 x 105 purified from a horse spleen) and PBLH (poly-benzyl-L-histidinel prepared in
a micro Langmuir trough. The sample thus prepared was imaged with AFM in a fluid cell under pure
(a)
(c)
(b)
(d)
(e)
Fig. 15. Hexagonal array of bacteriorhodopsin in purple membranes: (a) in a large scan area: (b) diffraction order
extending to 0.7 nm; (c), (d) and (e) averaged images of bacteriorhodopsin. Scale bars are 20 nm in (a), (2 nm ~t in (b) and
4 nm in the rest (after Miiller et al. [92]; reproduced with permission of authors and publisher).
A. Ikai/Sur[ace Science Reports 26 (1996) 261 332
299
Fig. 16. Spontaneously formed 2D crystalline array of cholera toxin B on a fluid phase lipid layer l aftcr Mou et al. [94]:
reproduced with permission of authors and publisherl.
water. Protein molecules were found in hexagonal arrays. The authors claimed that subunits were
resolved when images of individual molecules were compared with the known crystallographic data.
Imaging of ferritin molecules was done in a pH range of 4.5 6.5 where ferritin was expected to be
negatively charged (isoelectric point of ferritin being 4.5) and the PBLH layer to be positively
charged. At pH 5.3 where there was no attractive interaction between the tip and sample even in pure
water, the '~self-screening effect" between the negatively charged protein and the positively charged
PBLH layers was considered to be effectively working, resulting in an improvement of the image
quality. Ferritin molecules in hexagonally arrayed parts did not show intramolecular details but
those ~inhomogeneously anchored in two dimensions" displayed a dip in the center of the molecule.
Ohnishi et al. [97] studied a similar system prior to the above-mentioned work and established
a self-screening condition of the ferritin layer over that of PBLH. Under the self-screening condition,
the force between the tip and the sample could be reduced to less than 0.1 nN.
Ferritin was also studied by Yang et al. on a mica surface [98]. The randomly adsorbed ferritin on
mica never formed regular arrays but individual molecules sometimes showed an internal subunit
arrangement though the interpretation of such images was difficult. The subunit structure was more
readily apparent in their work on immunoglobulin M (IgMt which is known to be a pentameric
oligomer of a total MW of 9 × 105. Some of the selected images taken on a bare mica surface showed
clear images of the pentagonal subunit structure of IgM [98] as reproduced in Fig. 17.
An important comment from the authors was that a deleterious effect of loosely bound proteins on
the substrate surface must not be overlooked because when they were scraped off, they tended to
stick to a tip and contaminated it, reducing the resolution of the images. It is therefore important to
wash off all the loosely bound molecules before scanning is started. Use of a cryo atomic force
microscope has been suggested by Han et al. [99]. The instrument works between 77 and 220 K and
the images o f l g M obtained with it are indeed the best images of isolated protein molecules so far. At
300
A. lkai/Surface Science Reports 26 (1996) 261 332
a
b
c
d
e
f
g
h
i
j
50 nm
Fig. 17. The pentameric structure of IgM, panel e in particular, as identified with AFM (after Yang et al. [98]; reproduced
with permission of authors and publisher).
lower temperatures, proteins are supposed to be more rigid than at room temperature and thermal
fluctuations are smaller, both contributing to a better resolution. We decidedly need a cryo AFM for
structural studies and a conventional one with liquid cell for functional studies.
An attempt to image the surface of a 3D crystal of Ca-ATPase of sarcoplasmic reticulum with
AFM was not very successful not being able to resolve individual molecules but it will be a useful
technique in the future [-100].
An interesting application paper on the use of AFM in identifying the structure of a paired helical
filament deposited on some of the neurons of Alzheimer's disease patients has appeared [,101]. The
general view on the structure of this filament based on a TEM study of negatively stained samples
has been that of a pair of two strands composed of incorrectly phosphorylated ~ protein but a recent
study by scanning electron microscopic (SEM) observation of metal-coated samples proposed
a twisted ribbon model rather than a paired-helical one. The authors studied the filaments with AFM
in dry state and produced an image that was more compatible with the twisted ribbon model. In fact,
one of the images presented in the paper is strikingly clear in showing the twisted ribbon structure
(Fig. 18). No information about how the original data were treated is available from the paper.
AFM was used to study the internal structure of spider dragline silk by Li et al. [102] The 90 :~
section of the dragline silk shows two concentric structures which were termed as the inner and outer
cores. The 45 ° and longitudinal sections of the same fiber showed accordion-like pleated sheet
structure which the authors regarded as a basic structure to guarantee the tensile properties of
dragline silk. The proposed structure is very different from that of the silk from silk worm. The
similarity of the structure with the synthetic Kevlar fiber was mentioned which had also been studied
with AFM by the same authors [,103]. It is, I believe, important that the AFM be applied in newly
A. Ikai/Surlklce Science Reports 26 (1996) 261 332
301
i
200
100
...................
0
100
nm
20O
Fig. 18. The twisted ribbon structure of polymerized r-protein in neurons of Alzheimer's disease patients (after Poll:men
et al. [ 101 ]: reproduced with permission of authors and publisher).
developing fields such as this one to produce reliable images that might settle some of the
controversies regarding the structure of key elements.
Another fibrous biological system studied with AFM is collagen. When the native and reconstituted
collagen fibers were examined with the AFM they revealed a characteristic 67 ~69nm banding
pattern as has long been known from TEM studies of negatively stained samples [104]. A new line of
information that was available only from the AFM study was that the change in height over such
banding pattern was 4 nm. They used both contact and tapping mode AFM to image collagen fibers.
The flagella of Halobacterium halobium were imaged by Jaschke et al. [ 105] showing a periodical
substructure of repeating distance from 50 to 100 nm.
As early as in 1989, Drake et al. [106] published a highly influential paper on the time sequence of
fibrinogen fibrin clot formation on a mica surface under physiological fluid conditions. Since this
paper has been cited many times in various review articles, I will not go into details except to say that
the analysis of time dependent biological as well as chemical processes is a very promising field in
AFM and STM.
3.3.2. DNA
In 1989, Lindsay et al. published a paper on an attempt to image nucleosomal DNA with STM
and AFM [ 107] both operating in aqueous solutions. Especially, their pioneering effort to develop
an electrochemical AFM for the biological samples has been influential since then. DNA was
electrochemically deposited onto a crystalline gold surface which has a positive potential with
respect to the STM tip and irreversibly attached to the substrate. A tip coated with insulating
material except at the very end was used to avoid a faradaic current to interfere with the tunneling
current measurement. Nucleosomal DNA with 146 nucleotide base pairs was purified and used for
imaging. They found aggregates as well as individual molecules with corresponding lengths to the
theoretical value of 52 nm for B-DNA. AFM images were less clear than those obtained with STM at
this stage, but specimens with similar lengths were observed.
A. Ikai/Surface Science Reports 26 (1996) 261 332
302
Weisenhorn et al. [108,109] used a mica surface pretreated with cadmium arachidate and later
coated with LB film (1 : 1 mixture of dioctadecyl dimethyl a m m o n i u m bromide and dipalmitoylphosphatidyl glycerol) as a substrate for DNA imaging with A F M in water. A short 15 mer DNA of
solely pyrimidine bases was discerned on the corrugated surface of LB film. A vanguard of reliable
and realistic images of circular plasmid DNA was presented by Bustamante et al. [110] in 1992. They
imaged circular plasmid DNA and 623 base pair fSK14 restriction fragments. Under a relative
humidity of less than 45%, DNA was imaged without much displacement by the lateral force exerted
by a tip. The height and width of DNA showed a dependency on the scanning force in the following way.
At 20 nN: H -- 1.01 nm and W= 2.1 nm (21nm is more likely from the figure): at 110 nN: H = 0.62 nm
and W= 29 nm; and at 170 nN: H = 0.49 nm and W= 47 nm, and the change was largely irreversible.
A method of estimating the radius of a parabolic tip is presented using the following equation
W= 4(Re
+ Um)x/Rm(Uc -
Rm)
Uc
R~ and R m are defined in Fig. 19.
This equation can be substituted with the following much simpler one based on a spherical
approximation of the tip (Reviewer's comment):
W = 4 ~ .
Using the observed values of W= 6.9 nm for a DNA sample and R m = 1 nm, they obtained the
value of 2.9 nm for the radius of curvature for an electron beam deposited tip. If this is correct, an
electron beam deposited tip is indeed very sharp compared with conventional pyramidal ones.
