Wide-area scanner for high-speed atomic force microscopy
Hiroki Watanabe, Takayuki Uchihashi, Toshihide Kobashi, Mikihiro Shibata, Jun Nishiyama et al.
Citation: Rev. Sci. Instrum. 84, 053702 (2013); doi: 10.1063/1.4803449
View online: http://dx.doi.org/10.1063/1.4803449
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REVIEW OF SCIENTIFIC INSTRUMENTS 84, 053702 (2013)

Wide-area scanner for high-speed atomic force microscopy
Hiroki Watanabe,1 Takayuki Uchihashi,1,2 Toshihide Kobashi,1 Mikihiro Shibata,3
Jun Nishiyama,3 Ryohei Yasuda,3,4 and Toshio Ando1,2,a)
1
Department of Physics, College of Science and Engineering, Kanazawa University,
Kanazawa 920-1192, Japan
2
Bio-AFM Frontier Research Center, College of Science and Engineering, Kanazawa University,
Kanazawa 920-1192, Japan3
Department of Neurobiology, Duke University Medical Center, Durham, North Carolina 27710, USA
4
Max Planck Florida Institute, Jupiter, Florida 33458, USA(Received 25 February 2013; accepted 11 April 2013; published online 3 May 2013)
High-speed atomic force microscopy (HS-AFM) has recently been established. The dynamic processes and structural dynamics of protein molecules in action have been successfully visualized using HS-AFM. However, its maximum scan ranges in the X- and Y-directions have been limited to∼1 μm and ∼4 μm, respectively, making it infeasible to observe the dynamics of much larger samples, including live cells. Here, we develop a wide-area scanner with a maximum XY scan range of∼46 × 46 μm2 by magnifying the displacements of stack piezoelectric actuators using a leveragemechanism. Mechanical vibrations produced by fast displacement of the X-scanner are suppressedby a combination of feed-forward inverse compensation and the use of triangular scan signals withrounded vertices. As a result, the scan speed in the X-direction reaches 6.3 mm/s even for a scansize as large as ∼40 μm. The nonlinearity of the X- and Y-piezoelectric actuators’ displacements thatarises from their hysteresis is eliminated by polynomial-approximation-based open-loop control. Theinterference between the X- and Y-scanners is also eliminated by the same technique. The usefulnessof this wide-area scanner is demonstrated by video imaging of dynamic processes in live bacterial
and eukaryotic cells. © 2013 AIP Publishing LLC. [http://dx.doi.org/10.1063/1.4803449]
I. INTRODUCTION

Over the last two decades, various efforts have been
made to increase the imaging rate of atomic force microscopy(AFM).1–4 The bandwidth of feedback control that maintains
the tip–sample interaction force constant has been increasedto ∼100 kHz, enabling the capture of protein moleculeson video at sub-100-ms temporal and sub-molecular spatial
resolution. Importantly, the tip–sample interaction does not
disturb the function of fragile proteins and weak protein–protein interactions.4 High-speed AFM (HS-AFM) is now
started to be routinely used to study the dynamic processesof purified protein molecules under physiological conditions.5
For example, dynamic conformational changes of motorand membrane proteins at work,6–10 dynamic protein–protein
interactions,11–14 dynamics occurring in 2D protein crystals,15
and wiggling motion of intrinsically disordered proteins16
have been successfully visualized. However, to achieve thishigh-speed, low-invasive performance, the scan range has had
to be limited to approximately 1 μm, 4 μm, and 1 μm in the
X-, Y-, and Z-directions (at 100 V), respectively. This is because a smaller maximum displacement allows us to build acompact scanner, and hence attain higher resonant frequencies and scan speeds.
The limited scan ranges are sufficient for imaging protein molecules in action. However, in the biological sciences,
there is also an increasing demand that dynamic events occurring in live cells be directly visualized at high spatiotemporala) Author to whom correspondence should be addressed. Electronic mail:tando@staff.kanazawa-u.ac.jp.

0034-6748/2013/84(5)/053702/10/$30.00

resolution. Toward meeting this demand, wide-area scanners
with scan ranges of >10 × 10 μm2 have been developed for
HS-AFM.17–21 Their excellent performance has largely been
demonstrated by fast imaging of hard material surfaces. However, fast wide-area imaging is difficult for soft biologicalsamples. Fantner et al.20 recently performed fast wide-area
imaging of bacterial cells to study the effect of antimicrobial peptides on the surface of E. coli cells. The imaging rates
used there are 12.8 s/frame (scan rate, 20 lines/s; scan speed,
0.12 mm/s) for an area of 3 × 3 μm2 and 128 s/frame(scan rate, 2 lines/s; scan speed, 0.036 mm/s) for an area of
9 × 3 μm2 . Thus, despite these previous advances, AFM’s
capability for faster and wider bioimaging remains to be
pursued.
To achieve this capability, we need techniques for magnifying the displacement of a piezoelectric actuator, damping
mechanical vibrations, expanding the frequency bandwidth,
minimizing the nonlinear displacement, and minimizing theinterference between the X- and Y-displacements. In addition, we need a Z-scanner with a large maximum displacement as well as a high feedback bandwidth. In this study, wedeveloped these techniques. For a hard material surface, thetechniques achieve an imaging rate of 3 s/frame (scan rate,
85 lines/s) for a 40 × 40 μm2 area. This rate corresponds toa scan speed of 6.8 mm/s, which is faster than the previous
fastest record.19
To demonstrate the capability and usefulness of this new
wide-area scanner in bioimaging, we observed the bacteriolysis process of Bacillus (B.) subtilis subjected to lysozyme
and the dynamic processes of actin retrograde flow and84, 053702-1© 2013 AIP Publishing LLC

