JOURNAL OF MOLECULAR RECOGNITION
J. Mol. Recognit. 2007; 20: 448–458
Published online 27 September 2007 in Wiley InterScience(www.interscience.wiley.com) DOI:10.1002/jmr.843

Review

High-speed atomic force microscopy for observing
dynamic biomolecular processes
Toshio Ando1,2*, Takayuki Uchihashi1,2, Noriyuki Kodera1,2, Daisuke Yamamoto1,2,
Masaaki Taniguchi1, Atsushi Miyagi1 and Hayato Yamashita1
12Department of Physics, Kanazawa University, Kanazawa 920-1192, Japan
CREST, JST, Sanban-cho, Chiyoda-ku, Tokyo 102-0075, JapanThe atomic force microscope (AFM) is unique in its capability to capture high-resolution images of biologicalsamples in liquids. This capability will become more valuable to biological sciences if AFM additionallyacquires an ability of high-speed imaging, because ‘direct and real-time visualization’ is a straightforward
and powerful means to understand biomolecular processes. With conventional AFMs, it takes more than aminute to capture an image, while biomolecular processes generally occur on a millisecond timescale or less.
In order to fill this large gap, various efforts have been carried out in the past decade. Here, we review thesepast efforts, describe the current state of the capability and limitations of high-speed AFM, and discusspossibilities that may break the limitations and lead to the development of a truly useful high-speed AFM forbiological sciences. Copyright # 2007 John Wiley & Sons, Ltd.
Keywords: AFM; biomolecules; high-speed AFM; imaging; biomolecular processes
Received 26 April 2007; revised 14 July 2007; accepted 15 July 2007

INTRODUCTION
The atomic force microscope (AFM) was invented in 1986
(Binnig et al., 1986), four years after the invention of thescanning tunneling microscope (STM) (Binnig et al., 1982).
Unlike STM or electron microscopy, AFM is unique in its
ability to observe insulating objects in liquids. Although it
was examined not in water but in paraffin oil, this in-liquid
observation ability was first demonstrated at Hansma’slaboratory by the observation of atomic lattice structures ofgraphite and sodium chloride surfaces (Marti et al., 1987).
This unique feature of AFM was of considerable advantage
to biological sciences because biological samples show vital
activities only in aqueous solutions. Around 1988, cantilevers manufactured by using microfabrication techniques
became available (Binnig et al., 1987), and the optical levermethod for cantilever deflection detection was introduced(Meyer and Amer, 1988); this promoted AFM imaging of
biological samples (Gould et al., 1988; Marti et al., 1988a,b;
Drake et al., 1989; Lin et al., 1990). Remarkably, at this very
early stage, Paul Hansma and his colleagues alreadyobserved the dynamic behavior of biological samples inaction. For example, they observed at 1 min intervals the*Correspondence to: T. Ando, Department of Physics, Kanazawa University,
Kanazawa 920-1192, Japan. E-mail: tando@kenroku.kanazawa-u.ac.jp

Abbreviations used: AFM, Atomic Force Microscope; STM, Scanning
Tunneling Microscope; PM-AFM, Phase-modulation Atomic Force Microscopy; FM-AFM, Frequency-modulation Atomic Force Microscopy;
AM-AFM, Amplitude-modulation Atomic Force Microscopy; Q, uality
Factor; PID, Proportional-Integral-Differential; nc-AFM, Noncontact
Atomic Force Microscopy; SNFUH, Scanning Near-field Ultrasound Holography; ICSPM, Ion-conductance Scanning Probe Microscopy; SPM, Scanning Probe Microscopy.

Copyright # 2007 John Wiley & Sons, Ltd.

clotting process by fibrin molecules that was initiated by thedigestion of fibrinogen with thrombin (Drake et al., 1989).
After this experiment, some trial observations of biologicalprocesses had been actively made to explore the potential of
AFM as a novel tool in biological sciences (Häberle et al.,
1992; Ohnesorge et al., 1992). However, this enthusiastic
research activity seemed to have declined gradually, incomparison with the increase in the AFM-user population
due to the availability of commercial AFMs. This isunderstandable because at this stage only contact mode wasused (tapping mode was invented in 1993 (Zhong et al.,1993)), and therefore, biomolecules weakly attached tosurface were easily swept away by the scanning cantilevertip. At this stage, greater efforts were directed towardattaining appropriate conditions under which high spatialresolution images could be obtained (Butt et al., 1990a,b;
Edstrom et al., 1990; Egger et al., 1990; Gould et al., 1990;
Weisenhorn et al., 1990; Hoh et al., 1991; Appel et al., 1992;
Karrasch et al., 1993; Schabert and Engel, 1994). After thetapping mode was introduced, the research activity on
exploring biological processes was revived moderately,
although the imaging rate was as low as before. For example,
in 1994, Bustamante and his colleagues imaged DNA
diffusion on mica surface (Bustamante et al., 1994) andDNA bending on binding to l Cro protein (Erie et al., 1994),
and Hansma and his colleagues imaged DNA digestion with
DNase (Bezanilla et al., 1994) and DNA–RNA polymerase
binding process (Guthold et al., 1994). These two groups
continued these studies and obtained time-lapse images(30 s intervals) on the RNA replication reaction by DNA
and RNA polymerase (Kasas et al., 1997) and on the
one-dimensional diffusion of RNA polymerase along a DNA
strand (Guthold et al., 1999).