Nanodissection of supercoiled plasmid DNA is the topic of the paper by Henderson [111]. He
tried: (1) a simple deposition of DNA on an untrea.ted mica surface followed by first blotting, then
washing in an a m m o n i u m acetate solution, and finally air drying before scanning in AFM; (2)
Rm Sample
i
2 /-RcRm
Fig. 19. Definition of R c and
R m
in the convolution effect of tip and substrate (adapted from Bustamante et al. [110]).
A. lkai/Sur['ace Science Reports 26 (1996) 261 332
303
heating the washed sample to 80°C for 30-60 min in a vacuum oven before imaging. DNA molecules
after heating were much more stable against scanning compared with air dried ones. He excised
a part of the adsorbed DNA corresponding to roughly 100 150nm, 300-400 base pairs, by
temporarily increasing the scanning force and claimed that this was a much shorter fragment of
DNA than the shortest fragment that could be cut out using a glass needle and a micromanipulator.
The effect of cisplatin (cis-diaminedichloroplatinum), an anticancer reagent known to form a DNA
adduct, was studied for an oriented fiber of DNA rather than individual molecules [112]. The
authors claimed that cisplatin distorted the structure of DNA in fibrous assemblage but it is not
altogether clear from the given figures.
Thundat et al. [113] reported an interesting observation that the negative and positive contrast of
DNA images taken with A F M depended on the environmental humidity. At a higher relative
humidity (RH) of 60%, DNA was imaged lower in apparent height than the substrate surface
(negative image), and at a RH of 30% it was imaged higher than the substrate (positive image). This
effect was accompanied by the observation that at a high RH, the tip-sample interaction was
characterized by a strong adhesion force of larger than 100 nN, whereas at a low RH the force can be
as low as 10 nN or less. This was attributed to the capillary force of surface water and the negative
images obtained at a high RH were attributed to cantilever buckling due to an increased capillary
force around DNA molecules. The apparent diameter of DNA was much larger in negative images
than in positive ones. Lyubchenko et al. [114] used a modified mica surface with 3-aminopropyltriethoxy silane (APTES) to adsorb a double stranded renovirus genome DNA and imaged it with
AFM. Operating in the contact mode in air and, using a pyramidal tip, they reported that imaging
was very stable and that the DNA molecules were imaged as strings of an approximate width of
10 nm or more. The interaction between the modified mica surface and DNA was strong enough to
withstand repeated scans. The length distribution of the genome DNA was also reported.
Yang et al. [115] mixed DNA with cytochrome c to form binary complexes in solution and
adsorbed the complex to a carbon coated mica surface. Cytochrome c:DNA complex was stably
adsorbed and DNA was repeatedly imaged as 6 nm wide strands. Both circular cDNA and M 13
linear DNA were used. The Klenow fragment of DNA polymerase I was added together with d N T P
and a random priming reaction was started. Imaging of DNA:polymerase complex with AFM
revealed the attachment of at least five globules which were considered as enzyme molecules.
Li et al. [116] reported that incubating an aqueous DNA solution at 35"C helped in spreading
circular plasmid DNA molecules on a mica surface pretreated with magnesium acetate solution to
replace AI + + + with Mg + +. It was also important to use a very low concentration of DNA to avoid
aggregation. AFM was operated in air and DNA consistently appeared as circular strings of 8 nm in
width. They pariicularly emphasized that their method of preparing DNA sample required an
amount of DNA in the order of nanograms. The images of DNA in the cited paper are actually very
clear and probably one of the best examples of DNA imaged in air.
Vesenka et al. [117] studied the effect of RH in the environment when DNA was scanned in air.
DNA molecules were labeled with colloidal gold particles for identification and as a standard of
height measurement. The linkage of gold particles to DNA was formed by, first, labeling the 5'-end of
DNA with biotin-- d U T P using the Klenow fragment of DNA polymerase I and then mixing it with
streptavidin labeled colloidal gold particles. After application of gold:DNA complex on mica, the
surface was thoroughly washed with distilled and deionized water. It is, therefore, surprising that not
only the height of DNA but also that of gold particles was highly dependent on RH and the
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A. Ikai/Sutface Science Reports 26 (1996) 261 332
explanation given by the authors was based on the assumed presence of a thin layer of water with
dissolved small molecules. It is rather curious for the authors to invoke the presence of buffer salts as
the cause for an increased hydration layer under a high RH because the sample was washed with
water beforehand.
Binding of RNA polymerase on DNA as a transcription complex was studied by Rees et al. [118]
using 681 bp fragments of DNA containing 2P L promotor and E.coli RNA polymerase. The DNA
appeared bent (about 54 °) in an open promotor complex containing RNA polymerase and more
severely bent in an elongation complex (average 92 °) where the enzyme completed RNA synthesis up
to 15 nucleotides. The images were taken in air after drying the samples on mica.
Imaging of DNA with AFM then shifted to imaging under liquid conditions and in 1993 we saw
a rush of papers reporting the results of such procedures. Examples are seen in [119-124].
DNA was imaged under n-propanol in as early as 1992 by Hansma et al. [125]. Instead of imaging
DNA in air or water, they tried alcohols such as n-propanol, isopropanol, and n-butanol. DNA was
deposited onto a mica surface from a solution containing MgCI 2 and, after drying, immersed under
one of the alcohols mentioned above. Imaging was easier and cleaner than in air or water
presumably because the tip-sample interaction was weak and the insolubility of DNA in organic
solvents kept it stably attached to the mica surface. The authors also pointed out that, unlike in
water, the electrostatic interaction between mica surface and DNA was still effective due to low
values of the dielectric constant of the solvents. A plasmid DNA was imaged with good reproducibility and its width was as small as 3 n m when a supertip (electron beam deposited tip in SEM
apparatus) was used. Occasionally some reproducible bumps were observed along the double helical
strand of DNA and they were tentatively interpreted as representing a feature associated with the
double helical nature of DNA. Binding dynamics of the E. coli RNA polymerase complex to a short
fragment of DNA of 1258 base pairs that contained 2P R promotor was studied. Continued imaging
in a physiological buffer solution was performed using electron beam deposited silicon nitride tips.
DNA in a buffer containing 1 m M MgC12 was deposited on freshly cleaved ruby mica for 10s and
washed thoroughly with clean water. The sample was then dried under nitrogen and put in
a desiccator. It was then placed in a liquid cell and scanned with a contact mode AFM. Fig. 20 shows
a binding-unbinding-rebinding process of RNA polymerase complex on a single piece of DNA
imaged at roughly every 70 s [122].
Hansma et al. [119] imaged DNA in water and in propanol. DNA was extensively dehydrated by
baking in vacuum before being transferred to an aqueous environment containing MgC12 and
imaged with electron beam deposited (EBD) tips. DNA was imaged wider (19 _+ 4 nm) and higher
(2.5 _+ 0.5 nm) in aqueous environment than in propanol (9 _+ 3 nm and 1.6 + 0.5 nm, respectively)
presumably because the A F M tip was imaging hydration layers around DNA, according to the
authors. Some topographic features were recognized along the length of DNA but the authors did
not interpret them as reflecting the helical nature of DNA structure.
Hegner et al. [120] immobilized DNA on gold-coated mica using thiol modification. DNA of
a specific length was PCR amplified using a primer having 5'-oligodeoxynucleotide that was
esterified at the terminal phosphate with a 6-mercaptohaxanol group. The amplified double helical
DNA fragments were thus thiolated at both ends. An aqueous solution of thiolated DNA was
applied on a gold-coated mica surface for 40-60 min and dried without blotting so that the number
density of chemisorbed DNA could be increased. The dried sample was washed to remove free DNA
and submerged in either an aqueous salt solution containing dithiothreitol (DTT) or in propanol for
A. lkai/Sut~l~we Sciem'e Reports 26 ( 1996 ) 261 332
305
A
Fig. 20. RNA polymerase bound to a promotor containing DNA fragment in (B), unbound in (CI and rebound in (DI (after
Guthold et al. [122]: reproduced with permission of authors and publisher}.
AFM imaging. DNA was imaged with an expected length for B-DNA, its width being thinner at the
two ends where it was anchored to the substrate and wider in the middle, which, the authors believed,
allowed the freedom for DNA molecules to react with other molecules such as enzymes. The gold
surface that was imaged in the presence of D N A was highly corrugated despite the fact that the
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A. lkai/Surface Science Reports 26 (I 996) 261 332
original surface was flat with a mean roughness of 0.2-0.3 nm over 25 g m 2, which was attributed to
the instability of tip movement at a very low force (the lowest was 1 nN) reacting to discontinuities
and/or salt deposition on the gold surface. When a higher force was used for imaging, the substrate
surface was flat according to the authors. The result in propanol was better in the sense that DNA
was imaged thinner than in water and the substrate surface was clean. Inclusion of D T T in DNA
solution was necessary to disaggregate DNA but, the question whether it would have reacted with
the gold surface and caused the observed corrugation, remains.
