Japanese Journal of Applied Physics
Vol. 45, No. 3B, 2006, pp. 1897–1903#2006 The Japan Society of Applied Physics

High-Speed Atomic Force Microscopy for Studying the Dynamic Behaviorof Protein Molecules at Work
Toshio A NDO1;2;3 , Takayuki UCHIHASHI1;3 , Noriyuki K ODERA1 , Atsushi M IYAGI1 , Ryo NAKAKITA1 ,Hayato Y AMASHITA1 and Mitsuru SAKASHITA1
1

Department of Physics, Faculty of Science, Kanazawa University, Kakuma-machi, Kanazawa 920-1192, Japan
Frontier Science Research Organization, Kanazawa University, Kakuma-machi, Kanazawa 920-1192, Japan
3
JST/CREST, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan
2(Received July 3, 2005; accepted November 28, 2005; published online March 27, 2006)

In the quest for the mechanism of protein functions, various key techniques and instruments have been developed. This is an
era when scrutinizing a certain protein from various angles is becoming possible through combined knowledge of its structureand function. However, it is necessary to link these diﬀerent aspects of a protein along a time axis, but no technology isavailable for tracing a protein in action, at high spatial and temporal resolutions. Atomic force microscopy made it possible forthe ﬁrst time to view a nanometer-scale world in an aqueous environment. In 2001, we developed the ﬁrst-generation highspeed atomic force microscope (AFM) that could capture moving protein molecules on video at 80 ms/frame. Since then, we
have been carrying out various eﬀorts to increase its scan rate as well as to substantially reduce tip–sample interaction force.
The reduction in this force is a key to making the high-speed AFM practically useful in life sciences. Various new techniquesand devices developed in the past four years have brought the AFM to its second-generation stage. It can now capture weaklyinteracting protein molecules successively without disturbing their physiological function. Here, we report our eﬀorts madeover the past four years, the present capacity of the high-speed AFM, and our preliminary work on the next generation of the
instrument. [DOI: 10.1143/JJAP.45.1897]
KEYWORDS: AFM, protein, bio-imaging, dynamics, high-speed AFM

1.

Introduction

Proteins perform various sophisticated functions thatmanmade machines cannot achieve. Various techniques
have been developed to study their molecular mechanism.
The physiological action of individual protein molecules canbe observed using a ﬂuorescence microscope equipped witha high sensitive video camera. NMR and X-ray crystallography enable the determination of the atomic structure ofproteins, and the number of structure-determined proteins is
rapidly increasing. Genetic engineering enables us to
produce arbitrarily recombinant proteins, thereby making it
possible to study the molecular mechanism of a protein’s
activity at the amino acid level. It is now becoming possible
to study proteins thoroughly from various angles bycombining knowledge of its composition, structure, andfunction. We need to link these diﬀerent aspects of proteinfunctions in time. However, information on the nanometerscale dynamic behavior of proteins has been lacking for along time.
Atomic force microscopy made it possible for the ﬁrst
time to view a nanometer-scale world in an aqueous
environment. Biologists have hoped that it would allow theimaging of the dynamic behavior of protein molecules atwork. However, its scan speed is too slow to capture
biological processes at the molecular level in real time. Scan
speed is determined by the bandwidth of the feedback
operator that keeps the tip–sample interaction force constantby moving the sample stage up and down depending on
sample height. Since biological samples are soft and fragile,
this feedback operation is indispensable for minimizing suchinteraction force. In this feedback operation, various devices
are involved, such as a cantilever, a detector for sensing
cantilever deﬂection, a feedback circuit, and a z-scanner thatE-mail address: tando@kenroku.kanazawa-u.ac.jp