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FIG. 1. (a) Scheme of newly designed wide-area scanner for high-speed
AFM. (b) Enlarged view of the leverage-based displacement magnification
mechanism. Asterisks indicate the points that are 5 mm (1), 15 mm (2), and
23 mm (3) distant from the fulcrum. Displacements were measured at thesepoints. (c) Displacements caused by the extension of two different piezoactuators (Piezo-1, blue line; Piezo-2, red line) measured at three different points
along the lever indicated in (b). (d) Photograph of the wide-area scanner.

endocytosis in HeLa cells. Even for an area as wide as
20 × 20 μm2 , B. subtilis cells were successfully imaged at
15 s/frame (scan rate, 13.3 lines/s; scan speed, 0.53 mm/s)
with 200 × 200 pixels. We also showed that this wide-areascanner can be used to image proteins in a narrow area with
an imaging rate comparable to that achieved by our fast smallarea scanner.
II. MECHANICAL DESIGN OF WIDE-AREA SCANNER

To achieve large displacements in the X- and Ydirections, we magnified the displacements of the X- andY-piezoactuators using the third-class leverage mechanism.
Figure 1(a) shows a schematic of the scanner designed inthis study. The entire frame mechanism made of aluminum(A5052) was obtained by monolithic fabrication. The X- andY-piezoactuators are arranged symmetrically with respect tothe supporting base to which the Z-piezoactuator is attached.
This symmetrical configuration enables us to choose either
the X- or Y-direction as the fast scanning direction and rotate
the scan direction.22
The leverage mechanism employed is shown schematically in Fig. 1(b). The entire lever length is 25 mm, and thepoint of effort to which the piezoactuator is glued is 5 mmdistant from the fulcrum; i.e., the designed lever ratio is
5. We tested two different types of piezoactuators: piezoI (AE0203D16F, Tokin-NEC) with dimensions of 2 × 3
× 20 mm3 , a nominal unloaded displacement of 11.6 μm
at 100 V, and a resonant frequency of 69 kHz, and piezoII (AE0505D16F, Tokin-NEC) with dimensions of 5 × 5
× 20 mm3 and the same nominal unloaded displacement andresonant frequency as piezo I. Because piezo-II can generatea higher force (850 N) than piezo-I (200 N), the magnified
displacement by piezo-II under a loaded condition was ex-

Rev. Sci. Instrum. 84, 053702 (2013)

pected to be larger than that by piezo-I. However, this was not
the case. Figure 1(c) shows the displacements of the lever atthree different points along the lever [indicated in Fig. 1(b)]that were caused by piezo-I (blue line) and piezo-II (red line)
when 100 V was applied. At point 1, 5 mm distant fromthe fulcrum, the displacement caused by piezo-II was slightly
larger than that caused by piezo-I. However, at points 2 and3, 15 mm and 23 mm distant from the fulcrum, respectively,
the displacements by piezo-I were larger than those by piezo
II. This is because piezo II is glued to the lever over a wider
area; the wider glued area results in a nonlinear displacementof the lever. Thus, we chose piezo-I for the X- and Y-scanners
and achieved maximum scan ranges of 46.7 μm for the Xdirection and 45.7 μm for the Y-direction. These measured
values were ∼20% smaller than the displacement of 58 μm
calculated from the lever ratio and the unloaded displacementof piezo-I at 100 V.
A photograph of the fully assembled scanner is shown in
Fig. 1(d). The gap spaces in the scanner were filled with anelastomer consisting of polyurethane (EXSEAL Co., sponge
hardness = 5: similar softness to skin of face or arms). Thisfilling with the elastomer passively damps low-frequency vibrations of the scanner.