Hansma et al. [121] recently published images of DNA with the best resolution so far, revealing
a periodic corrugation along a DNA strand with repeats corresponding to the double helical
structure of DNA. Thus the capability of AFM to discern detailed structures of DNA related
complexes has been verified.
An early attempt to image DNA molecules with A F M can be found in [108], where a single
stranded DNA was covalently linked to a surface of LB film that was formed on a mica surface.
Images of DNA, immunoglobulin and the LB film itself were given but they were not clear from the
present-day standard. One of the impressive points in this early attempt of DNA imaging is that the
imaging was already done in water or aqueous buffers.
An early success in imaging uncoated single and double stranded DNAs was achieved on a mica
surface that was soaked in MgC12 and glow-discharged under reduced pressure [126]. Instead of
dropping a DNA solution on a mica surface, they inverted a piece of freshly cleaved mica onto a drop
of DNA solution placed on a piece of Parafilm sheet. The ~X-174 genome was chosen as a single
stranded DNA sample and it was stored in 0.5% formaldehyde. They described various attempts to
spread the single stranded DNA on a mica surface and even tried rinsing the DNA-coated surface
with hot water. A F M images were taken in propanol and butanol as well as in air using ion-milled
supertips which were prepared by depositing carbon on ordinary tips in a scanning microscope.
Single stranded DNAs were imaged with more lumps along the strand than double stranded lambda
phage D N A for unknown reasons. Imaging was more difficult on a single strand DNA than on
a double strand. Some parts of the single strand DNA were imaged with a width approaching 3 nm.
Shaiu et al. [127] labeled DNA with a spherical gold particle at one end and deposited the labeled
DNA on a mica surface, dried it with nitrogen gas and imaged it with a contact mode A F M either in
air or in propanol. Apparently the gold sphere at the end of DNA stuck to the mica substrate rather
firmly and allowed a more stable imaging of DNA than with an unlabeled DNA. The height of
nominally 5 nm gold spheres was estimated as 5.8 + 0.12 nm from the image and that of DNA was
0.54 + 0.12 nm in air of RH less than 10%. The height of the gold sphere was in a correct range but
the authors considered the error range too large to be used as a height standard though height
standards for A F M in this range are desperately needed. The wide range of the height value was
primarily due to a variation in the sample which was nominally claimed as 5 nm gold spheres. The
low value for the height of DNA was probably due to the deformation of soft DNA under the force
exerted by contact mode AFM.
The Fur (for ferric uptake repression) repressor is an example of DNA binding protein studied by
EM and AFM. Cam et al. [128] studied the location of the protein on DNA by Electron Microscopy
and AFM. In this case there was no bend in the DNA structure after Fur binding. The comparison of
A F M images with those of dark field EM images was generally favorable. The repressor protein
bound on DNA had a shape like a peanut shell with two globular domains. The domain structure
was clearer in the A F M image than in the dark field TEM image of a metal coated sample.
A. lkai/Surl'ace Science Reports 26 (1996 ) 261 332
307
An interesting class of compounds with a capability of binding to DNA and straightening it were
studied by AFM [129]. Apparently these compounds non-covalently bind to the major or minor
grooves of double stranded DNA and reduce the flexibility of DNA chains. Distamycin is one of such
natural products that straighten on abnormally bent kinetoplast DNA, whereas a compound called
microgonotropen-6b (MGT-6b) is a synthetic one composed ofa tripyrrole peptide (that binds to the
minor groove of DNA) and a polyamine, that binds to the phosphate backbone. The AFM images of
kinetoplast DNA with and without distamycin or MGT-6b showed a distinct difference in the shape
of the samples. Bending of kinetoplast DNA was straightened by binding 1 MGT-6b per run of A's,
that is four to six successive A bases, whereas approximately 10 times more MGT-6b must bind
before normal DNA was to be bent. The extent of bending was represented in terms of the
frequencies of the end-to-end distance of the imaged DNA with results showing a distinct shift in the
frequency histogram.
Bezanilla et al. [130] studied a dynamic process of degradation of DNA in the presence of DNase
I, an enzyme that hydrolyzes phosphodiester bonds of DNA without requirement for specific base
sequences. They adsorbed a double stranded DNA on a mica surface which was treated with NiCI 2
prior to application of a solution containing DNA and the loosely adsorbed DNAs were washed off
with high pressure water. The procedure has been found rather important to image DNA in buffer
[131]. The nickel ion was supposed to give a better anchor for negatively charged DNA on mica than
the previously used Mg + + ion. After an AFM scan was started in a tapping mode for imaging of
DNA, DNase | was added to the DNA solution and progressive splitting and degradation of DNA
were continuously monitored with repeated scans (see Fig. 21 }.
DNA strands were observed to be split at several sites and fragmented into short strands and
finally to disappear from the field after being scraped off from the substrate. Enzyme molecules of
MW = 31 000 were not observed even on DNA presumably because the reaction time of the enzyme
was much faster than the scanning time of AFM. The authors attributed their success to a loose
adsorption of DNA and more importantly to the choice of DNase I which has been known to act on
an immobilized DNA and without specific sequence requirements for the recognition of the cleaving
sites. When an endonuclease such as Pvu II or an exonuclease such as Exo lII was used, there was no
indication of DNA degradation on the adsorbed DNA because these enzymes need specific sites on
DNA to cleave and sliding motion on the part of the enzyme along the DNA strand has been
speculated to be involved in the process of searching such sites. The strongly adsorbed DNA on the
surface will not allow sliding of enzymes along its helix axis. The paper showed that if one chooses
a right kind of enzyme and right conditions for DNA adsorption, the process of DNA degradation
can be monitored.
The effects of adhesion and scanning force were examined on DNA and a linear fd phage by
Lyubchenko et al. [132] in air, in water and in propanol. They carefully measured the width and
height of DNAs in the images taken under various environmental conditions and concluded that the
AFM image resolution depended more on the adhesion than the force exerted by the tip on samples.
Z-DNA and G-wire DNA have been treated by Pietrasanta et al. [133] and Marsh et al. [134].
Z-DNA which was inserted as a d(CG)~I fragment into pAN022 DNA plasmid was biochemically
identified by using a specific antibody. The antibody was identified as a globular protrusion at a fixed
position in a linear DNA molecule. G-wire DNA is a member of the quadruple helical nucleic acid
family and its structure was imaged clearly with AFM.
308
A. lkai/Sur['ace Science Reports 26 (1996) 261 332
A
C f--
g~
Q
D
,%.
-¢.
•
E
F
g
v'ig. 21. Enzymic degradation of loosely anchored DNA on mica in a series of photographs. The DNA molecule marked
with an arrow in (A) is fragmented into small pieces and carried away (after Bezanilla et al. [130]; reproduced with
permission of authors and publisher).
3.3.3. Lipid membranes
Langmuir-Blodgett films of various compositions have been the targets of intensive studies with
AFM and STM. Here, literature citation will be limited to those with immediate biochemical
applications. Meyer et al. reported the molecular arrangement of the head groups of cadmium
arachidate LB film adsorbed on an amorphous silicate substrate [135]. A similar cadmium
arachidate LB film was formed on a crystalline silicon wafer and they found that crystalline arrays of
lipid head groups were only visible when the film was either three or five layers thick [136].
Single-layered areas remained amorphous to AFM imaging. Bourdieu et al. [ 137] studied the effect
of heating and annealing of barium arachidate LB film. Finally Hui et al. studied the stability of
biologically more relevant membranes of distearoylphosphatidylcholine, dipalmitoylphosphatidylethanolamine and dilinoleoylphosphatidylethanolamine[138]. Unlike the LB films derived
from arachidate, the phospholipid membranes studied by Hui et al. did not show crystalline
arrangements of the head groups. LB films of other types have also been studied [139-141].
3.3.4. Viruses and cells
Viruses have been good targets for imaging with AFM especially in an early phase of its
application to biology. Bacteriophage T4 was first imaged by Kolbe et al. [142] with a distinction of
the head and tail. The result was soon improved by Ikai and his collaborators with a successful
imaging of tail fibers of 2 nm in diameter. Zenhausern et al. and Imai et al. imaged tobacco mosaic
virus as well as bacteriophages [143-146] (see Fig. 22).
The dimension ofT4 phage in the air dried state was: the head (W= 140 nm, H = 50-60 nm); the
tail ( W= 40 nm, H = 17 20 nm), the tail-fiber( W= 7 8 nm, H = 1-2 nm) according to Ikai et al. [ 143].
