A high-speed atomic force microscope for studying
biological macromolecules
Toshio Ando*†, Noriyuki Kodera*, Eisuke Takai*, Daisuke Maruyama*, Kiwamu Saito*, and Akitoshi Toda‡*Department of Physics, Faculty of Science, Kanazawa University, Kakuma-machi, Kanazawa 920-1192, Japan; and ‡Olympus Co., 2951 Ishikawa-machi,
Hachiohji, Tokyo 192-8507, Japan
Communicated by Shinya Inoué, Marine Biological Laboratory, Woods Hole, MA, July 31, 2001 (received for review May 21, 2001)

The atomic force microscope (AFM) is a powerful tool for imaging
individual biological molecules attached to a substrate and placedin aqueous solution. At present, however, it is limited by the speed
at which it can successively record highly resolved images. We
sought to increase markedly the scan speed of the AFM, so that in
the future it can be used to study the dynamic behavior of
biomolecules. For this purpose, we have developed a high-speed
scanner, free of resonant vibrations up to 60 kHz, small cantilevers
with high resonance frequencies (450 – 650 kHz) and small spring
constants (150 –280 pN兾nm), an objective-lens type of deflection
detection device, and several electronic devices of wide bandwidth. Integration of these various devices has produced an AFM
that can capture a 100 ⴛ 100 pixel2 image within 80 ms andtherefore can generate a movie consisting of many successive
images (80-ms intervals) of a sample in aqueous solution. This isdemonstrated by imaging myosin V molecules moving on mica (seehttp:兾兾www.s.kanazawa-u.ac.jp兾phys兾biophys兾bmv㛭movie.htm).

O

ne of the advantages of the atomic force microscope (AFM)(1) is its capacity to image individual biomolecules in, say,a buffered solution containing ions at physiological concentrations (2, 3). Such capacity suggests that the instrument can beused to record the dynamic behavior of such molecules. In
practice, however, only very slow processes can be recorded (2,
4–6), because commercially available AFMs require minutes toform an acceptable image, and many interesting biologicalprocesses occur at much higher rates. To understand, andovercome, the factors that limit the scanning rate of an AFM, we
begin by considering relations between the characteristics of theconstituting components.
We consider only the ‘‘tapping mode’’ of AFM operation(Digital Instruments, Santa Barbara, CA). This is the modesuitable for imaging biological macromolecules, because verticaloscillation of the cantilever at (or near to) its resonance frequency reduces lateral forces between the tip and the sample (7).
The oscillating tip briefly taps the surface at the bottom of eachswing, resulting in a decrease in oscillation amplitude. During thex-y scan of the sample stage a feedback loop (see below) keeps
this decrease (and hence the tapping force) constant; this isnecessary for minimizing the deformation of soft samples. The
error signal—the difference between a preset signal and the rms
amplitude of the cantilever—is fed into a proportional-integraldifferential (PID) feedback circuit. The PID output is amplified
and then sent to the z-piezo actuator; this is repeated until theerror signal returns to zero. For the three-dimentional movement of the sample stage to follow the sample topography
accurately, the bandwidth of the feedback loop should becomparable to, or larger than, the frequency determined by thex-y scan velocity and the apparent width of the features on thesurface. To increase the imaging bandwidth, all elements in the
feedback loop have to be optimized. The first element in thisloop is the cantilever. A high resonance frequency in liquid isrequired to generate a high tapping frequency; also required isa low spring constant, so as to minimize the tip-sample interaction force. These conflicting requirements can be met only withsmall cantilevers (8, 9). Every circuit in the feedback loop must
12468 –12472 兩 PNAS 兩 October 23, 2001 兩 vol. 98 兩 no. 22