III. IMPROVEMENT OF SCANNER PERFORMANCE
A. Vibration damping for X-scan

Ideally, the trajectory of an X-scan is an isosceles triangle as a function of time. However, when the X-scanner isdriven by a voltage that changes in the form of an isosceles triangle wave, high harmonic frequency components containedin the vertices of the triangular wave excite the X-scanner,
resulting in the generation of vibrations at its resonant frequencies. Any X-scanners for wider-area scanning generally
have lower resonant frequencies and therefore are apt to vibrate when scanned quickly. Figure 2(a) shows the frequency
response of the wide-area X-scanner built as described above(red line, amplitude; blue line, phase). Here, the lateral displacement was measured using a laser displacement meterat the central base to which the Z-piezoactuator is attached.
Figure 2(b) shows an input triangular wave (black line) of
28 Hz and the measured displacement (red line). This frequency corresponds to an imaging rate of 9 s/frame for a 256
× 256 pixel image. At this scan speed, vibrations are not excited even around the vertices of the triangular wave because
higher harmonic components contained in the triangular wave
are negligible around the resonant frequency of the X-scanner(∼2 kHz). In contrast, during a scan at 85 Hz, vibrations of
2 kHz appeared, as shown by the red line in Fig. 2(c).
In practice, non-negligible vibrations are produced when
the fundamental frequency of the input triangular wave ishigher than ∼2% of the first resonant frequency of the Xscanner. To overcome this adverse effect, modified lateralscan waves, such as sinusoidal,19 spiral,23 cycloid,24 andLissajous25 waves have been proposed. Sinusoidal scanningis the simplest solution, but both the left and right end areasof an image are greatly distorted. Assuming that an approximately linear scan is obtained when the scan velocity is higher

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FIG. 2. Effects of vibration damping on displacement of X-scanner. (a) Frequency spectra of mechanical response of the X-scanner (red line, amplitude; blueline, phase). (b) Driving signal of 28 Hz with a non-modified triangular wave form (black line) and corresponding displacement (red line). (c) Driving signal of
85 Hz with a non-modified triangular wave form (black line) and corresponding displacement (red line). (d) Driving signal of 85 Hz with a triangular wave form
modified by inverse compensation (black line) and corresponding displacement (red line). (e) Driving signal of 256 Hz with a non-modified triangular wave
(black line) and corresponding displacement (red line). (f) Driving signal of 256 Hz with a triangular wave form modified by the inversed compensation (blackline) and corresponding displacement (red line). (g) Driving signal of 85 Hz with a rounded triangular wave form containing harmonics up to the ninth order(black line) and corresponding displacement. (h) Driving signal of 1 kHz obtained after modification by inverse compensation of a rounded triangular wave form
containing harmonics up to the ninth order (black line) and corresponding displacement (red line).

than 85% of the maximum velocity, the nonlinear scan area is
65% of the full image. Moreover, the resolution in the lessdistorted central image area is low (i.e., a smaller number ofpixels). Even after linearization of the obtained image by calculation, the lower resolution in the most important central
image area cannot be changed. Although the spiral, cycloid,
and Lissajous waves contain no higher harmonic frequencies,
the procedures for producing the scan signals, reconstructing the image, and compensating for the hysteresis effects ofthe piezoactuators are complicated. Moreover, the cantilevertends to be twisted by a lateral dragging force component
perpendicular to the lever arm, which hampers the sample
height measurement. Here, we employed two approaches tothe elimination of X-scanner vibrations: an inversion-based
feed-forward damping17, 26 and a modified triangular wave
with reduced higher harmonics.27, 28
The black line in Fig. 2(d) shows the modified triangular wave obtained after filtration of a triangular wave throughan inverse transfer function that was constructed using the frequency response shown in Fig. 2(a). Here, the triangular wave
to be filtered was constructed with the first 20 terms in the

Fourier cosine series of an isosceles triangle function. The vibrations previously observed [Fig. 2(c)] disappeared to a practical extent with this method [the red line in Fig. 2(d)]. Even
during scanning at a higher frequency of 256 Hz (corresponding to a imaging rate of 1 s/frame for 256 lines), vibrations
were suppressed significantly (but not to a practical extent) by
this damping, as shown by the red line in Fig. 2(f), comparedwith the result observed without damping [the red line in
Fig. 2(e)]. In principle, this damping method can significantly extend the bandwidth of the X-scanner, but in reality, the limited gain and current of a piezodriver at highfrequencies limit the bandwidth extension. Moreover, thefrequency response of the X-scanner occasionally varies tosome extent depending on the sample attached. Therefore,
for scan frequencies higher than ∼100 Hz, vibration damping to a practical extent cannot be achieved by this method
alone.
Another damping method we tested was simple roundingof the vertices of the triangular wave.27 An isosceles triangular wave is composed of a cosine wave with the fundamental
frequency and an infinite series of its odd higher harmonics.

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By reducing the number of higher harmonics, one can easily generate a triangular wave with round vertices. As shownin Fig. 2(a), the amplitude gain of the X-scanner starts to increase at 500 Hz. Assuming that the gain tolerance is less than
1 dB, the highest frequency of harmonics to be included in thedriving signal should be lower than ∼780 Hz, judging fromthe frequency response shown in Fig. 2(a). For scanning at
85 Hz, the highest harmonics should be less than the ninth
order (i.e., 745 Hz). Figure 2(g) shows a rounded triangularwave constructed from harmonics up to the ninth order (blackline) and the corresponding displacement of the X-scanner(red line). The displacement did not show noticeable vibrations. The nonlinear scan range is about 10% of the full scan
range, which is much smaller than that when a sinusoidal scanwave is used.
For actual imaging, we combined these two damping
methods (as well as compensating for the nonlinearity andinterference of the X- and Y-scanners’ displacements, as described later); the rounded triangular wave containing higher
harmonics up to the ninth order was filtered through an inversed transfer function constructed using the measured frequency response. By using this combination, the fundamental
frequency of X-scanning could be extended up to ∼1 kHz, as
shown in Fig. 2(h).
For Y-scanning, a sawtooth wave is typically used. The
quick return to the scan origin along the Y-direction, whichis performed after the completion of line scans in the Xdirection, easily generates vibrations. We avoided vibration
generation by slowing the return scan. Even when the return