moves the sample stage in the z-direction. Various eﬀorts
have been made to date for the purpose of enhancing
feedback speed. Viani et al. used small cantilevers withresonant frequencies of 150 kHz to image DNA and GroEL–
GroES complexes on mica, in liquid.1,2) However, theirframe rate was less than 1/s, because the conventionalpiezotube scanner they used had a low resonant frequency inthe z-direction. To overcome the low resonant frequency of
conventional sample-stage scanners, Sulcheck et al.3,4) and
Rogers et al.5) used a cantilever with an integrated zinc oxide
piezoactuator capable of deﬂecting itself to keep the tip–
sample interaction force constant. They achieved an imaging
rate of 4/s.3) However, the self-actuating cantileverinevitably becomes very stiﬀ, and hence is inappropriate
for the imaging of soft samples. Although it was performed
not with atomic force microscopy (AFM) but rather withnear-ﬁeld scanning optical microscopy, Humphris et al.
achieved an imaging rate >100 frames/s, using a scansystem that employs the mechanical resonance of an opticalﬁber probe to address the sample.6) However, this high speed
is achieved without any feedback operation to keep theprobe-sample distance constant. Therefore, this technique isapplicable only to the AFM imaging of hard samples. We
optimized all the devices involved in the feedback loop, andwere able to produce a high-speed AFM that made it
possible, in the tapping mode of operation, to capture
rapidly moving protein molecules on video at a frame rate of
12.5/s.7,8)
However, it became clear later that the high-speed AFM
could not capture weakly interacting protein molecules,
because the protein–protein interaction was largely disturbed
by the tip–sample interaction.9) The diﬃculty in developinga high-speed AFM for life sciences lies not only inenhancing scanning speed, but also in keeping the tip–
sample interaction force small. These two aspects are often
contrary to each other. We have been developing various

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techniques to make these aspects compatible.9,10) Now thesecond-generation high-speed AFM can capture weaklyinteracting proteins on video, without disturbing their
physiological action.
2.

Quantiﬁcation of Feedback Bandwidth

Since the tapping mode of operation is most suitable forobserving soft samples that are weakly attached to a
substratum, here we consider only this mode. The cantileveris oscillated at (or near) its resonant frequency. The
oscillating tip brieﬂy taps the surface of the sample at thebottom of each swing, resulting in a decrease in oscillation
amplitude. This decrease is used to determine sample height.
Sulcheck et al. qualitatively analyzed how feedback bandwidth in the tapping mode is aﬀected by various factors,
such as the set-point, sample height, resonant frequency andoscillation amplitude of the cantilever.4) From the qualitativedescription, we cannot get a prospect of how extensively wehave to improve various devices in order to enhance
feedback bandwidth. Here, we give a semi-quantitativedescription.
Suppose that a sample on a substrate has a periodicity and that the sample stage is moved horizontally at a velocity
Vs , the spatial frequency 1= is converted to a temporalfrequency f  Vs =. This is the feedback frequency atwhich the sample stage is moved in the z-direction. The mostrigid deﬁnition of feedback bandwidth is the feedback
frequency at which feedback operation is phase-delayed atless than 45 . This corresponds to a time delay 1=8 f . The
feedback loop contains various delaying steps. The main
delays are in the time of reading the cantilever’s oscillation
amplitude (it takes at least 1= fc ), (2) the cantilever’s
response time (Qc = fc ), and (3) the z-scanner’s responsetime (Qs = fs ), where Qc and fc are the quality factor andresonant frequency of the cantilever, and Qs and fs are thequality factor and resonant frequency of the z-scanner,respectively. These delays and additional delays (the total,) give the following relationship.
fc
Qc Qs fcf <
þ fc 
ð1Þ
1þ
þ
8

 fs

Feedback bandwidth is determined by either the smaller
term on the right sides of eqs. (1) and (2). Although it isdiﬃcult to obtain theoretically a precise analytical expression for feedback bandwidth as a function of various
parameters, we can obtain a quantitative expression experimentally. The frame acquisition time (T) is determined by
feedback bandwidth ( f ), the scan size (L), the number ofscan lines (N), and the apparent sample size (), as shown
below.
T ¼ 2NL= f
3.