have a wide bandwidth. The last element, the z-scanner, is apiezo actuator with a high resonance frequency, which in assembly must react very quickly without unwanted mechanicalvibrations. Viani et al. (10, 11), using the tapping mode, haveused small cantilevers with resonance frequencies ⬇150 kHz toimage DNA and GroEL–GroES complexes on mica, in liquid.
Their frame rate was less than 1 s⫺1 because they used aconventional piezo-tube with a resonance frequency of ⬇15 kHz
in the z direction, together with feedback electronics of a limited
bandwidth. Sulchek et al. (12), using a cantilever with an
integrated zinc oxide piezo actuator (13), achieved an imaging
bandwidth of 38 kHz and frame rate of ⬇4兾sec. However, theircantilever was too stiff to image soft samples. In the AFM
assembly described below we have tried to optimize each element for its use in imaging samples in solution, using fast tapping
operation, and an objective lens type of deflection detection
device suitable for small cantilevers. Assembly of these selectedcomponents results in an imaging bandwidth of ⬇60 kHz (fastest
among the high-speed AFMs that use the tapping mode and aresuitable for examining soft samples in liquid). Combination ofthis high speed with zooming-in capability (reduction of the scan
area to 240 ⫻ 240 nm2) allows viewing of liquid samples ofproteins on mica at a rate of 12.5 s⫺1.
Test Sample, Components, and Imaging
Sample Preparation. For test purposes, myosin V was extracted

from chick brains and purified as described (14). Myosin V
stored at 0°C in buffer A (25 mM KCl兾25 mM Imidazole, pH
7.6兾2 mM MgCl2兾1 mM EGTA兾0.2 mM DTT) was diluted to 7
nM with buffer A. A drop (⬇2 ␮l) of myosin V solution wasplaced on freshly cleaved mica (1 mm in diameter) for 3 min,rinsed with buffer A to remove unattached myosin V, and thenimaged in 2 mM ATP plus buffer A without DTT.
Cantilevers. The cantilevers, made from silicon nitride using
micromachining techniques, were 140 nm thick, 2 ␮m wide, and9–11 ␮m long. The rear side of the cantilevers was coated withgold 20 nm thick. All surfaces of the cantilevers were further
coated with osmium, about 2 nm thick. The tips were grown byelectron-beam deposition (15), with growth rate of about 5 nm兾s.
The tip length was adjusted to ⬇1.0 ␮m. The radius of the tip end
was 6–8 nm. The mechanical properties of the cantilevers were
tested by measuring the spectra of their thermal motion. The
resonance frequencies were 1.3–1.8 MHz in air, and 450–650
kHz in water, and the spring constants were estimated to be
150–280 pN兾nm.
Deflection Detection System. We used an optical lever method tomeasure the deflection of a cantilever. The optics must generate

Abbreviations: AFM, atomic force microscope; PID, proportional-integral-differential: S兾H,
sample兾hold.
†To

whom reprint requests should be addressed. E-mail: tando@kenroku.kanazawau.ac.jp.

The publication costs of this article were defrayed in part by page charge payment. This
article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C.
§1734 solely to indicate this fact.

www.pnas.org兾cgi兾doi兾10.1073兾pnas.211400898

BIOPHYSICS

Fig. 1. Schematic drawing of the AFM head integrated with an inverted typeof optical microscope. (a) ␭兾4 Plate. (b) Dichroic mirror. (c) Polarization beam
splitter. (d) Focusing lens. (e) Split photodiode. ( f) Collimator lens. (g) Laser
diode. (h) Illumination lamp. (i) Collimator lens. (j) Half mirror. (k) Viewing
system of the optical microscope. (l) Screw for adjusting the cantilever height.
(m) Piezo for exciting cantilever. (n) Objective lens. (o) Cantilever. (p) Sample
stage. (q) Sample stage of the optical microscope. All of the components (a–gand n) of the deflection detection system are hung down from the sample
stage of the optical microscope. The scanner (see Fig. 2 also) is mounted on thesame sample stage. The collimated laser beam is reflected up, incident on theobjective lens at an off-centered position. The outgoing beam thus tilted
about 10o from the vertical plane is focused onto the cantilever whose plane
is tilted about 10o from the horizontal plane. The beam reflected at thecantilever is collimated by the objective lens, separated from the incident
beam by the polarization beam splitter with ␭兾4 wave plate, and reflected
onto the split photodiode. The optical microscope allows us to view thecantilever and the focused laser spot. The specimen is supported at the bottomof the sample stage (p), and an inverted cantilever (o) probes the specimen
from below.