Rev. Sci. Instrum. 84, 053702 (2013)

scan to the origin is slowed, the time it takes is much shorter
than the time for which an image is captured.
B. Compensation for nonlinearity

All piezoelectric actuators have an intrinsic hysteresisthat distorts the AFM image owing to their nonlinear displacement as a function of the applied voltage. The position error
generally becomes 10%–15% of the full scan range,29 whichcauses considerable image distortion, especially for wide-area
imaging. Two types of control methods have been proposed tocompensate for the nonlinear hysteresis curve: closed-loop30
and open-loop31 compensations. The former enables more
precise control than the latter. However, the use of closed-loop
compensation makes the scanner assembly complicated because of the implementation of displacement sensors. Moreover, the bandwidth of high-precision displacement sensors is
too low for application to high-speed scanners. In the openloop method, the nonlinearity is compensated for by the useof a feed-forward inverse model. Here, we used open-loop
compensation based on the pre-measured nonlinear hysteresis curves.
Figure 3(a) shows hysteresis curves of the X-scanner for
displacement ranges of 40 μm (red line) and 24 μm (blueline). Figure 3(d) shows the effect of hysteresis on an AFM
image of a square grating with a pitch of 10 μm. The image was acquired without compensation at 15 s/frame for a
33 × 33 μm2 area. The image shows grating pitches withvariations of approximately ±20% and ±15% in the X- and

FIG. 3. Hysteresis of X-scanner and its compensation. (a) Hysteresis curves for two different ranges (blue line, 24 μm; red line, 40 μm). (b) Hysteresis curves
normalized with regard to both displacement and voltage. Black line shows a fitting curve with fourth-order polynomials. (c) Driving signal of 85 Hz with atriangular wave form modified by open-loop compensation for hysteresis (black line) and corresponding displacement (red line). Here, hysteresis compensation
was applied to a triangular wave form modified by inverse compensation. (d) AFM image of a test grating sample with a pitch of 10 μm obtained without
hysteresis compensation (imaging rate, 7 s/frame; pixels, 256 × 256). (e) AFM image of the same grating sample obtained using hysteresis compensation(imaging rate, 7 s/frame; pixels, 256 × 256). (f) AFM image of the same grating sample obtained using hysteresis compensation (imaging rate, 3 s/frame; pixels,
256 × 256).

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Y-directions, respectively. Figure 3(b) shows hysteresis
curves that are the same as those shown in Fig. 3(a) but normalized with respect to both the displacement and the applied
voltage. The normalized curves are identical irrespective ofthe displacement ranges, meaning that the nonlinearity of thepiezoactuator is independent of the scan range. We confirmedthat the normalized curves obtained for other scan ranges
are also identical. The normalized expansion and contraction
curves can be fitted by fourth-order polynomial functions, as
shown by the black lines in Fig. 3(b). Although higher-orderpolynomial functions give better fitting, the fourth-order fitting is good enough practically to compensate for the hysteresis. The driving voltage signal that was not processed forinverse vibration damping but compensated for the hysteresis
was first constructed using the inverse functions of the fitted
hysteresis curves. Then, this driving signal was processed forinverse vibration damping or for both inverse vibration damping and rounding the vertices of the signal. Figure 3(c) showsthe effect of hysteresis compensation on the displacement of
the X-scanner. The black line indicates a driving signal of
85 Hz constructed by this method (here, the process of rounding vertices is omitted), and the red line indicates the corresponding displacement of the X-scanner. The outwardly (ascending regime) and inwardly (descending regime) distorted
curves of the driving signal reflect the compensation for hysteresis. It is apparent that the X-scanner is displaced linearlyin both the expansion and contraction regimes. As a result, thedistortion of the grating image was much improved, as shownby the image in Fig. 3(e) captured at 7 s/frame. The variations in the grating pitch are now less than ±1%. As shown in
Fig. 3(f), even when an image is captured at 3 s/frame, the image distortion is eliminated, except for the rhombic distortionof the square grid holes, which is due to interference between
the X- and Y-scanners.
C. Compensation for interference between
X- and Y-scanners

Interference between the X- and Y-scanners occurs because the free ends of the levers used for displacement magnification do not move in a straight line. The green and redlines in Fig. 4(a) show the sample stage displacement in the Ydirection as a function of the X-scanner displacement (driving
voltage was applied only to the X-piezoactuator) up to 5 μm
(green line) and 30 μm (red line). The sample stage moved almost linearly in the Y-direction with a coupling ratio of 0.017(i.e., 17 nm per 1 μm displacement of the X-scanner). Notethat the two methods of active damping and the compensation for the hysteresis effect were applied in this experiment
as well as in the following experiments in this subsection. To
eliminate this interference, an appropriate fraction of the voltage applied to the X-piezoactuator was subtracted from thedriving signal for the Y-piezoactuator. As shown by the blackand yellow lines in Fig. 4(a), this method minimized the coupled movement of the sample stage in the Y-direction, irrespective of the displacement range of the X-scanner. The sample stage was also passively moved in the X-direction when
the Y-scanner was displaced, as shown by the green and redlines in Fig. 4(b). This interference was also minimized by