ð3Þ

New Devices

3.1 Active damping of scanner
The scanner is newly designed, employing a ﬂexure stage
instead of the ball-guide stage used in our ﬁrst-generation
high-speed AFM.7,8) As we reported previously, the structural resonance of the z-scanner is eliminated using twoz-piezoactuators placed in the counter direction. The
actuators are replaced with customized stack piezoactuators(3  3  3 mm3 , NEC-Tokin, Tokyo) with a self-resonancefrequency of 420 kHz and a maximum displacement of
900 nm (at 100 V). A glass sample stage, which has acircular trapezoid shape with a small top surface of 1 mm
diameter, is attached to the top of one of the z actuators via athin layer of nail enamel. To maintain balance, a dummy
stage that has the same mass as the sample stage is attachedto the counterbalance z-piezoactuator.7,8) The open-looptransfer function of the z-scanner is represented by curves (a)in Fig. 1. A resonant peak appeared at approximately 150
kHz, with Q ¼ 18. A tiny bump in the gain that appears at
approximately 50 kHz is due to the structural resonance ofthe z-scanner. Without counterbalance (curves b), it appears
with a much larger amplitude.

Here we have to note that the diﬀerential operator of the
proportional-integral-diﬀerential (PID) feedback circuit canpartially compensate the phase delays. Feedback bandwidthis not determined only by eq. (1), but also by sample height(h), the free oscillation amplitude (A0 ) of the cantilever, andthe setpoint (rA0 ). This is because the cantilever tip end
tends to detach completely from the sample surface when
scanning over a downhill, and its tendency is aﬀected bythese factors. Once detached, the tip end cannot re-land on
the surface quickly (like ‘‘parachuting’’), because the error
signal is saturated at ð1  rÞA0 . The time required for relanding is roughly proportional to   h=ð1  rÞA0 . Therefore, feedback bandwidth is also a function of . The
condition (as a ﬁrst approximation) that the oscillating tip
should touch even the steepest downhill of sample surface
at the bottom of every swing leads to the following
relationship.
f <fc 1 1
sin



ð2Þ

Fig. 1. Open-loop transfer functions of z-scanner (a) without active
damping and with counterbalancing, (b) without active damping andcounterbalancing, and (c) with active damping and counterbalancing.

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The response time of the z-scanner is 38 ms. This slow
response is due to the large Q value. When active Q-control
is applied to the z-scanner to reduce its Q value, it isnecessary to detect the displacement of the z-actuators,
which is not easy to do. However, the mechanical responseof the z-actuators is well approximated by a second-ordertransfer function. The input–output relationship is determined only by the transfer function. Therefore, instead ofmonitoring the displacement of each piezoactuator, we canmonitor output signals from a resonant system (‘‘mock’’ z
actuator) which is characterized by the same transfer
function as that of the z-piezoactuator.10) We constructed
the mock actuator from a LCR circuit. Using this active
Q-control, the Q value of the z-actuators is reduced to 0.5(curves c), resulting in a 36-fold enhancement in response
speed (response time = 1.1 ms).
A drawback is that the phase delay becomes pronounced
as shown in Fig. 1 (lower panel). We compensated this delay
by means of an inverse transfer function method,10) andsucceeded in extending frequency resulting in a 45 phasedelay from 51 to 131 kHz. Because this phase compensation
increases the gain in proportion to frequency, higher-orderresonant vibrations were increased. We solved this problem
by adding more mock z-actuators corresponding to higherorder resonant vibrations, and by using notch ﬁlters in series
to remove high-frequency noises (>1 MHz).
The same technique applied above to the z-actuators was
also used to remove the resonant vibrations of the x- andy-scanners. This resulted in almost no vibrations in thez-direction even when the frame rate was increased up to
30 frames/s.
3.2 Dynamic PID control
For the tapping force to be kept very small, thecantilever’s oscillation amplitude has to be kept very close
to the free oscillation amplitude. This condition often causes
a complete detachment of the cantilever tip from thesample/substratum surfaces. Once detached, re-landing is
attained very slowly, just as in ‘‘parachuting’’, because of‘‘feedback saturation’’ (i.e., the error signal saturates at a low
level, independently of how far the tip separates from thesurface at the bottom sweep). During parachuting, allinformation on sample topography is lost. Therefore, thefaster the scan is made over a steep-downhill sample surface,
the lower the setpoint has to be set to avoid feedback
saturation. Feedback saturation also occurs when the tip
encounters a steep-uphill sample surface, because cantilever
oscillation would be completely quenched in this encounter.
This malfunction is more seriously pronounced during fasterscanning. Fast scanning would thus not be compatible withharmless scanning.
When the setpoint (rA0 ) is very close to the free oscillation amplitude (A0 ), the saturated error signal ð1  rÞA0 isvery small. In addition, the conventional PID controller
operates mainly with the integral function. Therefore, thevery small saturated error signal prolongs the period ofparachuting. The gain parameters of the PID controller canbe increased to shorten parachuting period, but this often
results in feedback control instability. To overcome thisproblem associated with the conventional PID, we devised a
new PID controller (dynamic PID) that was practically freeFig. 2. Schematic of dynamic PID control technique. The gray line
represents the amplitude–distance curve. The broken line indicates a false
amplitude–distance curve produced when dynamic PID control operates.