a very small incident beam spot, because our cantilevers for rapid
scanning are much smaller than commercially available cantilevers. Schäffer et al. (16) designed an AFM using optics for smallcantilevers, wherein a laser beam reflected from the rear side of
the cantilever is collected and collimated with the same lenses as
are used for focusing the incident laser beam onto the cantilever.
We used the same method, but instead of using single lenses weused an objective lens (CFI Plan Fluor ELWD 20xC, Nikon) witha long working distance of 8 mm. As shown in Fig. 1, our AFM
is integrated with an inverted type of optical microscope (IX50,
Olympus, Tokyo). The focusing objective lens also is used to viewthe cantilever and the focused laser spot with the optical
microscope. The focused spot is 2–3 ␮m in diameter. In the
Ando et al.

Fig. 2. Scanner assembly. (Lower) The side view when looked at the scannerfrom ⫹x to ⫺x. The scanner has a two-layered structure that guarantees thatthe x-scan and the y-scan do not interfere with each other. For structuraldetails, see the text.

design of Schäffer et al., the entire optical train is tilted from thevertical so that the incident beam is normal to the plane of thecantilever. Instead of this, we used a much simpler design
wherein the vertical laser beam is incident on an off-centeredposition of the objective lens to make the outgoing beam axis
normal to the plain of the cantilever. The incident and reflected
light beams were separated by polarization using a beam splitterand a quaterwave plate. The laser beam, reflected, collimated,
and then slightly converged, is fed into the two closely spaced
photodetectors (S2721–02, Hamamatsu Photonics, Hamamatsu
City, Japan) whose photocurrents are sent to a differential
amplifier with a wide bandwidth (2.5 MHz).
Scanner. The scanner is shown in Fig. 2. Stack piezoelectric

actuators (AE0203D04, Tokin, Tokyo) are used in this scanner.
They have the resonance frequency of 260 kHz in free oscillation,
their maximum displacement is 4.5 ␮m, and their capacitance is
90 nF. The y-piezo displaces base-1 on which the x- andz-scanners are mounted, while the x-piezo displaces base-2 onwhich the z-scanner is mounted. The z-scanner has two z-piezo
actuators placed in the opposite direction to one another (thereason is described below). Base-1 is glued to the y-piezo, andbase-2 is glued to the x- and z-piezo actuators. A sample stage
PNAS 兩 October 23, 2001 兩 vol. 98 兩 no. 22 兩 12469

a differential amplifier to generate output that represents theamplitude of the source signal. The signal generation for the x-yscan, and the data sampling are made in different ways. One ismade by using a digital-signal-processor (DSP) control board
integrated with A兾D and D兾A converters. This allows flexible
controls for AFM imaging, but its operation rate is limited
because of our slow DSP system. The other is made by using an
external circuit that generates analog signals for the x-y scan andtiming pulses for data acquisition, and by using a fast A兾D board
for data sampling. This system allows a fast operation (the pixel
clock can go up to 1 MHz).
AFM Head Assembly. The sample stage of the inverted type of

Fig. 3. Circuit for fast amplitude measurement. The output sinusoidal signal
from the split-photodiode amplifier is fed to this circuit. The output of thiscircuit provides the amplitude of the sinusoidal input signal at periodicity of
the input signal. For details, see the text.