Rev. Sci. Instrum. 84, 053702 (2013)

FIG. 4. Elimination of interference between X- and Y-scanner displacements. (a) Sample stage displacement in the Y-direction as a function of Xscanner displacement. Displacement ranges of X-scanner are 5 μm (greenline) and 30 μm (red line). Yellow and black lines represent displacements in
the Y direction when interference compensation was applied for X-scannerdisplacements of 5 μm and 30 μm, respectively. (b) Sample stage displacement in the X-direction as a function of Y-scanner displacement. Displacement ranges of Y-scanner are 5 μm (green line) and 30 μm (red line). Yellow
and black lines represent displacements in the X direction when interferencecompensation was applied for Y-scanner displacements of 5 μm and 30 μm,
respectively. (c) AFM image of a test grating sample with a pitch of 10 μm
obtained without interference compensation (scan range, 33 × 33 μm2 ; imaging rate, 7 s/frame; pixels, 256 × 256). The square grids are distorted in arhombic shape. (d) AFM image of the same sample obtained using interference compensation (imaging rate, 7 s/frame; pixels, 256 × 256). (e) Enlarged
AFM image of the sample obtained without interference compensation (scanrange, 15 × 15 μm2 ; imaging rate, 7 s/frame; pixels, 256 × 256). (f) Enlarged AFM image of the sample obtained using interference compensation(scan range, 15 × 15 μm2 ; imaging rate, 7 s/frame; pixels, 256 × 256).

the same method, as shown by the yellow and black lines inFig. 4(b). As a result, the images of square grid holes withrhombic distortion shown in Figs. 4(c) [imaged under thecondition identical to that used for Fig. 3(e)] and 4(e) wereconverted to images with much less distortion, as shown in
Figs. 4(d) and 4(f), respectively.
D. Z-scanner

The feedback bandwidth is one of the most importantfactors in achieving fast imaging of soft biological samples

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FIG. 5. Tracing accuracy of feedback Z-scanning by the wide-area scanner. (a) Frequency response of the Z-scanner (gain, red lines; phase, blue lines) obtainedwith (dotted lines) and without (solid lines) the use of active Q-control damping. (b) Cross sections of topographic AFM images (256 × 256 pixels) of a grating
sample acquired at different imaging rates (top, 3 s/frame; middle, 10 s/frame; bottom, 20 s/frame). (c) Histograms of topographic height measured for the top
surface of the grating sample. The average heights measured are 276 nm (top, 3 s/frame), 279 nm (middle, 10 s/frame), and 276 nm (bottom, 20 s/frame). (d)Cross sections of the error-signal images that appeared when the grating sample was imaged at different imaging rates.

because delayed movement of the Z-scanner increases theloading force from the AFM tip to the sample. The feedback bandwidth depends on several delay factors such as themechanical properties of the Z-piezoactuator and the cantilever and imaging conditions.32, 33 Among the delay factors, the response delay of the Z-piezoactuator is the major
one because the resonant frequency of the Z-piezoactuator
suitable for observing a large specimen is much lower thanthat of small cantilevers optimized for HS-AFM. For the Zscanner, we used a stack piezoactuator with dimensions of
2 × 3 × 5 mm3 (AE0203D04F, Tokin-NEC) having a maximum displacement of 2.5 μm at 100 V, which was glued
to the top of the supporting base to be moved in the Xand Y-directions. As a counterweight, the same type of
piezoactuator was also glued to the bottom side of the supporting base. This additional piezoactuator is displaced sothat the impulsive force exerted on the supporting base by
the rapid displacement of the Z-scanner is counteracted.32
Figure 5(a) shows the frequency response of the Z-scanner.
Here, the displacement was measured at the cylindrical glass
stage (1.5 mm diameter) attached to the Z-scanner. The first
90 kHz and higher-frequency peaks were damped by using
the active Q-control technique,34 as shown by the dotted red(amplitude) and blue (phase) lines in Fig. 5(a). The peak
gain at the first resonant frequency was damped to about
3 dB. Although further damping was possible, we attenuateddamping to this extent to minimize the phase delay at lower
frequencies.