of feedback saturation.9) Here, ‘‘dynamic’’ means that thegain parameters of the PID can be changed dynamicallydepending on tip–sample interaction force. The dynamic
PID controller is constructed with a circuit named the
dynamic operator, which is inserted between the error signal
and the input of a conventional PID circuit. This dynamic
operator works as follows (Fig. 2). A threshold level (Aupper )is set between rA0 and A0 . When the cantilever oscillation
amplitude (A) exceeds Aupper , the diﬀerential signal A 
Aupper is ampliﬁed and added to the error signal. The error
signal that now contains an extra signal is fed to theconventional PID. This ‘‘false error signal’’, which is larger
than the ‘‘true error signal’’, produces a quicker feedback
action, thereby A quickly becomes smaller than Aupper , andthen feedback operation returns to the conventional one. By
these actions, the feedback does not practically saturate evenwhen the true error signal saturates at a low level. A similar
manipulation of the error signal can also be made when A issmaller than rA0 . In this case, a new threshold level (Alower )is set lower than rA0 (Fig. 2). When A becomes smaller than
Alower , the diﬀerential signal A  Alower is ampliﬁed andadded to the true error signal. This manipulation can keep
the cantilever tip from pushing against the sample toostrongly, especially when the tip encounters a steep-uphillsurface of the sample.
The feedback performance of the dynamic PID controller
was compared with that of the conventional PID controller,
using a mock sample surface consisting of four rectangularsteps with two diﬀerent heights: one height is equal to A0 ,and the other is 2 times higher than A0 . The scan speed is0.25 mm/s, and the setpoint is 0:9  A0 . With the conventional PID controller, the topographic image is very blurred,because of a long period of parachuting [Fig. 3(a)]. On theother hand, with the dynamic PID controller, the image isvery clear, indicating that there is no complete detachmentof the cantilever tip from the sample surface [Fig. 3(b)].
Next, the closed loop feedback bandwidth was measured as afunction of setpoint level. In this measurement, the sample
stage height was varied using sinusoidal wave signals, and
the PID output signal was monitored. With the conventional
PID controller, feedback bandwidth is gradually decreased
with increasing setpoint level from 0:7A0 to 0:95A0 . The
bandwidth at r ¼ 0:95 is about one-sixth the bandwidth at
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Fig. 3. Eﬀect of dynamic PID control on suppression of parachuting
landing of cantilever tip onto surface. AFM images obtained (a) with aconventional PID controller, and (b) with the dynamic PID controller.