HIGH-SPEED ATOMIC FORCE MICROSCOPY

In 1993, the scan speed limit of AFM was described (Buttet al., 1993). Although we found no publications, somestudies aiming at increasing AFM scan speed must havebeen initiated at least before 1995. In fact, we started todevelop high-speed scanners in 1994 and small cantileversin 1997. Hansma’s group also started to develop devices forhigh-speed AFM around 1995. They presented the firstreport on short cantilevers (23 mm by 12 mm) in 1996
(Walters et al., 1993), and subsequently a report on fast
imaging in 1999, in which small cantilevers and an opticaldeflection detector (Schäffer et al., 1996) designed for thesmall cantilevers were used to take a DNA image in 1.7 s(Viani et al.). Next year, they imaged the formation anddissociation of the GroES–GroEL complexes (Viani et al.,2000). However, due to the limited feedback bandwidth, thismolecular process was traced by scanning the sample stage
only in the x and z directions. A more complete high-speed
AFM system was reported by us in 2001 and 2002 (Andoet al., 2001, 2002), in which a high-speed scanner and fastelectronics were introduced in addition to small cantilevers(resonant frequency 600 kHz in water) and an opticaldeflection detector for the small cantilevers. An imaging rateof 12.5 frames/s was achieved, and therefore, the swinging
lever-arm motion of myosin V molecules was filmed as
successive images with a scan range of 240 nm. This study
inspired the study on high-speed AFM and seemed to bringabout a groundswell toward the full-scale development ofhigh-speed AFM and its application to biological issues thatwere difficult to solve by other techniques.
In this article, we review studies on high-speed AFM thatwere performed in the past decade. After a brief descriptionof the high-speed AFM capabilities required by biologicalsciences, we give a quantitative description of the imaging
rate and the feedback bandwidth; this is followed by adescription of the devices developed so far and a few
examples of high-speed biological imaging. In the last part,after summarizing the current state of our high-speed AFM
and its limitations, we discuss the future prospects ofpossible techniques that may break the limitations andthereby can fulfill all the requirements of biologicalsciences.

DEMANDS ON HIGH-SPEED AFM
It is quite understandable that during the early stage
immediately after the invention of AFM, researchers were
enthusiastic about the possibilities of observing biomolecular processes. Before the AFM era, it was absolutely
impossible to directly observe biological macromolecules(DNA, protein) in water, and therefore, the dynamic
behavior of biomolecules had to be deduced from indirect
data. From such inferences without a clincher, it was quitedifficult to reach a complete consensus on a model of aparticular biomolecular process. Biological functions
proceed through dynamic processes that occur on amillisecond timescale or less with biomolecules, biosupramolecules, organelles, and cells. Therefore, direct- andreal-time visualization is a powerful means of understanding
biological functions. Thus, biological sciences demand
Copyright # 2007 John Wiley & Sons, Ltd.

449

AFM (or any microscope) to have the ability to film dynamic
behavior of proteins that are purified and weakly attached toa substratum as well as those naturally embedded in living
cell membranes. Furthermore, AFM must capture thedynamic behavior of intracellular organelles. Although
the last demand appears to be an impossible one, a recent
study (Shekhawat and Dravid, 2005) is probably making it
possible.
Biomolecules are generally fragile and dynamic protein–protein interactions are more delicate. The force acting onprotein–protein interactions ranges approximately from
1 pN to 100 pN. Even with the single ‘rigor’ complex of amuscle–myosin head and an actin filament that hardly
dissociates in equilibrium, it is ruptured quickly by a pulling
force of 15 pN (Nakajima et al., 1997). The force producedby motor proteins during ATP hydrolysis is generally a few
piconewtons (e.g., see Schmidt and Montemagno, 2004).
Moreover, living cell membranes are extremely soft.
Therefore, biological science further demands that AFM
maintains the tip–sample interaction force at a very (ornegligibly) small level.

FEEDBACK BANDWIDTH ANDIMAGING RATE
Suppose that an image is taken in a time T for a scan range
W  W with scan lines N, the scan velocity Vs is then given
by Vs ¼ 2WN/T. For W ¼ 240 nm, N ¼ 100, and T ¼ 30 ms, Vs
becomes 1.6 mm/s. Suppose that a sample has a sinusoidal
shape with a periodicity l, the scan velocity Vs requires a
feedback operation at a frequency f ¼ Vs/l to maintain thetip–sample distance. When l is 10 nm and Vs is 1.6 mm/s, f
becomes 160 kHz. The feedback bandwidth fB should beequal to f or higher and therefore can be expressed asfB  2WN=lT(1)

The feedback bandwidth is a function of various factors
and difficult to express explicitly. Qualitative descriptions
were previously made for its dependence on various factors(Sulchek et al., 2002). Numerical simulations were also
made for this purpose, including the dynamics of tip–sample
interaction (Kokavecz et al., 2006a). However, neither gives
a quantitative and practical guideline for developing ahigh-speed AFM. As shown below, we employed a simple
way to derive an analytical expression for the feedback
bandwidth, slightly sacrificing exactness (Kodera et al.,2006). Because of the ‘chasing-after nature’ of feedback
control, the sample topography is always traced with a finite
phase delay w. The phase delay w is given by 2pfDt, where
Dt is the time delay of the feedback control. The time delay
depends on the imaging mode and is a function of various
factors. Here, we consider only tapping mode that is suitablefor imaging fragile samples. The main delays are the reading
time of the cantilever’s oscillation amplitude, the cantilever’s response time, the z-scanner’s response time, theintegral time (tI) of error signals in the feedback controller,
and the parachuting time (tp). ‘Parachuting’ means that thecantilever tip completely detaches from the sample surface
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T. ANDO ET AL.

at a steep down-hill region of the sample and thereafter takes
time until it lands on the surface again. It takes at least 1/2fc
to measure the amplitude of a cantilever that is oscillating at
its resonant frequency fc. The response time of second-orderresonant systems such as cantilevers and piezoactuators is
expressed as Q/pf0, where Q and f0 are the quality factor andthe resonant frequency, respectively. The feedback bandwidth is usually defined by the feedback frequency that
results in a phase delay of p/4. With this definition, thefeedback bandwidth fB is approximately expressed as
fc 1 Qc Qs fc
þfB ¼ = þþ fc ðtp þ tI Þ
8 2 p
pfs

(2)

where fs is the z-scanner’s resonant frequency; Qc and Qs thequality factors of the cantilever and z-scanner, respectively.
The parachuting time is a function of various parameterssuch as the sample height h0, the free oscillation amplitude
A0 of the cantilever, the amplitude set point r, the phase delayw, and the cantilever’s resonant frequency. Its approximate
analytical expression has been derived (Kodera et al.,2006) ast p  ½ðtan bÞ=b  1=fc(3)

where ß is cos1 ½2A0 ð1  r Þ=3h0 sinð’=2Þ. Equations (2)and (3) give us a quantitative guideline for the developmentof high-speed tapping mode AFM.