In the following imaging experiments in this subsection,
the two methods of active damping as well as the compensation for nonlinearity and interference mentioned above wereapplied. Figures 5(b) and 5(d) show cross sections along theX-scan direction of the topographic and error-signal images
obtained for a square grating (pitch, 10 μm), respectively. The
256 × 256 pixel images were acquired at rates of 3 s/frame(top), 10 s/frame (middle), and 20 s/frame (bottom), whichcorrespond to scan velocities of 6.8 mm/s, 2.0 mm/s, and
1 mm/s, respectively. Dynamic proportional-integralderivative (PID) control was used to reduce the parachuting
time during which the cantilever tip was completely detachedfrom the sample surface at its bottom swing;35 the gain
parameters of the PID controller were automatically altereddepending on the cantilever’s oscillation amplitude. From thehistograms of the topographic heights shown in Fig. 5(c), theheights are almost the same irrespective of the imaging rate:
276 nm (top, 3 s/frame), 279 nm (middle, 10 s/frame), and276 nm (bottom, 20 s/frame), although the height variation
becomes larger at faster imaging rates. This consistency
demonstrates that the feedback Z-scan is able to track thesurface because variations in the error signals appear onlyaround the grating edges, whereas the error signal is mostly
constant over 80% of the flat areas even at the fastest imaging
rate [Fig. 5(d)].
The maximum displacement of the Z-piezoactuator usedhere is limited to 2.5 μm. We may need a larger displacement depending on the sample to be observed. We can

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FIG. 6. HS-AFM imaging of B. subtilis using the wide-area scanner. (a) Topographic image of B. subtilis in a culture medium solution before the addition of lysozyme. Scan range, 20 × 20 μm2 ; imaging rate, 15 s/frame; pixels, 200 × 200. (b) Successive images of bacteriolysis process of B. subtilis
subjected to lysozyme. Lysozyme was injected at t = 240 s. Scan range, 5 × 5 μm2 ; imaging rate, 20 s/frame; pixels, 256 × 256 (enhanced online).
[URL: http://dx.doi.org/10.1063/1.4803449.1]

alternatively use a longer piezoactuator with dimensions of
2 × 3 × 10 mm3 (AE0203D16F, Tokin-NEC), which canbe displaced up to 4.3 μm at 100 V. However, we have tosacrifice the imaging rate because the resonant frequency ofthe longer piezoactuator is lowered to approximately a half
(50 kHz). When this piezoactuator was used, the maximum
possible imaging rate was reduced to ∼7 s/frame for 256
× 256 pixel imaging of the same grating sample.
IV. IMAGING OF BIOLOGICAL SAMPLES

To demonstrate the capability of the wide-area scanner,
we imaged live bacterial and eukaryotic cells using the scanner under control by all the techniques mentioned above.
A. Large-area imaging

1. Imaging of Bacillus subtilis

We first observed the process of bacteriolysis of a rodshaped B. subtilis (approximately 700 nm in diameter and2 μm long) subjected to lysozyme. B. subtilis has been widelyused as a model system for investigating the roles of autolysins in the cell wall metabolism of endospore-forming
Gram-positive bacteria, and the structure of peptidoglycan
and spores36–38 because of its nonpathogenicity and amenability to genetic manipulation. It is well-known that B. subtilis issusceptible to the antibacterial action of lysozyme, which hydrolyzes 1,4-beta linkages between N-acetyl glucosamine andN-acetyl muramic acid, leading to degradation of peptidoglycan in the cell wall.37 Here, we captured this degradation process to demonstrate the capability of the wide-area HS-AFM
scanner.
The bacterial cells were immobilized on a glass stage
coated with poly-L-lysine (0.1 mg/ml). HS-AFM imaging was conducted in a culture medium (Penassay Broth).
Figure 6(a) shows a wide-area image of the cells captured
over an area of 20 × 20 μm2 at 15 s/frame (200 × 200 pixels).
The mean height of the cells was 730 nm, indicating accu-

rate tracing of the cell topography. Figure 6(b) (see video)
shows some of the images of the bacterium before and after
the addition of lysozyme, successively captured at 20 s/frame(256 × 256 pixels). Before the injection of lysozyme (t = 0s), the cell surface appears smooth. At t = 240 s, lysozyme(final concentration, 80 μM) was injected into the observation solution, and therefore, the imaging was somewhat disturbed. Then, after t = 320 s, the surface morphology gradually became varied, and wrinkle structures perpendicular tothe long cell axis appeared. At the same time, the cell gradually swelled (t = 1060 s, 1460 s, and 1580 s). Finally, at
t = 1720 s, the cell exploded, probably because of excessive
osmotic pressure from inside of it, resulting in partial exposure of the intracellular contents. We also observed the morphological changes induced by lysozyme (final concentration,
200 μM) at a faster imaging rate, 2 s/frame, for 120 × 90 pixels (Fig. 7). The cell surface roughness increased rapidly witha time lag of ∼90 s after the injection of lysozyme at t = 32 s,
as shown in Fig. 7(a). The corresponding images are shownin Fig. 7(b) (see video). Here, the surface roughness was expressed by the root-mean-square roughness of the area indicated by the white rectangle in Fig. 7(b). The roughness wasalmost constant (∼93 nm) before the injection of lysozyme,
whereas it increased to ∼125 nm after the injection. The increase in the roughness was well fitted by a single exponentialfunction with a time constant of τ = 36.8 s, as indicated by
the solid line in Fig. 7(a).
2. Imaging of HeLa cells