A. 1kai/Sur~we Science Report,s 26 ( 1996 ) 261 332
309
Fig. 22. AFM image of bacteriophage T4 dried and imaged in air clearly showing the tail fibers of 2 nm in diameter {after
lkai el al. [143]: reproduced with permission of authors and publisher).
When compared with the known structure of the phage, the height of the head was 70-80%, and its
width almost twice as wide as the expected values. The height of the tail and tail fibers was within
80% of the expected values. When the virus particles were coated with metal and scanned with an
STM, the height of the head turned out to be within _+ 10% of the expected value [146]. Occasionally
phage particles with a very low head height were observed corresponding to "empty" head particles
that were devoid of DNA normally packed in the head. One advantage of AFM over other
microscopic methods like electron microscopy is its ability to directly give information on the height
of the specimens. As to the biological structures, this argument does not apply even for well-packed
structures such as the phage head.
Imaging of cells either in dried or wet state has been tested for various different purposes.
Braunstein and Spudich [147] succeeded in imaging live rat basophilic leukemia (RBL-2H3) cells
which are known to be activated by cross-linking the IgE receptors on the cell surface. They
presented EM and AFM images of detergent extracted cells in an unactivated and an activated state
showing a difference in the network structure. They imaged the surface of an intact nucleus and
found that nuclear pore complexes were clearly imaged on its surface. Images of the complexes are
very clear and promise the usefulness of AFM technology in the study of cell biology. They aimed at
using A F M in cell biological studies to correlate the intracellular processes that could be visualized
with, for example, the differential interference contrast optics of a light microscope with the events
taking place on the cell surface, the latter were to be imaged with AFM. For this purpose, they took
310
A. lkai/Sutface Science Reports 26 (1996) 261-332
time sequence images of the surface of non-activated and activated cells of RBL, one image per less
than one minute ( 4 - 5 H z scan per one raster scan), and claimed that they observed gradual
movements of granules and surface waves with respect to the stationary spots in the image. To be
honest, it is rather hard to follow their detailed explanations of the movement of individual
structures on the given images but there were certainly some movements. Stationary images are easy
to evaluate but dynamic images are difficult because the processes to be identified by the readers
were either too small as in the case of granules or not well defined to unfamiliar eyes as in the case of
waves.
Another paper that caught a granular motion in the cellular processes was presented by Fritz et al.
[148] using human platelets during activation. They studied the activation process of human
platelets from the initial formation of filopoda to the fully spread form with A F M as reproduced in
Fig. 23.
Fig. 23. Platelet activation process caught with AFM. In a series of photographs from (A)-(H), each paired with an error
signal mode image on the top, changes in large and small corrugations are recorded (after Fritz et al. [148]; reproduced
with permission of authors and publisher).
A. lkai/Sur[itce Science Report,s 26 ( 1996 ) 261 332
311
Platelets are, according to the authors, activated after stimulation by agonists such as thrombin,
ADP, or a foreign wettable surface and changes its form from a spherical one of 3 jam in diameter to
a flat and spread form of 7 10 jam. During the activation process, certain granular structures are
known to move to the cell surface and fuse with the membrane, secreting chemical agents that
increase the surface area of the platelet. They studied the redistribution of granules during the
activation of individual platelets on a cover glass by AFM. Their AFM images of platelets clearly
showed: t 1~adhesion of the cells with an extension of lamellipoda, (2) appearance of large granules in
the cortical area of the adsorbed cell; and (3) an eventual disappearance of them with an increase of
the size of the cell. In a completely activated state, it was possible to identify the cytoskeletai structure
by probing from the outside of the cell. In the course of platelet activation, it was possible to image
a spreading process of the cell front by repeated extensions of sharp fibrous structures. It is very
promising that an activation process of platelets could be studied with such clarity. They also
reported that there was almost no attractive interaction between the tip and cell surface.
Migrating cells and the nanometer level microscopic processes associated with the cellular
locomotion were studied by Oberleithner et al. [149] using MDCK (for Madin Darby canine
kidney) cell line that was transformed with an alkaline treatment. Cells were grown on acid-cleaned
uncoated cover slips and imaged in a liquid cell with a constant superfusion of culture medium that
was kept at 37°C. Since the cells had a flattened morphology and were larger in diameter than the
largest scan area of the AFM used, the retracting tail part was separately imaged from the advancing
front part. They limited the scan force to equal or less than 10 nN and the scan rate between 10 and
12 Hz. Magnified images were obtained with a scanning force of 1 nN. Their observation in the
lamellipodial area of the advancing front of the cell revealed appearance and disappearance of many
dimples of the order of 90 nm in diameter and a depth of 6 nm. The density of the dimples was
roughly 10_+ 3 per jam2 and the total number of 2630 such dimples were calculated from
a lamellipodial area measurement. The authors attributed these dimples to have been formed by
endocytotic activities of the cell membrane, and argued that the membrane material internalized by
this activity was supplied to the membrane structure at the leading edge of the migrating cell. They
confirmed by calculating the size of each invagination and their number and concluded that if the
quantitative amount of the membrane material thus withdrawn from the lamellipodial area was
re-inserted to the membrane at the moving front, the material basis of the cell movement at 1 jam/min
could be accounted for. The dynamics of appearance and disappearance of the invagination was
estimated to have a time constant faster than 1 min because some of the dimples were only imaged
once and a successive imaging of the same area 52 s later was not able to locate it. The observed
endocytotic activity was dependent on the calcium concentration in the medium. When the calcium
concentration was abruptly reduced, formation of dimples was stopped and the cell surface became
smooth, and smaller (30 nm in diameter and 8 nm in depth) lipidic pores were observed at a density of
1 per 40 jam 2. The appearance and disappearance of such lipidic pores were again relatively fast and
a pore imaged once could not be imaged 15 s later.
It is very encouraging that the nanometer order microprocesses associated with the cell migration
were imaged with AFM. Such microprocesses cannot be observed with a conventional light
microscope, and an electron microscope, though equipped with a better resolution, cannot be
operated on live specimens. This, therefore, is one particular area where the AFM technology should
make significant contributions. What is now apparent from the study described above is that the
scan rate of AFM should be much faster than the present limit on the soft and convoluted samples
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A. lkai/Sur[ace Science Reports 26 (1996) 261 332
such as live cells. The scan rate of A F M itself can be set faster than 10 Hz but at a faster scan rate the
image quality is reduced and the damage to the cell becomes non-negligible.
M D C K cells were also the focus of the study by Grimellec et al. [ 150]. They studied the cell surface
morphology of the M D C K cells in dry as well as under liquid conditions. In a dried state, AFM
could image granular surface features of the cell but when imaged in living conditions and with
a scanning force of 5 nN or less, the cell surface was almost featureless due to the presence of a highly
branched polysaccharide layer called glycocalyx over the cell membrane according to the authors.
They then treated the cell with neuraminidasc, an enzyme known to digest glycocalyx layers by
hydrolyzing the ct-ketosic linkages of N-acetyl neuraminic acid to the neighboring unit. After such
a treatment, the cell surface was imaged as densely covered with globular particles of an apparent
diameter of 10-60 nm and a height of 2 4 rim. The lateral size was too large for these protrusions to
be membrane proteins, but subsequent treatment of them with a protein degrading enzyme, pronase,
ahnost completely eradicated them, strongly suggesting that they were in fact membrane associated
prolcins.
l h e use of enzyme reactions as described above to identify the chemical nature of the imaged
structures will remain as an important methodology in the absence of a spectroscopic capability in
the A F M technology. In electron microscopy, labeling of target molecules with specific antibodies or
those linked with colloidal gold particles has been extensively used along with enzyme treatment of
the specimens. When the immunological reaction was not strong enough, a stronger interaction
between avidin and biotin is used, often in combination with antibody-antigen reactions for
a greater specificity. Examples of such studies have been reported by Neagu et al. E151] and Saoudi
et al. [152]. Neagu et al. used the immunolabeling technique to locate CD3 containing receptors on
the surface of blood lymphocytes prepared from human blood. Cells were incubated with a mouse
monoclonal anti-CD3 antibody labeled with fluorescein isothiocyanate and then incubated with
goat anti-mouse antibodies linked to 1 and 3 nm colloidal gold particles. The labeled cells were fixed
on a glass substrate with a paraformaldehyde acetone and the silver enhancement procedure of gold
particles was performed. The cells were examined for fluorescence labeling as well as for silver
enhanced gold staining with a fluorescence microscope and AFM, and corresponding images
obtained by both techniques were compared for the identification of labels on cell surfaces. The
surfaces were rather strongly corrugated and it was difficult to locate small gold particles without
silver enhancement. They calibrated the size of silver enhanced gold labels on a poly-lysine coated
glass surface. Correspondence between fluorescently brighter parts of the cells and the surface
corrugation obtained with an error signal mode of A F M allowed an identification of CD3 specific
labels. They claimed that labeling with antibodies that were linked to 1 nm gold particles had a more
uniform and denser labeling than antibodies with larger gold particles. It was difficult to differentiate
colloidal gold from the native protrusions on the cell surface because the surface of the leukocytes
was strongly convoluted as noted above. The authors tried labeling of transferrin receptors with
specific antibodies linked to superparamagnetic beads of 5 nm in diameter because the surface of red
blood cells was smoother than that of the leukocytes. The density of the labels on red blood cells was
surprisingly low and individual beads were clearly identified with A F M imaging. Their conclusion
on the capability of A F M in an identification of gold labeled antibodies on the cell surface was that
the silver enhancement procedure was an indispensable step and the image taken with silver
enhancement should be compared with those taken without the procedure to identify the true size
and location of the small gold particles.