is attached to one of the z-piezo actuators via a thin layer ofvacuum grease. When the z-piezo is displaced quickly, hydrodynamic force is generated as the reaction from the sample
solution to the sample stage. To minimize this reactive force, aglass of a circular-trapezoid shape with a small top surface of
1-mm diameter is used as the sample stage. To hold the massbalance, a dummy stage that has the same mass as the sample
stage was attached to the other z-piezo actuator. Base-1 sits on
three steel ball bearings, and its top plane is pushed down byplate-1 through three steel ball bearings. The balls are 1 mm indiameter. Base-2 is also held between base-1 and plate-2 in asimilar manner. This way of holding base-1 and base-2 between
two flat surfaces reduces vibrations of these bases in thez-direction and allows smooth displacements in the desired
directions. However, these arrangements are not sufficient tominimize such vibrations. Quick displacements of a z-piezo exert
impulsive forces on base-2, which causes vibrations of base-2, andin turn vibrates the z-piezo itself. Such impulsive forces arecountered by the simultaneous displacements of the two z-piezo
actuators of the same length, in the counter direction.
Electronics. The custom-made piezo-drive amplifiers have a

1-MHz bandwidth for a 90-nF capacitive load, in 2 Vp-p output.
These amplifiers are used to drive the z-piezo actuators and todrive another piezo actuator (custom-made; with a 2-MHz
resonance frequency) that excites the cantilever in the tapping
mode AFM operation. The x- and y-piezo actuators are driven
by amplifiers (HJPZ-0.15Px3, Matsusada Precision, Tokyo) witha 25-kHz bandwidth for a 90-nF capacitive load, in 100 Vp-p
output. The PID and two types of rms-to-dc circuits were
custom-made. The bandwidth of the PID circuit is 300 kHz. An
analog type of rms-to-dc converter with a fourth-order low-passfilter can accept up to a 1-MHz input signal and requires fivewaves of the signal to output an accurate rms value; in otherwords, five tip-sample contacts are needed to measure thetapping amplitude. To increase the conversion rate, we made anovel circuit with 1-MHz bandwidth, which requires only one
wave to generate the amplitude signal (Fig. 3). The input(source) signal is divided into two. One is sent to two sample兾
hold (S兾H) circuits. The other is sent to a 90o phase shifter, andits output is fed to a zero-cross comparator to generate rectangular wave signals. These rectangular signals are sent to a
mono-stable multivibrator to generate pulses at the top andbottom peaks of the input source signal. The pulses from themono-stable multi vibrator are used as triggers to hold the source
signal by the S兾Hs. The outputs from the two S兾Hs are sent to12470 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.211400898

optical microscope was replaced with a stainless steel plate 10
mm thick. Every mechanical and optical component of the AFM
is mounted on or hung from this plate. This design reduces
relative movement between these components, despite vibrations of the optical microscope itself, which is susceptible to theambient sounds or mechanical noises because of its low resonance frequency. Because we use confocal optics for focusing thelaser beam onto a cantilever, as well as for viewing the cantilever
and the incident spot with the optical microscope, the rear sideof the cantilever must face downward (and hence, the tip muststick upward). This setup is opposite to that widely used incommercial AFMs. The cantilever is attached to a transparent
glass plate via a short cantilever holder glued to the glass plate.
The scanner, with a sample stage facing downward, is mounted
over the cantilever (see Fig. 1).

Imaging Bandwidth. When a piezo actuator is oscillated with its

one end being fixed, its resonance frequency is half the resonancefrequency when it is oscillated with both ends free. So, thez-piezo actuators must have a resonance frequency of 130 kHz,
without considering the mass attached to the free end of theactuators. The z-scanner in the whole scanner assembly musthave a lower mechanical resonance frequency, considering themass (of the order of g), the size (of the order of mm), and theYoung’s modulus (of the order of 1010–1011 N兾m2) of thematerials such as super invar steel and resin used in the piezo
actuators. Therefore, the z-scanner is the component that is mostdifficult to optimize for high-speed scanning. We tested thez-scanner’s ability to follow a frequency modulation of thedriving signal, without a feedback system, while the tip of a
cantilever remained in contact with a mica surface. The sensor
output is shown as a function of the driving frequency (Fig. 4).
When the z-piezo for the counterbalance was not operating, thez-scanner showed resonance vibrations around 8.5 kHz, 34 kHz,and 100 kHz (Fig. 4a). The resonance at 100 kHz is caused by theresonance of the z-piezo actuator itself. The extra amplitudes
gained by resonance at the lower frequencies are, however,relatively small compared with the oscillation amplitude without
resonance, indicating the value of our scanner design in suppressing the resonance vibrations. When the counterbalancer
was operating (Fig. 4b), the extra amplitudes at the lower
resonance frequencies were reduced, and we further noticed
much less variation in the amplitude over frequencies lower than
60 kHz, compared with those without counterbalance.
The bandwidth of the whole feedback loop system was assessed by measuring the system’s ability to follow a modulationof the set point while the tip was in contact with a mica surface(12). Sinusoidal signals were sent to the set point input, and theoutput of the PID was fed to a lock-in amplifier to measure theamplitude and the phase relative to the input sinusoidal signals.
The output amplitude was almost constant up to 60 kHz, andthen gradually decreased with increasing frequency. This gradual decrease is caused by the resonance oscillation of thez-scanner around 100 kHz. The phase difference was zero overthe frequencies tested (up to 100 kHz). These results indicate
Ando et al.