We tested whether large-area imaging could be conducted even for live eukaryotic cells, which are much softer
than bacteria. HeLa cells, one of the most commonly usedimmortal cell lines derived from human cancer cells, weremaintained on a poly-L-lysine-coated glass stage at 37 ◦ C in
5% CO2 using Dulbecco’s modified Eagle’s medium (LifeTechnologies) supplemented with 10% fetal bovine serum(Life Technologies). HS-AFM imaging was performed in

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FIG. 7. Time course of roughness change of the outer surface of B. subtilis subjected to lysozyme. (a) Time course of root mean square (RMS)
roughness. Lysozyme was injected at t = 32 s. Solid line shows the resultof best fitting (time constant, τ = 36.8 s) by a single exponential function for the time course between t = 112 s and t = 300 s. (b) Corresponding AFM images of the outer surface of B. subtilis at different time
lapses. Values of RMS roughness were calculated for the area encircled by
the white broken line shown in the image at t = 86 s (enhanced online).
[URL: http://dx.doi.org/10.1063/1.4803449.2]

HEPES-buffered artificial cerebrospinal fluid (H-ACSF) at
32 ◦ C. The fine structures of HeLa cells were successfully
imaged by HS-AFM at 5 s/frame (200 × 200 pixels). Sheetlike structures with ∼100 nm height were observed at thecell edge regions. Interestingly, the HS-AFM movie clearly
showed a continuous flow of small objects directed from thecell periphery toward the center of the cell body, as shown in
Fig. 8(a) (see video).

Rev. Sci. Instrum. 84, 053702 (2013)

It is well known that actin filaments polymerize at theleading edge of eukaryotic cells and flow back into the cellbody in the so-called actin retrograde flow.39, 40 To determine
the origin of the flow observed by HS-AFM and its relationship to the actin retrograde flow, cytochalasin D (Sigma), a
chemical inhibitor of actin polymerization, was bath-applied
to HeLa cells. A HS-AFM movie showed that the continuous
flow stopped completely after the application of cytochalasin
D (final concentration: 20 ng/ml) [Fig. 8(b)] (see video), indicating that the flow observed in the HS-AFM movies is theactin retrograde flow.
Next, we conducted real-time observations of endocytosis by HS-AFM. Endocytosis is a cellular process in whichcells bring molecules or substances into the cell interior fromthe outside of the cell by engulfing them with the cell membrane. The engulfment typically begins with the formation ofnanoscale pits on the cell surface. The small GTPase Rab5 is
known to be crucial in regulation of endocytosis. Its constitutive active mutant Rab5(Q79L) stimulates endocytosis,41–43
and replacement of the C-terminus of Rab5 with CAAX box
derived from k-Ras enhances the plasma membrane localization of Rab5. Figure 8(c) shows successive AFM images ofa HeLa cell in which mEGFP-Rab5(Q79L)-CAAX is overexpressed. Here, mEGFP was co-expressed with Rab5(Q79L)
and Rab5(Q79L)-CAAX in the HeLa cells to evaluate theoverexpression level by fluorescence microscopy. As seenin Fig. 8(c) (see video), each of the two pits encircled bybroken lines was capped by a membrane protrusion thatgrew with time from the membrane surrounding each pit(5 s/frame, 200 × 200 pixels). This process resembles the recent observation of endocytosis-mediated pit closure on the

FIG. 8. HS-AFM imaging of dynamic processes occurring in HeLa cells. (a) Topographic image of HeLa cell at an edge region before the addition ofcytocharasin D. Thin black lines indicate flow of small objects. Imaging rate, 5 s/frame; pixels, 200 × 200. (b) Topographic image of HeLa cell at anedge region after the addition of cytocharasin D. Black dots show that no flow is occurring. Scan range, 5 × 5 μm2 ; imaging rate, 5 s/frame; pixels, 200
× 200. (c) Successive images showing the dynamic process of endocytosis. HeLa cells were transfected by mEGFP-Rab5(Q79L)-CAAX. The imaging was
performed 24 h after transfection. Scan range, 5 × 5 μm2 ; imaging rate, 5 s/frame; pixels, 200 × 200. (d) Frequencies of pit appearance in HeLa cells
transfected by mEGFP, mEGFP-Rab5(Q79L), and mEGFP-Rab5(Q79L)-CAAX (enhanced online). [URL: http://dx.doi.org/10.1063/1.4803449.3] [URL:
http://dx.doi.org/10.1063/1.4803449.4] [URL: http://dx.doi.org/10.1063/1.4803449.5]

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Rev. Sci. Instrum. 84, 053702 (2013)

FIG. 9. Narrow-area imaging by HS-AFM equipped with the wide-area scanner and effect of the gain-variable piezodriver on imaging. (a)–(d) HS-AFM imagesof two actin filaments aligned in parallel obtained with different piezodriver gains [(a) ×1; (b) ×2; (c) ×5; (d) ×10]. Scan range, 200 × 100 nm2 ; imaging rate,
0.15 s/frame; pixels, 120 × 60. (e) and (f) AFM images of outer surface of B. subtilis before (e) and after (f) injection of lysozyme. Scan range, 500 × 500 nm2 ;imaging rate, 2 s/frame; pixels, 256 × 256.