DEVICES FOR HIGH-SPEED AFM
Small cantilevers
The resonant frequency fc and the spring constant kc of arectangular cantilever with thickness d, width w, and length
L are expressed asd
fc ¼ 0:56 2
L

rﬃﬃﬃﬃﬃﬃﬃﬃ
E12r

follows Du=Dz ¼ 3=2L, where Dz is the end displacement
and Du is the end angle change of a cantilever of length L. A
high resonant frequency and a small spring constant result ina large ratio ( fc / kc), which affords the cantilever a highsensitivity to the gradient (k) of force exerted between the tip
and sample. The force gradient shifts the cantilever’s
resonant frequency by approximately 0.5kfc /kc. Therefore,
small cantilevers with large values of fc / kc are very usefulfor phase-modulation AFM (PM-AFM) and frequencymodulation AFM (FM-AFM). With respect to the feedback
bandwidth, the most important aspect is that the amplitudedetection time and the cantilever’s response time decrease ininverse proportion to the resonant frequency. To realize asmall spring constant and a high resonant frequency
simultaneously, cantilevers with small dimensions mustbe fabricated. The small cantilevers recently developed by
Olympus are manufactured using silicon nitride and coatedwith gold of thickness 20 nm. They have dimensions oflength 6 mm, width 2 mm, and thickness 90 nm, which
result in the resonant frequency of 3.0 MHz in air and1.2 MHz in water, spring constant 0.2 N/m, and Q 2.5
in water. However, these cantilevers are not commerciallyavailable yet. Cantilevers integrated with piezoelectric films
have advantages, because they can self-sense their deflectionand even self-actuate (Manalis et al., 1996). However, more
complicated fabrication processes are required, and hence,at present it is very difficult to manufacture small cantilevers
with such capabilities.
The tip radius of microfabricated small cantilevers
developed by Olympus is 17 nm (Kitazawa et al.,2003), which is not sufficiently small for high-resolution
imaging of biological samples. We usually attach an
additional tip using electron beam deposition and sharpen
it (4 nm in radius) by plasma etching in argon gas.
However, this piece-by-piece attachment is time consuming.
Batch procedures for attaching a sharp tip to each cantilever
have been attempted either by using direct growth of a single
carbon nanofiber (Tanemura et al., 2006) or a carbon
nanotube at the cantilever tip (Cheung et al., 2000).

(4)
Optical beam deflection detector

3

kc ¼

wd
E
4L3

(5)

where E and r are the Young’s modulus and the density ofthe material used, respectively. The Young’s modulus andthe density of silicon nitride (Si3N4), which is often used as amaterial for soft cantilevers, are E ¼ 1.46  1011 N/m2 andr ¼ 3,087 kg/m3, respectively. For a given spring constant,the resonant frequency increases with decreasing mass of thecantilever. The total thermal noise depends only on thespring constant and the temperature. Therefore, a cantileverwith a higher resonant frequency has a lower noise density.
In tapping mode, the frequency region used for imaging is
the imaging bandwidth (its maximum is the feedback
bandwidth) centered on the resonant frequency. Thus, acantilever with a higher resonant frequency is less affectedby thermal noise. In addition, shorter cantilevers give ahigher optical deflection sensitivity, because the sensitivity
Copyright # 2007 John Wiley & Sons, Ltd.

To focus an incident laser beam onto a small cantilever, alens with a high numerical aperture (resulting in a shortworking distance) has to be used. With such a lens, the beamreflected back from the cantilever has to be collected withthe same lens. The incident and reflected laser beams can beseparated using a quarter wavelength plate and a polarization splitter. Two groups reported similar optical deflection
detection systems with this basic design (Schäffer et al.,
1996; Viani et al., 1999; Ando et al., 2001).

Amplitude detection
Conventional rms-to-dc converters use a rectifier circuit anda low-pass filter, which requires at least several oscillation
cycles to give an accurate rms value as output . To detect the
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HIGH-SPEED ATOMIC FORCE MICROSCOPY

cantilever’s oscillation amplitude from a half oscillation
cycle, a method to capture the peak and bottom voltages
and then produce their difference as the amplitude
was developed (Ando et al., 2001). A different method(the Fourier method) to output the amplitude from a single
oscillation was proposed (Kokavecz et al., 2006b).
The Fourier method calculates the Fourier sine andcosine coefficients for the fundamental frequency fromthe deflection signal and then produces the square root of thesum of their second powers as the amplitude. This method isprobably less susceptible to noise but requires analogue ordigital calculation systems with bandwidth at least twoorders of magnitude higher than the cantilever’s resonant
frequency.
High-speed scanner
The scanner is the device that is most difficult to optimize forhigh-speed scanning. High-speed scan of mechanicaldevices with macroscopic dimensions tends to produce
unwanted vibrations. Three techniques are required tominimize unwanted vibrations: (a) a technique to suppress
the impulsive forces that are produced by quick displacement of the actuators, (b) a technique to increase theresonant frequencies, and (c) an active damping technique toreduce the quality factor.
The first issue was solved by a counterbalancing
technique (Ando et al., 2001, 2002). For example, for thez-scanner that moves at much higher frequencies than the xand y-scanners, two identical piezoactuators are placed attheir supporting base in the counter directions and displaced
simultaneously with the same length. An alternative way is
to support a piezoactuator at both the ends with flexures.
This method was applied to the x-scanner and z-scanner andworked very well (unpublished data). Recently, we tried a
different method. The z-piezoactuator is held only at the four
corners of its surface perpendicular to the displacement
direction. The piezoactuator can be displaced almost freely
in both the counter directions, and therefore impulsive forces
are barely exerted on the holder; thus, only a very low
mechanical excitation is produced to the scanner mechanics.
This holding method has an additional advantage that theresonant frequency is not lowered by the holding, although
the maximum displacement decreases by half.
The resonant frequency of a piezoactuator is determinedalmost solely by its maximum displacement (in other words,
by its length). However, it can be effectively extended by aninverse compensation method as described later. The
structural resonant frequency is enhanced by the use of acompact structure and a material that has a large ratio of theYoung’s modulus to the density. However, a compact
structure tends to produce interferences between thethree-scan axes. A ball-guide stage (Ando et al., 2001) is
one choice for avoiding the interferences. An alternative way
is to use flexures (blade springs) that are flexible enough to
be displaced but stiff enough in the directions perpendicular
to the displacement axis (Kindt et al., 2004; Ando et al.,2005). It should be noted that the scanner mechanics except
for piezoacutators has to be produced by monolithic
processing in order to minimize the number of resonant
elements. An asymmetrical x–y configuration has been
Copyright # 2007 John Wiley & Sons, Ltd.