that the component that has the lowest bandwidth in the systemis the z-scanner, and therefore, the bandwidth of the system isabout 60 kHz.
The minimum imaging time (t) per frame with N2 pixels of size
p is given byt ⫽ N 2 p兾wf s ,[1]

where fs is the bandwidth of the feedback loop, and w is theapparent width of features on the surface. w is given by convolution of the tip with radius R and the sample with radius r, andis approximated as w ⫽ 公rR (17). A scan range of 200 nm and1002 pixels, thus a pixel size of 2 nm, can be scanned in 33 ms forw ⫽ 10 nm and fs ⫽ 60 kHz. Because the corresponding pixel timeis 1.65 ␮s, this scan condition requires the tapping and data
sampling frequencies to be equal to, or larger than 606 kHz (thereciprocal of the pixel time). One step of the x-y scan has to becompleted within this pixel time.
Imaging. We examined whether imaging can really be made at (ornear to) the maximum rate expected above. Myosin V directly
attached to mica, in solution containing 2 mM ATP, was imaged
successively (240-nm scan range; 1002 pixels) for 4 sec (50frames). The scan rate was 1.25 kHz, corresponding to a tip speedof 0.6 mm兾s, and the frame rate was 12.5 s⫺1. In the myosin V
molecule located on the center of the images (Fig. 5), its twohead兾neck regions, one long tail, and the globular tail end areclearly resolved. The tail seems to be about 100 nm long, longer
than a previous estimate based on its primary structure and theelectron microscopic images (18). The angle of the long tailrelative to the head兾neck regions changed between the eighthand ninth frames (see the third and fourth images in Fig. 5).
After this quick change, the tail and the tail end are slowly
Ando et al.

moving. The reconstructed movie and the other movies that
dramatically show the changes in the locations of the myosin
heads, neck, and tail regions can be viewed at http:兾兾
www.s.kanazawa-u.ac.jp兾phys兾biophys兾bmv㛭movie.htm. We
tried faster imaging (25 frames兾sec for 240-nm area with 100 ⫻100 pixels), but it was not successful because of the inertia of themass attached to the free end of the x-piezo. This inertia
produces mirror images on the left half area of the total image.
Note that even in Fig. 5 mirror images are noticed (indicated byarrow-4 in the second image).
Discussion
The components that limit the imaging rate of our AFM are first
the x-scanner, and second the z-scanner and the cantilever. An
upper limit of the imaging rate that is reachable by improving
these components can be estimated. We used super invar steel
for base-2. To minimize the inertia of the mass, we may be able
to use hard ceramics for base-2, which can reduce the mass to aquarter, and therefore can increase the imaging rate four times.
Cantilevers smaller than those we made can be constructed. For
instance, a silicon nitride cantilever, 42 nm thick, 6 ␮m long, and2 ␮m wide, may be possible, although this will be probably thesmallest size, considering how it has to be used in practice. This
small cantilever with a gold coat will have the likely resonancefrequency of ⬇2.5 MHz in air and the spring constant of ⬇200pN兾nm. The resonance frequency in water must be around 1.2
MHz, about two times larger than that of our present cantilevers.
In the tapping mode operation with this cantilever the pixel
imaging time can be reduced to 0.83 ␮s, which corresponds to 17
PNAS 兩 October 23, 2001 兩 vol. 98 兩 no. 22 兩 12471

BIOPHYSICS

Fig. 4. Oscillation of the z-scanner as a function of the driving frequency. The
amplitude of the driving signal was kept constant while sweeping its frequency. The data were obtained with (b) or without (a) operating the z-piezo
for the counterbalance.