surface of COS-7 cells revealed by scanning ion conductance microscopy.44 Figure 8(d) shows histograms of theobserved rates of pit formation for three types of cells ineach of which mEGFP, mEGFP-Rab5(Q79L), or mEGFPRab5(Q79L)-CAAX is overexpressed. The nanoscale pits
were infrequently observed in HeLa cells transfected only by
mEGFP (1.9% of a total of 2689 frames of 27 cells), whereas
the frequency of nanoscale pit appearance was significantly
increased by overexpression of mEGFP-Rab5(Q79L) (19%of a total of 3994 frames of 30 cells) and further increasedby overexpression of mEGFP-Rab5(Q79L)-CAAX (36% of
a total of 3090 frames of 24 cells), consistent with a persistent stimulation effect of Rab5 mutants on endocytosis.41–43
Thus, we demonstrated here that wide-area HS-AFM can also
be used to visualize dynamic processes occurring in very soft
eukaryotic cells.
B. Small-area imaging

The large-area imaging capability achieved here allows
us to easily position the AFM tip on a specific area of interest within a pre-imaged large object. If the wide-area scanner has a high-resolution imaging capability even when it isused to image a small area, it becomes possible to image dynamic events involving tiny objects such as molecules in narrow areas within a pre-imaged large object. To achieve thiscapability, we have to improve the resolution of the wide-areascanner without reducing the wide-area positioning capability. The extension coefficient of the X- and Y-piezoactuators
used for the wide-area scanner is large (435 nm/V). To effectively use this large coefficient, the gain of the piezodriver should be high (we used a ×10 gain). However, thefast digital-to-analog convertor has a limited resolution (12bits for a 10 V range in our case). Consequently, the lateral
resolution is limited to approximately 11 nm. To solve thisproblem while maintaining the wide-area positioning capability, we developed a new piezodriver with two-channel inputs(now commercially available: M-2345, MESS-TEC Co.). The
first channel with selectable gains (×1, ×2, ×5, and ×10) is
used for scanning (hence, for imaging), whereas the second
channel with a fixed gain (×10) is used for positioning. The

gains of the first channel are selected depending on the size ofthe area to be imaged.
We tested this piezodriver’s capacity for high-resolution
imaging by imaging actin filaments anchored on a mica surface [Figs. 9(a)–9(d)]. The HS-AFM images in Figs. 9(a)–9(d) were obtained with driver gains of ×1, ×2, ×5, and ×10,
respectively, whereas the scan area and imaging rate are fixedat 200 × 100 nm2 (120 × 60 pixels) and 0.15 s/frame, respectively. As seen in Figs. 9(a) and 9(b), not only the helicalstructure with a 36-nm pitch but also actin monomers within
the filaments are resolved. On the other hand, when the drivergain was increased to ×5 and ×10, the image quality deteriorated significantly because of lower bit resolutions.
Figure 9(e) shows a high-resolution image of the cell surface of B. subtilis not subjected to lysozyme. The image wasobtained at a piezodriver gain of ×1 and an imaging rate of
2 s/frame for a scan area of 500 × 500 nm2 (200 × 200
pixels). Fine structures on the cell wall were clearly visible,
whereas they could not be resolved by large-area imaging,as shown in Fig. 6(b) (t = 0 s). Soon after the injection oflysozyme, the fine structure on the cell surface became blurryowing to degradation of the cell wall, and a wrinkle structure with a periodicity of about 300 nm appeared, as shownin Fig. 9(f).
V. CONCLUSIONS

We developed techniques to achieve both fast and widearea scanning capabilities. The wide-area scanner developed
can be scanned over ∼46 × 46 μm2 . Unwanted vibrations
produced during rapid scanning were eliminated by inverse
compensation combined with the use of rounded vertices ona triangular scan wave constructed with a limited numberof Fourier components. The nonlinear displacement arisingfrom the hysteresis of the piezoactuators and interference between the X- and Y-scanners was also eliminated. As a result, a test grating sample was imaged at 3 s/frame for a scan
area of 40 × 40 μm2 . Finally, the fast scan capability of thewide-area scanner was demonstrated by imaging of dynamic
processes including lysozyme-induced bacteriolysis, actin
retrograde flow, and endocytosis. It was also demonstrated

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that the wide-area scanner retains its capacity for high-speed,
high-resolution imaging even when used for a small imaging
area. Thus, the range of samples to which HS-AFM can be applied is now greatly extended, from single molecules to live
cells.

Rev. Sci. Instrum. 84, 053702 (2013)
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ACKNOWLEDGMENTS

This work was supported by the Human Frontier Science Program, a Grant-in-Aid for Basic Research (Project
ID: 24227005, 23115008), the Knowledge Cluster Initiative
Project funded by the Ministry of Education, Culture, Sports,
Science and Technology in Japan, JST/ALCA, and by a program on Development of Systems and Technology for Advanced Measurement and Analysis funded by JST.

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