451

employed to gain a high resonant frequency for thex-scanner (the fast scan direction) (Ando et al., 2005).
However, a symmetrical x–y configuration has an advantage
of being capable of rotating the scan direction (Kindt et al.,2004). As a material for the scanner, aluminum or duralumin
is often used. However, magnesium and magnesium alloys
seem better candidates for it because of their larger
mechanical damping coefficients and larger ratios of theYoung’s modulus to the density.
The active Q-control is well known as an active damping
technique and has been often used to control the quality
factor of cantilevers (Anczykowski et al., 1998; Sulchek
et al., 2000; Tamayo et al., 2000, 2002). When this control isapplied to the z-scanner, its displacements have to be
detected. However, it is difficult to do so. Kodera et al.
(2005) developed a new method in which instead ofdetecting the displacements, output signals from an electric
circuit characterized by the same transfer function as thez-scanner were used to damp the z-scanner. With thistechnique, they achieved a bandwidth of 150 kHz and aquality factor of 0.5, which resulted in a response time of
1.1 ms. This method worked well for the z-scanner with asimple transfer function but not for one with a complicatedone having multiple resonant peaks. An alternative method
can be used for the active damping. The z-scanner is driven
through a circuit with a transfer function 1/G(s), where G(s)is the transfer function of the z-scanner. However, for acomplicated G(s), it is very difficult to design an electric
circuit with 1/G(s). We invented a circuit that can
automatically produce a transfer function that is approximately the same as the inverse transfer function of a given
transfer function (Morita et al., 2007; Yamashita et al.,2007). This approximation becomes better with the use ofoperational amplifiers with higher bandwidths. In addition,
this approximation becomes better with nested circuits evenwhen the amplifiers’ bandwidth is not high enough. Thismethod works not only for reducing the quality factor but
also for enhancing the apparent resonant frequency, so longas the driver for the piezoactuators has sufficient gain at highfrequencies. Using this method and holding a piezoactuator(resonant frequency ¼ 370 kHz) at its corners, we achieved az-scanner bandwidth of 500 kHz with a maximum displacement of 1 mm (unpublished data).
The active damping of the x- and y-scanners is easy
because their scan speed is not high and their scan waves are
already known, and therefore, a feedforward controller for
damping can be implemented in a digital mode. For
example, the x-scanner is moved in isosceles triangles X(t)with a periodicity Tx. Its Fourier transform Fx(v) is given by
"

þ1
1
2 X
1
Fx ðvÞ ¼ 2pX0 dðvÞ  2
dðv  kv0 Þ
2
p k¼1 k2#(6)

ðk : oddÞ;where X0 is the maximum displacement and v0 ¼ 2p/Tx.
Suppose that the transfer function Gx(iv) (¼ jGx ðivÞjexp½iFðvÞ) of the x-scanner is experimentally measured,
the inverse Fourier transform of Fx(v)/Gx(iv) gives thedriving signal X’(t) for moving the x-scanner exactly inisosceles triangle waves. However, in practice, we need only
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T. ANDO ET AL.

the first 15 terms of Fx(v). Therefore, the driving signal isexpressed as
X 0 ðtÞ ¼

X0
2Gx ð0Þ


29
4X0 X
1
1
cos½kv0 t  Fðkv0 Þ (7)p2 k¼1 k2 jGx ðikv0 Þj

ðk : oddÞ;To achieve a very fast scan in the x-direction, a resonant
scan system with a tuning fork was used by Miles’ group(Humphris et al., 2005; Picco et al., 2007). The scanner uses
mechanical resonances instead of avoiding them. Due to thelarge quality factor of the tuning fork, the scanner oscillates
sinusoidally with high stability. However, this method has
two disadvantages. The scan speed cannot be changed easily,
and the scan speed and the pixel size vary depending on theposition in the x-direction.
Cantilever actuation
The tip–sample distance can be controlled not only by thez-scanner but also by actuating the cantilever. The latter has
the potential of affording a higher feedback bandwidth
because small cantilevers can have higher resonant
frequencies than those of the z-piezoactuators. In addition,
driving only a small cantilever beam produces much lesshydraulic pressure on the sample, compared with driving thesample stage or the whole cantilever chip. However, we haveto note that direct driving of a cantilever with any methods
for controlling the tip–sample distance has the drawback of
reducing the deflection detection sensitivity because a larger
dynamic range is required for the sensing. Magnetic
actuation of a cantilever coated with a magnetic material
has been used for exciting the cantilever at its resonant
frequency (Han et al., 1996). It can also be used forcontrolling the tip–sample distance (Jayanth et al., 2006).
However, magnetic coating often reduces the cantilever’s
resonant frequency and makes the cantilever stiffer. Opticalactuation has also been used for exciting a cantilever at its
resonant frequency (Umeda et al., 1991; Marti et al., 1992;
Ramos et al., 2006; Verbridge et al., 2006). In liquids,
optical actuation derives from a thermal expansion effectrather than optical pressure (Ramos et al., 2006). Because
heat transmission is slow, photothermal driving of a
cantilever cannot be made quickly. To solve this problem,
we again applied the inverse transfer function compensation
as mentioned above. This compensation resulted in an
apparent transfer function very close to the pure mechanicaltransfer function (Yamashita et al., 2007). With the natural Q(2.5) of our small cantilevers (resonant frequency ¼ 1.2 MHz in water, spring constant 0.2 N/m), thecantilever responds to the power-modulated laser in 0.66 ms.
By this control of the tip–sample distance together withother devices optimized for high-speed scanning, myosin V
was imaged at video rate (30 ms/frame) for the scan range of
240 nm with 100 scan lines (Yamashita et al., 2007). A
drawback of this photothermal driving is its small deflection
efficiency. With a laser of 980 nm, it was 1 nm/mW when
Copyright # 2007 John Wiley & Sons, Ltd.