,4. lkai/Smf~we Science Reports 26 ( 1996 ~ 261 332
313
Seoudi et al. [152] used a gold labeling technique to thin sections prepared from sunflower
cotyledons infected by Sclerotinia sclerotiorum. The purpose was to locate the presence of enzymes
derived from the infectious fungus using anti-fungal enzyme antibody. The thin sections were fixed
with paraformaldehyde and glutaraldehyde and dehydrated in alcohol and embedded in resin. The
fixed sections were labeled with a rabbit anti-fungal enzyme serum followed by colloidal gold (20 nm
in diameter) conjugated anti-rabbit secondary antibodies. This was an interesting attempt to use
AFM in tissue labeling work. Resin embedded samples were rigid and easy to scan with AFM and
the labeling with the antibodies took place only on the surface of the section. Thus AFM was ideal to
locate gold labeled antibodies.
Phagocytosis of latex spheres (450nm in diameter) by mouse peritoneal macrophages was
studied by Beckmann et al. [153] with the aim to "bolster" the cytoplasm, i.e. to make it appear
bloated for emphasis. They reported that they were able to image the phagocytosed particles
from the outside of the cell even for the living cells growing in the culture medium. The force
distance curve was typical for soft material but the theoretical equation used by Tao et al. [154]
could not be fitted to the observed curve. When the cells were fixed with glutaraldehyde and dried,
the protrusion due to the presence of latex particles became more prominent but the detailed
topography of the cell surface was not discussed. According to the authors, cytoskeletal elements did
not seem to be involved in the phagocytotic activity of particles of this small size. Zymosan particles
of an oblong shape were also clearly imaged after phagocytosis by maerophages. The purpose of this
study was thought to be an improvement of the images of membrane surface by "bolstering" the
cytoplasm, but the result did not seem to show an expected advantage of using phagocytosed
particles for the betterment of resolution of the cell surface. Perhaps, the use of gold labeling together
with the bolstering technique adopted in this article will enhance the resolution of the membrane
surface.
Fine images of subcellular structures of rat m a m m a r y carcinoma cells in dry state were reported
by Pietrasanta et al. [155]. Using fluorescence microscopy for the spectral identification of the
structures, they obtained the nucleoli, microspikes, microvilli, and after selective removal of plasma
membrane and proteins, they identified mitochondria in the perinuclear space, together with the
cytoskeletal networks. Mitochondria which were clustered in the perinuclear regions were imaged
with a snake-like morphology but beyond that the image was not informative.
The shape of the uremic red blood cells was investigated by Zach6e et al. [156]. Red blood cells
from uremic patients were collected and the characteristic echinocytes were identified with a light
microscope and subsequently imaged with AFM in dried state, Echinocytes are deformed red blood
cells having 10 30 spicules (spike like protrusions) evenly distributed over the cell surface. AFM
revealed the presence of 11 large spicules all over the cell surface and a large crater in the center of
the cell, repeatedly observed in many echinocytes, but its identity was not determined. Since
images were taken on a dried sample, their true dimensions and the shape of the cell itself were
unknown.
The early events in the neurite extension process of neuroblastoma cells induced by retinoic acid
were studied by Bonfiglio et al. [157]. They kept human neuroblastoma cells on glass with the
culture medium. They studied the effect of all-trans-retinoic acid which is known for its powerful
morphogenic and differentiating properties. After establishing that AFM can image fine structures
such as neurites on the cell surface they studied time dependent changes of the cell surface structure
as reproduced in Fig. 24.
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A. lkai/Sur/hce Science Reports 26 ( 1996 ) 261-332
b
a
5o
4o
3o
2o
lO
o
10
o
C
20
30
40
50
0
,,
5
10
15
20
d
14
14
12
12
I0
10
8
8
6
6
4
4
2
2
0
0
0
2
4
6
8
10
12
14
0
2
4
0
2
4
10
12
14
6
8
10
12
14
e
14
12
10
0
6
8
Fig. 24. The time course of neurite extension from the cultured neuroblastoma cells in response to the addition of retinoic
acid at a final concentration of 5 pM (after Bonfiglio et al. [157]; reproduced with permission of authors and publisher).
A. lkai/Sulface Science Reports 26 (1996) 261 332
315
This is a good example where AFM was used to obtain new information rather than to confirm
previously obtained data. The resolution of AFM was just right to fill the gap between the optical
and electron microscopes and especially it was effective to have complemented the data from EM by
the capability to work on the living cells.
Imaging of neurons in dry state was also reported by U m e m u r a et al. [158]. They found that the
surface of the cells was covered with globular particles in a range of 20-50 nm in diameter. They were
likely to be protein particles on the cell surface but the authors did not pursue biochemical methods
to establish the identity of these particles.
Plant cells are more difficult to prepare for AFM. They are protected by hard cell walls and do not
stick to ordinary substrates such as glass or HOPG. It is, therefore, not difficult to imagine that hard
spherical objects with little tendency to stick to the substrate are not easy to locate even with AFM,
let alone to image them. Kasas and Ikai [75], and Gad and Ikai [76] challenged this problem and
came up with two different solutions, both using almost spherical yeast cells as sample specimen.
Kasas and Ikai used a Millipore filter to trap yeast cells (Saccharomyces cerevisae) in pores matching
in size under a slightly negative hydrostatic pressure through the filter. Yeast cells were then imaged
in a captured state in the pores with little freedom to move. On the surface of yeast cells, clear images
of bud scars were obtained. This is a very stable way to immobilize round objects for AFM
investigations.
Gad and Ikai [76] improved the above method by immobilizing the yeast cells on the surface of
thinly layered agar. Yeast cells were evenly distributed on an agar surface with a surprisingly high
density and with freedom to grow and bud daughter cells. When the cells outgrew the agar layer by
approximately half its height, they were easily displaced with a scanning tip. The growth process of
a daughter cell from its mother cell was clearly imaged with a precise extent of agar coverage over the
cells. Reference experiments using autoclaved and chemically fixed cells did not detect any
meaningful change in the cell position or its dimensions, confirming that the observed emergence of
live cells from the agar surface was indeed a growth process and not due to the recession of the agar
surface. The budding and birth scars were also very clearly imaged and the distortion of their images
due to the high profile of the cells was demonstrated. The results on immobilized yeast cells are given
in Fig. 25.
3.3.5. Miscellaneous subjects
Humic substance was studied by Osterberg et al. using X-ray solution scattering and A F M [159].
This heterogeneous mixture of variously dehydrogenated biomaterial has been a difficult target of
scientific study. Some insights have been obtained by a combined use of solution scattering and
AFM. The size of the major components in humic substance prepared by a relatively mild treatment
of the soil was estimated from solution scattering to be in the range of 100-300 nm. AFM images of
dried material revealed the presence of quasi-globular particles o f a corresponding diameter and
their dried surface had a characteristic wrinkled feature which was taken as representing the fractal
nature of the material suggested from the solution study.
An application of AFM to the study of the interaction of dentin and dentin adhesives with tooth
hard tissue was undertaken by Schaad et al. [160] and Cassinelli and Morra [161].
The growth of crystals and its dissolution kinetics were studied with AFM including those of
amino acid crystals [162 164]. Crystal surfaces were very clearly imaged but the atomic resolution
has not yet been obtained.
316
A. Ikai/Surface Science Reports 26 (1996) 261 332
h
e
d
Fig. 25. AFM images of immobilized live yeast cells. Cells 1,2, and 5 are growing in the time sequence images from (a) to (f),
whereas cells 3 and 4 are budding daughter cells (after Gad and Ikai [76]; reproduced with permission of authors and
publisher).