bandwidth is almost constant, irrespective of setpoint level.
Thus, the dynamic PID control is a superior techniqueenabling the fast-scanning tip to touch samples very lightly.
3.3 Cantilevers
The small cantilevers that we developed together with
Olympus in 2001 had a resonant frequency of 600 kHz inwater, and a spring constant of approximately 150 pN/nm.7)
Since the lever did not have a probe tip, the tip had to begrown using electron beam deposition (EBD). Recently,
Olympus has developed smaller cantilevers integrated withbeaklike tips [Fig. 4(a)].11) The resonant frequency was
800 kHz–1.2 MHz in water, and the spring constant ranged
from 100 to 250 pN/nm. Although we still have to grow an
EBD tip on a beaklike tip, a much shorter EBD tip is
suﬃcient [Fig. 4(b)], compared with that on the previous ﬂatlevers.
3.4 Other devices
Although not completed yet, here, we brieﬂy mention how
we have been working toward the third-generation highspeed AFM. To enhance feedback bandwidth, it is important
to increase the resonant frequency of the z-scanner, butavailable piezoactuators with enough displacement arelimited in the resonant frequency. We estimate that theachievable maximum resonant frequency of a piezo-stage
scanner will be limited to be about 300 kHz, only twice thatof our z-scanner. Self-actuating cantilevers are an alternative
to the piezo-stage scanner,3–5) but a cantilever with an

integrated zinc oxide piezoactuator inevitably becomes too
stiﬀ. Driving a cantilever coated with a thin magnetic ﬁlm
directly by a magnetic ﬁeld is also an alternative.12)
However, a magnetic-ﬁlm coating suﬃcient for the cantilever to be driven reduces the resonant frequency of thecantilever. To drive a cantilever by a diﬀerent means, wemeasured the cantilever deﬂection caused by the photothermal expansion of gold coated onto a Si3 N4 cantilever
surface. The sensitivity was about 10 nm/mW in an aqueous
solution, when a 405 nm violet laser was used with our smallcantilevers. Because of the slow heat transmission, theresponse time of the deﬂection was about 30 ms. However,
inverse transfer function compensation was apparently able
to eliminate the slow heat transmission. Since the maximum
displacement of the cantilever driven by photo-thermal
expansion has been limited so far, and the ‘‘cantilever
scanner’’ requires a large dynamic range of the deﬂection
detection sensor (which reduces sensor sensitivity), it isapparent that this technique has to be used together with theconventional piezo-stage scanner. Photo-thermal expansion
can also be use to directly excite a cantilever at its resonant
frequency.13) Unlike acoustic excitation, it does not have the‘‘forest-of-peaks’’ eﬀect. This superior attribute facilitates
the active control of the cantilever to reduce Q value, which
enhances the response speed of the cantilever (and hence
feedback bandwidth). Another device that we have been
trying to include in the third-generation high-speed AFM is afeed-forward controller.14) Topographic diﬀerences between
two adjacent line scans are not large, as long as the sample ismoving slowly. Therefore, line-by-line feed-forward control
can facilitate performance of the feedback-control task.
Using stationary samples, we conﬁrmed that a signiﬁcant
extension of feedback bandwidth was achieved by the feedforward control. However, when the sample is moving
quickly, the feed-forward control is supposed to act occasionally in an undesirable direction. Yet, we have notnoticed an ill eﬀect expected from such an undesirable
action even with rapidly moving samples. This may be dueto the small time diﬀerence (less than 1 ms) between twoline scans.
4.

Bioimaging

In our ﬁrst-generation high-speed AFM, the tip–sample
interaction force was too large to image fragile protein

Fig. 4. Electron micrographs of small cantilever with beaklike tip (a) without an EDB tip, and (b) with an EBD tip grown on beaklike
tip.

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Fig. 5. Successive AFM images of biological samples captured using high-speed AFM. All the numbers indicate frame numbers. (a)Microtubules being destroyed during imaging (scale bar, 100 nm; 640 ms/frame), (b) actin ﬁlaments gliding over myosin V-coatedsurface (scan range, 1 mm; 1 s/frame), (c) 3D images of myosin V molecules pulling actin ﬁlament leftward (note that the right distal
end of the actin ﬁlament is moving leftward; 180 ms/frame), (d) dynein C changing its stem orientation during ATPase reaction(160 ms/frame; the most left-hand image is an electron micrograph15) of dynein C), (e) myosin V changing its conformation after
ATP is released from caged ATP by ﬂash photolysis (the UV ﬂash is applied between the third and fourth frames, 80 ms/frame), and(f) GroES binding to GroEL layer after ﬂash-photolysis of caged ATP (‘‘F’’ denotes that the UV ﬂash is applied just prior to theframes; scale bar, 100 nm; 1 s/frame).