applied to our small cantilevers (the laser power measured
was not the one irradiated onto the cantilever but that at theoutlet of the focusing lens). A laser of 405 nm gave a higher
efficiency of 10 nm/mW. However, this wavelength cannot
be used because biological samples are damaged by theirradiation. Wavelengths between these two have not beentested yet.
Dynamic PID control
Various efforts have been carried out to increase the AFM
scan speed. However, little attention has been directed
toward the reduction in the tip–sample interaction force.
This reduction is quite important for biological AFM
imaging as mentioned in ‘Demands on High-speed AFM.’
The most ideal scheme is the use of noncontact AFM(nc-AFM); however, the potential of high-speed nc-AFM
has not been exploited at all so far. We still do not know if thehigh-speed and noncontact conditions can be achieved
together in principle. We will discuss this issue later. Theremay be several methods to reduce the force in tapping mode:(a) using softer cantilevers, (b) enhancement of the quality
factor of small cantilevers, (c) using a shallower amplitude
set point (i.e., r is close to 1). None of these methods seemcompatible with high-speed scanning. Softer cantilevers canbe obtained only by sacrificing the resonant frequency. The
most advanced small cantilevers developed by Olympus
seem to have almost reached the limitation in appropriatelybalancing the high resonant frequency and the small spring
constant. The tapping force decreases with increasing Q of
the cantilever. However, its response speed decreases withincreasing Q. A shallower amplitude set point promotes‘parachuting’ during which the error signal is saturated
at 2A0(1  r), and therefore, the parachuting time increases
with increasing r, resulting in decrease in the feedback
bandwidth. This difficult issue was solved by the inventionof a new PID controller called ‘dynamic PID controller’
whose gains were automatically changed depending on the
cantilever’s oscillation amplitude (Kodera et al., 2006).
Briefly, a threshold level is set between the peak-to-peak freeoscillation amplitude 2A0 and the set point amplitude 2A0r.
When the cantilever oscillation amplitude exceeds thisthreshold level, a false error signal is added to the true error
signal, which shortens the parachuting time or avoids
parachuting. In fact, the dynamic PID controller can avoid
parachuting even when r is increased up to 0.9, andtherefore, the feedback bandwidth becomes independent of r
so long as r is set less than 0.9. Thus, high-speed scanningand gentleness with the sample became compatible witheach other to some extent.
Drift compensation
With a given cantilever, the tapping force can be reduced byusing small free-oscillation amplitude and a shallow set
point. However, under this setting, the amplitude attenuation
due to the tip–sample contact becomes very small. For
example, with 2A0 ¼ 5 nm and r ¼ 0.9, the attenuation of thepeak-to-peak amplitude becomes 0.5 nm. When the cantilever’s free oscillation amplitude changes due to drift in the
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HIGH-SPEED ATOMIC FORCE MICROSCOPY

cantilever excitation efficiency, the oscillation amplitude
during scanning also changes. The AFM feedback system
misunderstands this change. For example, when the
excitation efficiency is lowered, the feedback systeminterprets the resulting decrease in the oscillation amplitude
as the tip interacting with the sample too strongly. Therefore,
the feedback responds by withdrawing the sample stage
from the tip, which is an incorrect direction. Withoutstability in the excitation efficiency or A0, successive
imaging under a small tapping force cannot be realized. It isdifficult to eliminate causes for the drift. In addition, we
cannot detect the free oscillation amplitude A0 while
imaging. This problem was first challenged by Kindt et al.
(2002). They calculated feature richness from sample
images obtained in tracing and retracing with slightly
different set points. The richness was maintained byregulating the set point in the tracing regime. This method
is effective but may not be applicable to high-speed imaging
because the calculation is time consuming. A different
method for drift compensation was invented by Schiener
et al. (2004). The second harmonic amplitude is sensitive totip–sample interaction, and hence drift in A0 is reflected in
the second harmonic amplitude averaged over a period
longer than the image-acquisition time. They regulated r to
maintain the constant difference 2A0(1  r). However, thiscontrol varies the tapping force and feedback bandwidth. We
also used the second harmonic amplitude but regulated
the power of exciting the cantilever in order to maintain theconstant A0(1  r). By using this control together withthe dynamic PID control, stable high-speed imaging withmaintained weaker tip–sample interaction became possible(Kodera et al., 2006).

Electronics
In high-speed AFM, large amounts of data have to behandled in real time to display image data while saving thedata onto the computer hard disk. Recent commercialdata-acquisition systems and personal computers are fastenough to do this handling. Quantitative descriptions of thisissue were given by Fantner et al. (2006). More problematic
devices are the feedback controller, active damping systems,
and the drivers for z-piezoactuators. Although analog
feedback controllers are faster than digital ones, implementation of analog mode feedback-control algorithms thatare more complicated than those of the dynamic PID control
is difficult. Various advanced feedback or feedforwardcontrol algorithms have recently been proposed for highspeed AFM (Schitter et al., 2001; Schitter et al., 2004a,b;
Salapaka et al., 2005). However, by using digital systems,
the efficiency of these algorithms has been demonstrated
only in the case of relatively slow AFMs. At present, it is notcertain whether proposed control algorithms can be
implemented in the digital mode for a real high-speed
AFM. The driver for z-piezoactuators (the capacitive load isa few nanofarads (nF) or less) should have a large current
capacity, a high slew rate, 100 V output, high bandwidth(>1 MHz), a small output resistance, and low noises. It is noteasy to fulfill all these requirements. Rost et al. (2005)
developed a z-driver with a bandwidth of 400 kHz and a slew
Copyright # 2007 John Wiley & Sons, Ltd.