3.4. Force measurements
3.4.1. Basic physical interactions
In 1991, Butt discussed the electrostatic, Van der Waals, a n d h y d r a t i o n forces in electrolyte
solutions [,165]. He a p p r o x i m a t e d the electrostatic force between a tip a n d charged substrate as
follows.
F~
_
2n)LDR[-( 2 . 2,--2~,';D
+O-s) c
.. +20.TO.Se D.'54:,],
(3)
~o ~
where a T a n d a s are the surface charge densities of the tip a n d sample surface, eo a n d e represent the
v a c u u m permittivity a n d dielectric c o n s t a n t of water. D is the distance between tip a n d sample
surface, a n d R is the radius of tip apex. 2 D is the Debye length which for a m o n o v a l e n t salt of
c o n c e n t r a t i o n C a n d at 22°C is given by
0.304
•~ o - x ~
nm
(4)
A. lkai/Sur]'ace Science Reports 26 ( 1996 ) 261 332
317
Butt investigated the electrostatic force acting between the tip and substrate or between the
tip and sample in aqueous buffer solutions, where Van der Waals and electrostatic interactions dominate over other types of forces [166 168]. He also measured the local charge density
of the purple membrane containing bacteriorhodopsin and adsorbed on an alumina substrate
[166].
The interaction force between the tip and surface was assumed to be the sum of electrostatic and
Van der Waals interactions. The electrostatic interaction is influenced by the concentration of ionic
species in the solution whereas Van der Waals interaction is not. The electrostatic force was further
divided into two terms, one due to the electric stress tensor and the other due to the osmotic pressure
caused by the change in the ionic concentration around the tip surface interaction area. Butt
elaborated on both numerical and approximate analytical solutions for the electric stress tensor part
and found significant differences between the results obtained by the two methods when the distance
between the tip and the surface was less than 0.5 nm.
As has been pointed out in a previous work using a macroscopic surface force measurement
apparatus, the interaction between a charged surface and charged tip was found to be more often
repulsive than expected from the sign of the surface charges. At short distances, a negatively charged
tip may be repulsed from a positively charged surface because the tip with lower electrical
permittivity displaces the water near the surface, creating an energetically less favorable state than
before. According to Butt, only when the absolute values of the surface charge densities are the same,
the interaction between the tip and the surface becomes attractive. In the case of atomic force
microscope using a conductive tip, the electrical permittivity of the tip is infinite and the opposite
situation can be expected where the interaction between a charged surface and a conducting tip
under an applied potential will be attractive regardless of the sign of the surface charge. The
magnitude of electrostatic force in various concentralions of added salts was never larger than
10 "'N but since the force curve measurement in AFM is sensitive enough to measure such forces,
one must be aware of a theoretical prediction given by Butt. The hydration force between a tip and
surface is not taken care of in the continuum approximation adopted by Butt. Other shortcomings of
macroscopic models were also discussed.
Butt also pointed out that it is important to notice that not only the electrostatic but also the Van
der Waals force are greatly reduced in water because of the high dielectric constant and refractive
index of water as has been noted in [77]. The electrostatic force is still more reduced in ionic
solutions due to the Debye shielding effect. In water, moreover, there is no capillary effect. These
advantages expected for the measurement in water, and especially in physiological buffer solutions
are good news for the biological application of AFM.
Hob et al. [169] pointed out a possibility of hydrogen bond formation between the two oxidized
silicon surfaces, one on a silicon nitride tip and the other on a glass substrate in water. The adhesion
type interaction of the order of a few to a few tens o f n a n o Newton between the tip and glass substrate
was prominent in pure water below pH 10 and persisted in 10mM sodium phosphate below pH 7.
They also reported the possibility of measuring the strength of such hydrogen bonds in multiple
quantized steps involving the smallest force of 1.2 × 10 ~1N, i.e. 12pN [170].
Weisenhorn et al. [171] compared a non-specific interaction between a non-conductive tip and
substrate to that of a conductive tip and substrate and found anomalous force curves in pure water.
It is therefore important for biologically oriented researchers to extensively check force distance
curves between the tip and substrate before the measurement of tip-sample interactions, it is
318
A. lkai/Surface Science Reports 26 (1996) 261 332
noteworthy from a practical point of view that the adhesive force between a tip and mica substrate
they observed was dependent on the depth of the tip indentation.
Radmacher et al. [ 172] devised a way to measure viscoelastic properties of a sample with the force
modulation mode of A F M which images the sample surface according to the difference in the
viscoelastic properties. The amplitude change of the vibrating cantilever oscillation represented
chiefly the elastic properties, while the phase shift of the oscillation those of the viscosity. They
imaged the surface of D P D A (for diaminodiethylene glycol-pentacosadiynoic acid)monolayer film
and several other films with their force modulation mode AFM. Radmacher et al. [173] later
proposed a force mapping method of A F M where they measured force curves at 24 000 data points
in a single scan frame and displayed them in a special 3D display diagram.
3.4.2. Inter- and intra-molecular forces
With the arrival of AFM, it has become possible for the first time in history to measure interaction
forces between single pairs of biological macromolecules in terms of newton/molecular pair, rather
than in terms of binding constants or equivalently the free energies of interaction derived thereof.
The first of such measurements has been presented by Florin et al. [174] for the avidin biotin
system, the most popular binding system in biochemistry. They coated agarose beads with avidin
and derivatized a silicon nitride tip with biotin. When the AFM was operated in the force curve
measurement mode, they were able to detect a downward deflection of the cantilever which was
proportional to the force exerted on the cantilever. Generally speaking it is not possible to tell
whether the cantilever was measuring the interaction force of a single pair of biotin and avidin or that
of multiple pairs, but Florin et al. were successful in identifying that the force required for the final
release of the tip from the substrate were multiples of 160pN. The repeating unit of 160pN was
considered to be most likely corresponding to the force required for dissociating a single pair of
avidin and biotin (Fig. 26).
Dammer et al. showed that they could measure the force between two molecules of peptide glycans
prepared from marine plant [175]. In this instance, the tip and the substrate were both covered with
the same kind of peptide glycan molecules. First the peptide glycan molecules were imaged under
A F M as comb-like molecules in good agreement with the electron microscopic image. Then the
interaction force was measured in much the same way as in the previous example (see Fig. 27).
Since the molecules had an elongated shape, it is not pdssible to confine the measured force to
a restricted part between the two molecules, but the force obtained at a reduced density of molecules
was interpreted as the interaction force between a single pair ofglycans. They calculated that a single
pair interaction force was strong enough to hold about 1600 cells against the gravity.
D a m m e r et al. reported another case of force measurement using antibodies raised against biotin
[ 176]. Both the tip and the substrate were gold coated and fuctionalized with organic monolayers of
dithio-bis(succinimidylundecanoate) that bears thiol groups to react with gold and a succinimidyl
group to react with amino groups of proteins. The antigen was ligated to a protein, bovine serum
albumin, and covalently adsorbed on the functionalized surface of the tip. Antibodies were also
covalently adsorbed to the functionalized surface of the substrate. Antigens and antibodies on the tip
and the substrate were allowed to form specific non-covalent bonds while they were in touch during
the force curve measurement. A quantized amount of 60pN was resolved from the graphical
representation of the magnitude of the force required at the final lift-off of the cantilever. One
questionable point is that the measured force curves suggested that some part of the system was
A. Ikai/Surface Science Reports 26 (1996) 261 332
A
~ , _ _. ~ ' ~
319
Approach 5 nNI
_.- 100rim
action
B
o
M.
s°./
. . . . . . . .
! °°"m
C
.
.
.
.
.
00 50n °
Displacement
Fig. 26. The force curve used for the measurement of the force required to separate avidin biotin pairs (after Florin et al.
[174]; reproduced with permission of authors and publisher, copyright [-1994] American Associationfor the Advancement
of Science).
stretched up to 100 nm before the final lift-off. If this stretching involved that of antibody molecules,
the measured force may have involved partially denatured proteins.
Lee et al. [177] measured the force between complementary strands of synthetic D N A (polydeoxyribonucleotides) in 0.1N NaC1, pH 7.0 and 25°C. The synthetic polydeoxyribonucleotides had
complementary base sequences of(ACTG)s and (CAGT) 5 and they were thiolated at their 5'- and 3'ends. The two kinds of polynucleotides were, respectively, immobilized over the self-assembled
monolayers of 7-aminopropylaminoethyltrimethoxysilane on silica probe and substrate surface
with succinimidyl 4-(p-maleimidephenyl)butyrate. First, the probe-substrate distance was decreased
as in the ordinary force-distance curve measurement and a contact between the probe and surface
was established. As the distance was increased in the retracting realm of the force curve, a large
hysteresis pulling the cantilever d o w n w a r d was observed and finally the cantilever was released with
an abrupt j u m p to the horizontal position. The force sensed by the cantilever at the time of this
release was calculated and plotted in terms of the frequency. Four peaks were noticed in such plot
corresponding to 8, 12, 16, and 20 base pair disruption, according to the authors. It was not directly
determined as to how m a n y D N A molecules were interacting between the tip and the substrate but
the fact that the force vs. frequency plot had four peaks was taken as an indication that most of the
measurements were made for a single pair of complementary DNA.