Fig. 5. Successive images of myosin V on mica in buffer solution. The same
area of 240 ⫻ 240 nm2 was imaged 50 times with 100 ⫻ 100 pixels. Only nine
successive images are shown. The tip speed is 0.6 mm兾s (scan rate, 1.25 kHz),and the frame rate is 12.5兾s. The tapping frequency is 620 kHz, the amplitudeof the cantilever’s free oscillation is 12 nm, and the set point is about 11.5 nm.
In the first image, the head兾neck regions, the long tail, and the globular tailend are marked with arrow 1, arrow 2, and arrow 3, respectively. A mirror
image of one of the head region is marked with arrow-4 in the second image.
The still panels here appear noisier than the dynamic images in the movie on
the web site http:兾兾www.s.kanazawa-u.ac.jp兾phys兾biophys兾bmv㛭movie.htm.
The two head兾neck regions are overlapped in the z direction, and therefore,
they are not well resolved and do not show a typical ‘‘Y’’ shape. Dynamic
images of myosin V that show ‘‘Y’’ shape are also presented at http:兾兾
www.s.kanazawa-u.ac.jp兾phys兾biophys兾bmv_movie.htm.

ms of the imaging time, for 1002 pixels. If the second harmonic
oscillation can be used, the imaging time can be reduced to 8.3
ms. The speed limit of our z-scanner arises from the resonancefrequency of the piezo actuator. Piezo actuators with higher
resonance frequency are available, but their maximum displacement is shorter. Considering the necessary displacement size,
say, 1 ␮m, the maximum resonance frequency will be about 1
MHz when one end of such a piezo actuator is fixed. This is about
seven times larger than that used in this study. Considerations
made here suggest that an upper limit of the imaging time seems
to be 10⬃20 ms, as long as we use the optical deflection detection
method as well as the tapping mode operation.
The long tail of a myosin V molecule changed its orientationin less than ⬇30 ms (just before the change, the tail end locatedat y ⬇ 214 nm (i.e., at almost the end of the frame scan), while
just after the change the tail end located at y ⬇ 64 nm). On theother hand, after the quick change the tail moved slowly. This
marked contrast suggests that this quick orientational change
may be driven by ATP. However, at present we must reserve
judgment, because we could not observe repeated orientationalchange with the same myosin V molecule. To decide this, wehave to record images for a much longer time, because theATPase rate of myosin V alone is very small (⬇0.05兾s). At
present our AFM system cannot store many images at once. In
the near future we may be able to resolve this problem.
In life science, it has been a dream to view the nanometer-scale
dynamic behavior of individual biopolymers in solution. The
capacity of acquiring successive images every 80 ms will allow alarge expansion in the scope of biological processes that can be
examined in real time. In the near future we should be able tosee the behavior of processive motors [such as kinesin (19) andmyosin V (20)] walking along their tracks, or of molecularchaperones assisting a polypeptide chain to fold (21). Such direct
observations will be a great help in understanding the mechanisms by which biomolecular machines operate.

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12472 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.211400898

We thank Keichi Mukai and Ryuki Tsunekawa for their help in making the
AFM head, Takeshi Sakamoto and Tatsuya Kinosita for preparing myosin
V, and Professor Manuel Morales (University of the Pacific) for his
continuous encouragement and for reviewing this manuscript. This work
was supported by the Proposal-Based New Industry Creative Type Technology R&D Promotion Program from the New Energy and Industrial
Technology Development Organization (NEDO) of Japan to T.A.

Ando et al.