453

rate of 75 V/ms for capacitive loads up to 1 nF. We sacrificed
the output voltage (50 V) and thereby gained rms noises<2 mV, a high slew rate of 1000 V/ms, and a bandwidth of
3 MHz for capacitive loads up to 2 nF.

High-speed phase imaging
Tapping mode AFM (AM-AFM) has the capability to image
compositional variations in heterogeneous surfaces in
addition to surface topography (Tamayo and Garciá,
1996; Bar et al., 1997; Magonov et al., 1997). The phasedifference between the excitation signal and the cantilever
oscillation is affected by several surface properties. With
energy-conservative tip–sample interaction, as mentioned
before, the resonant frequency shifts by approximately
0.5kfc/kc. The frequency shift results in a phase shift
because the excitation frequency is fixed. For a given
frequency shift, the phase shift increases with Q. With
conventional cantilevers, the frequency shift is generally
around 50 Hz. Therefore, phase-contrast imaging had beenpossible only with a large Q (hence only at a small imagingrate). Because the ratios fc / kc with the most advanced smallcantilevers are 1000 times larger than that withconventional cantilevers, we can expect a shift of50 kHz. Therefore, even with a small Q, a relatively large
phase shift occurs, which suggests a possibility of detecting
the phase shift without using a very sensitive—yet very
slow—phase detector such as lock-in amplifiers; hence,
there is a possibility of high-speed phase-contrast imaging.
Even with non-conservative interaction (Cleveland et al.,
1998; Martı́nez and Garciá, 2006), we can expect theoccurrence of a relatively large phase shift with smallcantilevers, because their oscillation is damped weakly inwater compared with conventional cantilevers; therefore,
energy dissipation by tip–sample interaction can besignificantly reflected on the cantilever oscillation. In order
to explore the possibility of high-speed phase-contrast
imaging, a fast phase detector was developed by Uchihashi
et al. (2006) based on a previous design (Stark &Guckeberger, 1999). This can detect phase shifts within asingle oscillation cycle and importantly at any timing withina cycle. This flexibility of detection timing is of great
importance. Firstly, we can choose the timing when thelargest phase shift occurs in the cycle, and therefore
unlike conventional phase-contrast imaging, we do not haveto use small amplitudes. In conventional phase-contrast
imaging, the phase signal is averaged over many oscillation
cycles, and therefore the cantilever oscillation amplitude has
to be reduced to 1 nm so that the tip is almost always
oscillating within a region where the force field exists. Dueto the small amplitude, phase-contrast imaging is difficult
for samples with large roughness. The second importance ofthe flexibility of detection timing is that it can be
distinguished whether the interactions conserve energy or
dissipate it. The phase shift due to an energy-conservativeinteraction decreases with time very fast, while thephase shift caused by energy–dissipative interaction is
maintained over the oscillation cycle. In fact, images ofstyrene–butadiene–styren block copolymer films having
different contrasts were obtained in liquids, depending on
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T. ANDO ET AL.

the detection timing within the oscillation cycle. Remarkably, this phase imaging was carried out at 80 ms/
frame (Uchihashi et al., 2006). This new technique forphase-contrast imaging will allow us to study dynamicchanges in the physicochemical nature of protein moleculesin action.

BIOIMAGING
Full-scale high-speed imaging studies on biological processes have not been carried out in the past decade because
more efforts have been directed to develop high-speed AFM
apparatuses. Various attempts to capture biological processes have been made mainly for testing the ability ofhigh-speed AFM, which were feedbacked to the development of high-speed AFM. For example, in 2003, we imaged
at 0.5 s/frame, the unidirectional movement of a chimerakinesin along a microtubule in an ATP-containing solution(Ando et al. 2003). In this kinesin, the C-terminal tail ends
were replaced with gelosolin in order to avoid strong bindingof its intrinsic tail ends to the mica surface. The observedkinesin moved unidirectionally along a microtubule, while
being attached weakly to the mica surface. We could notobserve kinesin that was moving along microtubules without
touching the mica surface. This is because tip–sample
interaction was too strong even with assistance from a
prototype dynamic PID controller for keeping a constantshallow set point. The tip removed kinesin that had been
attached only to the microtubules. Without assistance by the
dynamic PID controller, the microtubules were destructed.
Under conventional PID control, actin filaments on myosin
V-coated surfaces could also not be clearly imaged without
destruction. It was more difficult to observe actin filaments
gliding over the myosin V-coated surface in the presence ofATP. In this case, actin filaments could not appear in thescanning region because actin filaments were easily
removed from myosin V by too strong tip–sample
interaction. With assistance by dynamic PID control, actin
filaments gliding on the mica surface that was densely
coated with myosin V were captured on video (Ando et al.,2005, 2006). However, when the myosin V density was
lowered, the gliding movement was hardly observed. Withan improved dynamic PID controller together with an
optical deflection sensor with low noises, the set point could
be set at >0.9; thus, actin filaments gliding on a surface
sparsely coated with myosin V were successfully imaged(Ando et al., 2005, 2006). Accidentally, a short actin
filament entered the observed region. Its entire length waswithin the region and therefore, all the myosin V moleculesinteracting with this filament were identified. All the
myosin V heads interacting with the filament were oriented
in one direction, which was similar to a well-known
arrow-head structure in muscles. From this oriented
structure, the polarity of the actin filament was identified.
The filament moved toward the minus (pointed) end, which
was the natural direction. However, conformational changes
in the interacting myosin V heads were not evident during
the unidirectional movement of the filament. Some AFM
movies can be seen at http://www.s.kanazawa-u.ac.jp/phys/biophys/roadmap.htm.
Copyright # 2007 John Wiley & Sons, Ltd.