320
A. lkai/Surface Science Reports 26 (1996) 261 332
:i:i::i::t::i:?+++H!+
:i i:2Li
Fig. 27. The force curve measurement on peptide glycan interactions. Force curves from (A) to (D) are representative cases
where glycan molecules are pulling down the cantilever and releasing it at the end. (E) is an explanation of cantilever
deflection in the approaching and retracting realm (after Dammer et al. [-175];reproduced with permission of authors and
publisher, copyright [19951 American Association for the Advancement of Science).
Mitsui et al. and Ikai et al. ]-178,179] reported a method to measure the force required to
mechanically unfold or stretch protein molecules by pulling apart the tip and substrate with
covalently sandwiched protein molecules between them. They used a mica substrate coated with
epitaxially grown crystalline gold, and first immobilized thiolated protein molecules on the
substrate. As the force~:listance curve measurement was started with a gold-coated tip, the
retracting realm of the force curve showed a hysteresis which was not observed when underivatized
protein was used or when the thiol groups on protein molecules were chemically blocked (see
Fig. 28).
As the density of the immobilized protein was reduced, the d o w n w a r d deflection of the force curve
began to have simple forms and could be interpreted as representing mechanical stretching events of
a single protein molecule. The distance between the tip and substrate immediately before the bond
rupture formed two peaks at 150 and 300 nm when a distance vs. frequency plot was made. F r o m the
primary structure of the protein used in this study, the former length corresponded to the stretching
of a single subunit and the latter to that of two subunits, according to the authors.
5 nm/div, t
I
I
~
I
t
50 nm/div.
b
5 nm/div
r
i
i
i
i
i
50 nm/div.
!\
',
tE
ilr
6 nm/div.
j
\ D
I
601nrn/dl v"
i
Dmax
Fig. 28. Force curve measurement in a single molecule stretching of proteins. Force curves taken over (a) a bare gold
surface, and (b) gold surface covered with protein without (b) and with (c) reactive sulfhydryls. Figure (ct contains definitions of
the length of stretched protein, D, and a maximum of D as Dmax. Figure (d) is a simple force curve obtained at a reduced
density of active sulfhydryl groups (after Mitsui et al. [178]; reproduced with permission of authors and publisher).
322
A. Ikai/Surface Science Reports 26 (1996) 261 332
d
6 nm/div.
I
60 nm/div.
I
i
I
l
I
I
t
I
Fig. 28 (continued).
The four experiments cited immediately above are interesting examples of how the A F M will be
used in the near future in biophysics and biochemistry, that is, to measure the force at a single
molecular level, be it that of an inter- or intra-molecular interaction. Such measurements will allow
us to calculate various mechanical parameters of biological macromolecules and supramolecular
structures at the single molecular level, which has never been possible before the advent of AFM.
Force measurements using laser tweezers have revolutionized the field of muscle and motile proteins
[180] and, with its vast potential capability, it is not unreasonable to expect that A F M will have the
same impact in wider disciplines of biological sciences because of its superbly better resolution, both
lateral and vertical, and of its much wider range of the available force.
A F M has an ability to respond to time-dependent changes of surface properties. If the process is
slow, a sequence of time lapse images will be taken and the result will be analyzed in terms of a slow
kinetics. Such slow processes have indeed been recorded and provided a technology to register
time-dependent biological phenomena taking place at nano to micrometer scales. The fastest scan
rate of A F M at the present state of technological advancement probably is in several seconds per
image and much too slow to catch dynamic processes of biochemical interests. Radmacher et al.
[181] reported a potentially interesting observation of cantilever response to the active enzyme
reaction taking place on a substrate surface. They placed lysozyme molecules on mica under
a physiological buffer solution. They kept the A F M tip over a monolayer oflysozyme molecules in
a liquid cell without scanning and recorded small deflections of the cantilever as a function of time.
There was a certain level of random vibrations of the cantilever even on a bare mica and the presence
of lysozyme on mica did not significantly change the amplitude of such random vibrations. When
substrate molecules (4-methylumbelliferyl-N,N',N"-triacetyl-chitotriose) for lysozyme was introduced into the liquid cell, the cantilever started to show larger vibrations with short durations.
Amplitudes of such spikes were rather constant and in the vicinity of 1 nm as reproduced in Fig. 29.
A. Ikai/SurJace Science Reports 26 1996) 261 332
A
323
Mica
1
0
.~
,...
,.,
, : ,..-~-~-.p,- . ~ . ~ • ...~...,~, - ~ . q . . , . - ~ . - ~ , . . : . ~ .
]B
,,.~..
Protein in buffer
1
A
o
,~. ~ ~:~: .'." ~ - : . . ~
.~..z,:.
~ - ~
C
.0
1-
"~ • ' ;
.....
'"
, ":,~;.'~'~i ~-'~%. ;
....
',-"
~
~',~ :-:,'~:
......... ,;:-:i
D
:i~
:i
Protein with inhibitor
1-
•
'~.
•L~-:.-:..."-
o-
.:,.,..~
Protein with substrate
• :;.
o-
t
~
:
.
.-.
E
::,
.
. , |
~; ...... -.'~:!::',~,
Protein with inhibitor and substrate I
1-
.~...~,~~,
0-
~.~.~..,.~..~
0
..,..'.~,~,~.~
• o~
.,..
-
_..-....
..:
|
..~..m;y:~.,~-,~ 1
I
I
[
I
1
2
3
4
Time
-
5
(s)
Fig. 29. Spike-like vibrations of a cantilever placed over lysozyme and substrate. The time vs. height fluctuation plots were
taken over (A) a mica surface, (B) a mica covered with lysozyme, (CI an enzyme substrate was added to (Bt, (DI an enzyme
inhibitor was added, and (E) both substrate and inhibitor were added. Vibrations with significantly large amplitude took
place only in (C) (after Radmacher et al. [181], reproduced with permission of authors and publisher, copyright [1994]
American Association for the Advancement of Sciencel.
The occurrence of spikes was clearly correlated to the co-presence of the enzyme and substrate.
The time scale of the spike appearance was not estimated and probably difficult to do so, because the
data were recorded at a frequency of 1 kHz, and four adjacent data points were averaged out.
Whether the response time of the cantilever is longer than the conformational change of the enzyme
is an important point. Introduction of an enzyme inhibitor almost entirely eliminated the occurrence
of spikes higher than 0.5 nm. The authors proposed that the AFM tip was somehow recording
certain dynamic processes associated with the enzyme function. The most straightforward interpretation is that the tip was monitoring the height change that is associated with the conformational
change suggested for the lysozyme action. Possibilities other than the conformationai change of the
enzyme were mentioned but not discussed fully. This is the first case of suggesting the real possibility
of following submillisecond conformational changes of protein molecules at the single molecule
level.
This work was later expanded to include dynamic oscillations of several other proteins using
a new technology of tracking a single target molecule under the tip [182]. The oscillation that
a cantilever sensed when it was placed over a single protein molecule was not limited to enzymes but
also recorded over an antibody molecule and a microtubule.
Using AFM as a measuring device of mechanical properties of small area of bulk material or those
of small structures in micro to nanometer range will be a very interesting field in the future. In this
area Radmacher et al. [183] again made a pioneering work by measuring force curves on a thin
gelatin film placed over a mica surface. Gelatin gel (5% in water) was prepared on mica and
324
A. lkai/Surface Science Reports 26 (1996) 261 332
submerged under pure water or water-propanol mixtures. The force distance curve was recorded
with a conventional A F M apparatus and the result is reproduced in Fig. 30.
The concave force curves obtained on the gelatin sample in various mixtures of water and
propanol were analyzed according to the theoretical model of Sneddon [184] in the form of
a logarithmic plot of the distance of indentation vs. the force. The resulting graph was consistent with
the spherical tip model of Sneddon [184,185] which predicted a slope of 2 in such plot when the
indentation was small, and shifted to a line with a slope of 23 as the indentation became deeper. There
is some confusion in earlier papers as to the quotation of(1 - v2) in Sneddon's equations as (1 - v),
where v represents Poisson's ratio. In the same manner, Eq. (13.100)in [5] is misquoted by a factor of
2 if R is to be defined as the radius of curvature of a paraboloid tip.