assemblies such as microtubules and actin ﬁlaments.9) Thesesamples were gradually disassembled during imaging[Fig. 5(a)]. Our eﬀorts made to solve this problem led to
the new active damping method and the dynamic PID
controller, as mentioned above. Owing to these devices, it
became possible not only to image fragile samples without
disassembly, but also to capture weakly interacting protein

molecules without disturbing their physiological functions.
This is demonstrated by imaging actin ﬁlaments moving, inthe presence of ATP, over a surface that is densely coatedwith myosin V molecules [Fig. 5(b)]. The velocity of actin
ﬁlaments gliding was very similar to that observed by
ﬂuorescence video microscopy. To view the dynamic
behavior of individual myosin V molecules when they are

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propelling actin ﬁlaments unidirectionally, the surface wassparsely coated with myosin V. A short actin ﬁlament
entirely within the imaging region was observed. This
fortunate situation enabled us to identify which myosin V
molecules are propelling the actin ﬁlament. Two myosin V
molecules were interacting with the short actin ﬁlament, andonly one of the two heads of each myosin V molecule was
attached to the actin ﬁlament. The actin ﬁlament moved
leftward in a stepwise manner; the step size was 36 nm,
which corresponds to one half of the long helical repeat of an
actin ﬁlament. During the stepwise movement, the interacting heads did not seem to change their conformations largely[Fig. 5(c)], which was not the behavior expected from thewell-studied swinging-lever arm hypothesis in which a large
swinging movement of the myosin’s neck domain is
supposed to propel an actin ﬁlament.
Dynein C is a single-headed dynein. A stalk and a stem
are protruded from the ring-shaped head, and the stalkinteracts with microtubules at its globular distal end.
Electron micrographs of dynein C revealed that the angle
between the stalk and the stem diﬀered between the apo state(no-nucleotide) and the ADP-Vi bound state.15) The nucleotide-dependent change in the structure is hypothesized todrive microtubule movement. However, ADP-Vi is a nonnatural substrate, and therefore it is not certain that such achange in angle really occurs during the ATPase cycle.
High-speed AFM imaging of dynein C in the presence of
ATP revealed that it does certainly occur [Fig. 5(d)]. The
stalk and head seemed stationary, but the stem went backand forth between two positions. The stem-stalk angle
changed between 137 and 184 ; the former angle corresponds to the apo state, and the latter was larger than that of
the ADP-Vi bound state (159 ). The frequency of the change
in angle approximately coincided with the ATPase rate.
Therefore, it is very certain that the stem movement
observed is driven by the ATPase reaction. This is the ﬁrst
time that a cyclic protein’s conformational change has been
captured on video in real time.
The ATPase rate of dynein C is relatively high. However,the ATPase rate of myosin V is very low, about 0.05/s.16) It
is thus diﬃcult to capture cyclic conformational changes in
myosin V during the ATPase reaction. To facilitate such anobservation, we introduced a UV photolysis system to the
AFM; ATP is quickly released from caged ATP upon UV
light application.17) As shown in Fig. 5(e), myosin V bent at
the head-neck junction immediately after UV irradiation,and then returned to the original straight form. This
technique is very valuable for recording substrate-induced
conformational changes in proteins, because protein molecules weakly attached to the substratum are always moving
due to thermal agitations, therefore, in steady-state recording, it is not easy to distinguish Brownian movement fromthe substrate-induced conformational change.
The UV photolysis of caged ATP was applied to the
GroEL–GroES system. GroEL is a chaperonin that assists its
substrate polypeptides to fold into their functional threedimensional (3D) structures, and GroES is a co-chaperonin.
GroEL consists of two homo-heptameric rings that arestacked back to back,18) whereas GroES is a single homoheptameric ring.19) GroES binds to GroEL only when GroEL
is in the nucleotide-bound state. GroEL was attached to themica surface with an end-up orientation, forming a highly
packed single layer [Fig. 5(f)]. The central channel of the
GroEL ring was therefore visible. Before UV application,
GroES was ﬂoating in the solution containing caged ATP,and therefore not visible. High-frequency pulses of a UV
laser light (355 nm) were applied within 5 ms while y-return
scanning was performed. Immediately after this, GroES
attached to the top of GroEL, forming protrusions on the
GroEL layer. Since the concentration of the ATP producedin the small UV-irradiated volume quickly decreased due todiﬀusion, the second application of UV pulses resulted in
more protrusions. Using this technique, the nucleotideinduced cis–trans conformational transition of individual
GroEL molecules was also studied.20)
5.