Recently, a prototype high-speed AFM, which is an
improved version of our first design (Ando et al., 2001,
2002), became commercially available (Nano Live VisionTM
manufactured by Olympus and distributed by RIBM). Its
users recently filmed dynamic processes of GroEL–GroES
interaction that were regulated by the ATPase reaction of
GroEL immobilized onto the mica surface in an end-up
orientation (Yokokawa et al., 2006a). Moreover, theyobserved the formation and dissociation of a streptavidin–
biotinylated DNA complex (Kobayashi et al., 2007) and
one-dimensional diffusion of a restriction enzyme along a
DNA strand which was followed by the cleavage reaction(Yokokawa et al., 2006b).
The dynamic processes mentioned above are alreadyknown or expected from a series of biochemical andbiophysical studies, and therefore, the filmed images may
not give new insights to the molecular mechanisms.
However, the new imaging tool has not matured yet, andhence, is not recognized yet as an established one.
Therefore, in the present stage, biological processes thathave been expected or known to occur have to be
demonstrated by high-speed AFM imaging. In addition,techniques for preparing samples and substrates for their
attachment have to be developed, which are often different
from those for still imaging. Along with the gradualaccumulation of successful imaging of known molecular
processes, high-speed AFM will be realized as a reliable tool
while newly filmed data on unexplored biological processes
will be accepted widely.
We have been seeking to image single myosin V
molecules walking along actin filaments, using the mostrecent version of high-speed AFM. Single molecules
myosin V moves processively along actin tracks (Sakamoto
et al., 2000). The hand-over-hand walking of myosin V
is already established (Yildiz et al., 2003; Forkey et al.,
2003; Warshaw et al., 2005; Syed et al., 2006) but its
detailed behavior is still unknown. The tail part of myosin V
was removed by digestion (Koide et al., 2006) because it
tended to attach to the mica surface. However, in a low ionicsolution, the truncated myosin V (HMM) still tended toattach to the mica surface. Therefore, we elevated the ionicstrength, although this lowered the affinity of myosin V
heads for actin. Because of the weak affinity, the oscillating
cantilever tip with the usual free amplitude (5 nm)
disturbed actin–myosin V interaction. Therefore, we
reduced the free amplitude down to 1 nm, sacrificing
the feedback bandwidth. Under these conditions, we could
successfully capture walking myosin V on video at 0.1 s/frame, in which the leading and trailing heads altered theirpositions with a walking stride of 72 nm (Kodera et al.,manuscript in preparation). In addition, the lead lever-arm
bent just before the rear head detached from the actin
filament. The detached rear head rotated around the junction
between the two lever arms and then attached to a frontward
actin. Just after the attachment, the new lead head moved
farther by 5 nm along the actin filament. We have been also
seeking to image GroEL–GroES interaction dynamics in an
ATP-containing solution, in which biotinylated GroEL is
immobilized to streptavidin 2D-crystal sheets in a side-onorientation (Taguchi et al., 2001). Due to this orientation,
both the GroEL rings were accessible to GroES that were
floating in the solution. Because floating GroES did not
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HIGH-SPEED ATOMIC FORCE MICROSCOPY

interfere with imaging, a high-concentration of GroES could
be used, unlike single-molecule fluorescence microscopy.
The negative cooperativity between the two GroEL rings
was confirmed; GroEL alternated its rings between the
GroES associated and dissociated states. However, interestingly, releasing one GroES associated complex andforming another did not necessarily occur simultaneously.
Two controversial intermediates, bare GroEL and GroEL(GroES)2, were detected just prior to the switching. The
latter intermediate appeared with a large probability(Yamamoto et al., manuscript in preparation). The ternary
GroEL–(GroES)2 complex could be distinguished from the
GroEL–GroES complex; the former looked like a footballwhile the latter looked like a bullet.

THE PRESENT STATE ANDPROSPECTS OF HIGH-SPEED AFM
Our current high-speed AFM is characterized by delay times
contained in the feedback loop: the time of 0.4 ms forreading the amplitude of a cantilever oscillating at its
resonant frequency of 1.2 MHz and with Q 2.5 in water,
the cantilevers’ response time of 0.66 ms, the response timeof 0.32 ms of the z-scanner with the effective resonant
frequency of 500 kHz and Q 0.5, no parachuting with theamplitude set point 0.9, and fast electronics withnegligible delays as compared with the other delays.
Together with the phase-compensation effect (40%) of
the differential operation of the dynamic PID controller, thesum of these delay times results in a feedback bandwidth of
125 kHz (see Eq. 2). Here, we neglected the integral time oferror signals because it is hard to estimate. An experimental
estimate of the feedback bandwidth also resulted in125 kHz, when the ratio 2A0/h0 ¼ 5 was used. Therefore,
from Eq. 1, this bandwidth corresponds to an imaging rate of
25 frames/sec (40 ms/frame) for W ¼ 250 nm, N ¼ 100, andl ¼ 10 nm. With contact mode or no electronic feedback
operation, the imaging rate becomes much higher. However,both tapping mode and electronic feedback operation arecertainly indispensable for imaging biological processes.
Therefore, for faster imaging, the scan size or the number ofscan lines has to be reduced at present.
How can we further increase the speed without reducing
the scan size or the number of scan lines? In the current state,
as seen above, the cantilever’s resonant frequency has adominant effect on the limited feedback bandwidth. It israther easy to enhance the resonant frequency by the use of
thicker cantilevers. However, they become stiffer and henceexert larger tapping forces on the sample. A unique solutionthat fulfills all desired conditions seems to be realization ofhigh-speed nc-AFM. Noncontact imaging has been realized
only with AM-AFM or FM-AFM in vacuum, where
cantilevers with large quality factors are used. However,the large quality factor significantly reduces the cantilever’s
response speed. We must seek a noncontact condition that iscompatible with cantilevers having a small quality factor, if
this is possible in principle. Although it has not been
explored theoretically and experimentally and hence notevidenced yet, it may be materialized by using ultrasonic
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455