Radmacher et al. measured the viscoelastic properties of protein molecules using a similar method
they used for gelatin films obtaining values around 0.5 G P a [186]. They also used an adhesion
mapping technique to identify proteins on a mica surface.
Ikai et al. [179] measured force curves on human chromosomes in a physiological buffer and
applied the equation derived by Sneddon for a conical indentor. The force curves obtained on
a chromosome were surprisingly reversible even for an indentation reaching 30% of the total height
of chromosomes in buffer solution. An approximate value of Young's modulus calculated from the
data assuming Poisson's ratio of 0.4 was in the order of 105-106N/m 2. The apparent Young's
modulus showed a sharp increase when the pH was lowered from 5 to 4 and the surface of
chromosome became very sticky at the same time, presumably due to the change in DNA properties.
Such changes in the physical nature of nanometer-sized structures have become to be appreciated
with the development of AFM.
Tao et al. [187] discussed the possibility of measuring the microelastic properties of biological
material using force~tistance curves measured on three different samples, namely, stainless steel,
bone and rubber. The results for stainless steel and rubber were used to calculate the spring constant
"-- 100~/oPropanol
I
I
I
I
I
I
100
200
300
400
500
600
sample height [nm]
Fig. 30. The force curve obtained on gelatin in various solvent conditions from 100% water to 100% propanol (after
Radmacher et al. [183]; reproduced with permission of authors and publisher).
A. Ikai/Surface Science Reports 26 (I 996) 261 332
325
of a cantilever using the mathematical derivation by Sneddon [184] and the result for the bone was
an example of biological material. The rigidity constant of the rubber used in the A F M experiment
was calibrated with macroscopic apparatus giving a value of 1.1 × l0 TN / m 2 for G = E/2(1 + v),
where E and v stand for Young's modulus and Poisson's ratio of rubber. The analysis of the
experimental force curves gave a value of 1.3 × 107 N / m z for the same rubber. The agreement
between the two experiments was good and provided yet another way to calibrate the force constant
of a cantilever. A question remained how to approximate a pyramidal tip with a conical shape since
Sneddon's mathematical formulas are given for a conical, paraboidal, and spherical indentor, or
more in general for axisymmetric bodies. Tao et al. approximated a pyramidal tip with a cone of
semivertical angle of 72 ~'.
There seems to be some confusion among the authors in citation of the mathematical formulas
derived by Sneddon. His formulas for the relationship between the depth of the indentations made
on a flat surface and the force applied on indentors with a cylindrical, conical, paraboidal, and
spherical indentor will be recited in the following:
(1)
4GaD
p - __
1 --
(5)
P
for a flat cylinder with the radius of a, where P is the total force acting on the cylinder, D is the
depth of penetration of the punch and G is equal to E/2(1 + v) and v is Poisson's ratio.
(2)
4Gcot
p - - - D
~(1 -
2
(6)
v)
for a conical punch, where ~ is the semivertical angle and meant to be the angle as shown in
Fig. 31.
(3)
8G
p _ --(2kD3)
3(1 - v )
(7)
1/2
/'
D
/
¥
Fig. 31. Definition of the angle ct and other parameters in Sneddon's equation for a conical indentation as interpreted and
adapted by the author after Sneddon [184].
326
A. lkai/Surface Science Reports 26 (1996) 261-332
for a paraboloid of revolution, which in the case of shallow penetration may be used for
a spherical punch with a diameter equal to 2k, that is the radius of curvature ofa paraboloid at
its tip.
(4) For a spherical punch of radius R, D and P are parametrically related through a, the radius of
the fitting circle between the sphere and the elastic solid. The equation is referred to the
original paper cited above.
It is of great interest that the mechanical parameters of biomacromolecules and supramolecular
complexes can be measured using AFM. The mechanical parameters and surface properties, such as
adhesive and frictional ones, will undoubtedly become important parameters to understand the
basic mechanics of biological structures at the nanometer level with the advancement of nanofabrication.
Friction measurements on a sample surface may prove to be of interest in the future. Friction
measurements such as reported by Overney et al. [188] on multi-component organic films will find
wide applications in biological researches if the tip is somehow chemically modified so that the
friction between biologically interesting surfaces can be measured.
3.4.3. Chromosomal manipulations
In the preceding section, I have already introduced several papers that referred to pulling, pushing
and cutting biological samples with a tip. These processes are now called micro-manipulation and it
is gaining m o m e n t u m in the application of scanning probe microscopy to biotechnology. Cutting
DNA strands or chromosomes can be and has been done on a substrate but in rather crude ways.
Biochemistry stresses on the molecular specificity and cutting with A F M in a biochemically
meaningful way must have an unparalleled specificity with respect to the position of cutting.
Otherwise there are better methods of cutting DNA in solution. Learning from successful manipulations of single atoms with STM in the field of surface science of inorganic materials, we must be able
to cut out a desired section of target molecules or supramolecular structures, pick up excised
fragments, move them to desired positions, detach them from the tip, and finally place them securely
on the target site. At the same time, cut positions in the original molecules or structures must be at
least repaired or even renovafed with newly endowed functions. It is finally desirable that one can
multiply the results of manipulation so that the tremendous effort that went into the tedious process
of nano-manipulation would be replicated efficiently. The chromosome presents itself as an ideal
target for such operations and there are already several attempts to conduct micro-operations on
chromosomes [189-192].
4. Conclusion
With the STM and AFM, one can not only image molecules and molecular complexes but also
touch them and act on them. This sense of actually touching a single molecule gives a totally new
feeling to researchers, prompting them to explore the world of atoms and molecules with a sense of
innovation. With an STM, one may eventually visualize reactive parts in a molecule by correlating
the tunneling property with the chemical reactivity. It is now being elucidated as to why some
functional groups are more readily conductive to the tunneling current while other functional
A. Ikai/Surface Science Reports 26 (I996) 261 332
327
groups are less so. Imaging hydrocarbons and their derivatives in vacuum and at liquid-solid
interfaces is now routinely done and soon the oligopeptides and oligonucleotides will be common
samples for STM study. The electronic properties of multiple repeating peptide bonds which have
been discussed only in theoretical terms will become accessible to experimental analyses and may
prove to be of significant interest in the design of new proteins.
Imaging DNA at its atomic resolution now looks like an achievable goal, thanks to the continued
effort of many devoted researchers. Imaging, of course, comes first for any microscope technology
including AFM. It is also exciting to see wider applications of new technologies in biomedical science
and, in this respect, AFM will prove its use more and more in force measurement. To establish such
a usage as a legitimate scientific discipline, however, much is still left to be desired, for example, in the
instrumentation of AFM, sample preparation for trustworthy measurements, and in theoretical
background of nanomaterial mechanics and manipulations. The present status of the force curve
measurement mode of commercial AFMs is basically an auxiliary operation for checking the
imaging conditions. Thus the operational freedom in tip movement is severely limited and the
quality of microfabricated cantilevers is widely varied requiring calibration of the force constant for
each individual tip. Improvements will, for sometime to come, be made individually in interested
laboratories but eventually when the force curve measurement becomes a routine operation in every
laboratory with AFM, commercial instruments should carry every feature that researchers want to
have.. Sample preparation methods must also be improved to avoid unnecessary inactivation or
denaturation of biological samples and genetic engineering techniques should be used to create
sample molecules that will ensure meaningful single molecule measurements amenable to theoretical
analyses. The activity of such samples in the adsorbed state to the substrate should be assayed,
preferably, molecule by molecule. After all these requirements are met and a quantity of biochemically relevant information is presented, a new discipline of"Single Molecule Biochemistry" will see
its day.
Acknowledgements
I wish to thank Professor Osamu Nishikawa of Kanazawa Institute of Technology for providing
me the precious opportunity to write this review article and for helping me to explore the exciting
field of STM and AFM on nanometer scaled structures. I thank Professors O. Nishikawa, Ichiro
Hatta and Fumio Arisaka, and Dr. Hideo Arakawa for critically reading the manuscript and giving
me invaluable advice to improve its quality. I also extend my thanks to the authors of the original
articles cited in the figure legends in this review for providing me original artwork and/or
photographs. This work was supported by a Grant-in-Aid for Research in Priority Areas from the
Japanese Ministry of Education, Culture and Science to A.I. (No. 05245102). Financial support from
Nissan Science Foundation and from N E D O is also appreciated.
Note added in proof
I would like to add an article by Hutter and Bechhoefer [193] as a calibration method of cantilever
force constant using thermal vibration analysis.
328
A. Ikai/Surface Science Reports 26 (1996) 261-332
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