Discussion

Our second-generation high-speed AFM has the capacity
to dissect dynamic processes of individual protein molecules
at work. The compatibility of a high scan rate and askimming scan over fragile samples is successfully achieved. Scan rate has reached a level at which video-rate imaging
is nearly possible. Tip–sample interaction force is greatly
reduced to a level where it does not disturb weak protein–protein interactions. The active damping that we havedeveloped for the sample-stage scanner10) is widely applicable, not only to any type of scanning probe microscope,
but also to instruments where fast and precise mechanicaldisplacement is required. The dynamic PID operator wehave developed is realized with a simple analogue circuit,and hence works very fast. Various types of robust feedback
techniques have been proposed,21,22) and the usefulness ofsome of them has been demonstrated with conventional
AFMs. However, most of them require intricate calculations
by a computer or a DSP system, and even with a fastcomputer/DSP, the required calculation time seems to be
too long for digital feedback operators to be implemented in
the high-speed AFM.
What new developments will the next generation highspeed AFM for life sciences require? The cantilever andz-scanner are devices that must be improved to achieve ahigher scan rate. At present, their respective response speeds
are similar to each other, because the cantilever’s Q value
cannot be reduced from its natural Q value in water (2:0)
due to the lack of means to excite the cantilever directly.
This is one reason we have recently been trying to use thephoto-thermal actuation of a cantilever. We are now
developing smaller cantilevers with a resonant frequencyof 2 MHz in water. When such cantilevers are combinedwith photo-thermal actuation to excite their resonance withactive damping as well as to deﬂect them to control tip–
sample distance, feedback bandwidth will reach 200 kHz.
This bandwidth will enhance the frame rate up to 40 –50 frame/s (with the scan size of 250 nm). The line-by-line
feed-forward control will become more useful for observing
moving proteins, when a higher scan rate is available. The
higher the scan rate the smaller are the height changes
produced by the movement during the time interval of two
adjacent line scans. By adding the feed-forward control tothe above-mentioned improvements, we may be able toreach a frame rate of 100 frames/s. Not only changes inprotein shapes, but also a change in the map of their

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physicochemical properties, such as electrostatic properties,
are involved in proteins’ physiological functions. AFM canmap such properties in the non-contact phase-imaging
mode.23,24) Small cantilevers for high-speed imaging havea large resonant frequency as well as a small spring constant.
These mechanical properties sensitize phase detection
markedly. Therefore, in phase detection, we do not have torely on a large Q value of the cantilever and slow phasemeasurement instruments. This will enable high-speed phaseimaging in the near future.
The observation of the nanometer-scale dynamic behaviorof protein molecules in action has just been started recently.
Numerous biological processes are waiting to be imaged by
the high-speed AFM, such as processive motors moving
along their tracks, molecular chaperones assisting a polypeptide chain to fold, and a ribosome synthesizing a polypeptide
according to the nucleotide sequence on mRNA. In addition,
the not-yet-studied subject of how proteins dynamicallychange their physicochemical properties while functioning is
waiting to be studied. Together with the knowledge
attainable from established techniques, the protein dynamics
that will be revealed for the ﬁrst time by the high-speed
AFM will uncover the molecular mechanisms of proteinfunctions.
Acknowledgement
This work was supported by CREST/JST, Special
Coordination Funds for Promoting Science and Technology(Eﬀective Promotion of Joint Research with Industry,
Academia, and Government) from JST, and a Grant-in-Aid
for Basic Research (S) from the Ministry of Education,
Culture, Sports, Science and Technology of Japan.

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