interference between the cantilever tip and the sample thatare ultrasonically excited at different frequencies f1 and f2( f1, f2  fc). The difference jf1  f2j is set close to the
cantilever’s resonant frequency. This configuration is thesame as that employed for scanning near-field ultrasoundholography (SNFUH) that has recently been developed forhigh-resolution sub-surface imaging (Shekhawat andDravid, 2005). A high frequency acoustic wave is launchedfrom the bottom of the sample and propagates through thesample. Materials with different elastic moduli embedded in
the sample modulate the phase and amplitude of the
propagating acoustic waves. These modulations are reflectedon the acoustic interference that occurs at the cantilever tip
and therefore reflected on the cantilever oscillation aroundits resonant frequency. When the sample in a solution is
only protein molecules attached to a uniform substratum,
the acoustic wave front in the solution may trace thesample topography. This wave front may be detected by
the cantilever tip that is not in contact with but is close to thesample surface.
The second possibility of high-speed noncontact imaging
may derive from ion-conductance scanning probe microscopy (ICSPM) that has already satisfied the noncontact
condition (Hansma et al., 1989). Due to the progress of
fabrication techniques to produce very sharp glass capillaries with a small pore at the end, the spatial resolution hasreached a few nanometers (Ying et al., 2005). Immobile
protein molecules of 14 nm on living cell membranes havebeen successfully imaged (Schevchuk et al., 2006).
However, in order to materialize high-speed ICSPM, wehave to find a method to increase the bandwidth ofion-conductance detection because ionic currents through asmall pore are very low.
High-speed nc-SPM in liquids will extensively expand
the range of biological systems to be visualized, because thenoncontact condition has the potential of enhancing theimaging rate. So far high-speed AFM imaging has beenpossible only for biological samples on a relatively hardsubstratum. Noncontact imaging will make it possible tovisualize the molecular processes that occur on very softliving cells. Membrane proteins unanchored to membrane
cytoskeletons diffuse very fast within the membranes when
they are not surrounded by protein clusters. High-speed
nc-SPM will be able to capture such proteins clearly. This
capability is very valuable and applicable to a wider range of
biological issues. Extracellular stimuli (such as drugs,
endogenous ligands, or electric one) are first received bymembranes’ proteins (receptors or ionic channels) and thentransmitted to the interiors to produce various cellularactions. Therefore, high-speed nc-SPM will also be used for
drug assessment and discovery.
Although not for high-speed nc-AFM, control algorithms
to reconcile a large quality factor of the cantilever withhigh-speed imaging have been proposed (Sahoo et al., 2003;
Sahoo et al., 2005; Jeong et al., 2006). The position andvelocity of the oscillating cantilever are continuously
monitored (or discretely monitored with small time-bins).
From these quantities measured, an estimator calculates thetip–sample interaction force of each tapping cycle. A
model-based predictor uses the estimated force to control thetip–sample distance in the next tapping cycle. Theirexperiments with conventional AFMs implemented with
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the new controllers demonstrated regulation of thetip–sample interaction force at each tapping cycle,
irrespective of the time delay of the cantilever’s response.
However, in order to apply this method to a real high-speed
AFM, extremely fast digitization and calculations are
required.
It seems quite difficult to enhance the bandwidth ofpiezoactuator-based scanners further. For a piezoactuatorwith a maximum displacement of 1 mm, the resonant
frequency is approximately 500 kHz at most. Therefore, we
require different types of actuators for further enhancementof the bandwidth. Microfabrication techniques can probably
produce small actuators with both a large displacement and ahigh resonant frequency. Recently, an aluminum membrane
actuator/sensor with a diameter of 150 mm was developed byDegertekin and his colleagues (Degertekin et al., 2005;
Onaran et al., 2006). This membrane is driven by anelectrostatic force exerted between the membrane and thecounter electrode with a separation of 2 mm. This design was
not created with the intention of producing a high-speed
z-scanner but it has such a potential.
At present, we have no technology that allows us to study
the structural dynamics of intracellular organelles at highspatial and temporal resolution. Recently demonstrated
intracellular imaging by SNFUH (Shekhawat and Dravid,2005) may afford such technology. It seems easy to combine
SNFUH with the high-speed scanning techniques developed
so far. It is still unclear whether this new imaging mode has
resolution in the z-direction. However, the images obtainedwith ultrasonic waves launched at different angles should
contain information along the z-axis. Therefore, there may

be a way to reconstitute a 3D image from multiple images
obtained at different launching angles.

SUMMARY
By focusing on the research activities on high-speed AFM in
the past decade, we reviewed various studies on the
instrumentation and imaging of biomolecular processes.
Various devices have been created in these years based on
the efforts of pioneers in this field, which led to high-speed
AFM that can capture successive images at video rate
without disturbing the biological processes to some extent.
This ability was demonstrated, for example, by filming
myosin V molecules walking along actin filaments in a
hand-over-hand fashion. One of the dreams of biologicalsciences had finally materialized. However, the force exerted
between the oscillating tip and sample is in a level that is justsmall enough not to break weak protein–protein association.
For high-speed AFM to become truly useful to studies on awide variety of biological systems, reduction in the force isessential. We proposed possible methods for achieving thisend. If the noncontact condition is realized in high-speed
imaging, small cantilevers that have higher stiffness andhence higher resonant frequencies can be used forbioimaging, which leads to higher imaging rates. In addition,it allows us to study biomolecular processes that occur onliving cell membranes, which tremendously expands theusefulness of high-speed AFM in biological sciences. We
need to carry out more efforts to exploit this difficult
challenge toward achieving our dream.

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