Method of mechanical holding of cantilever chip for tip-scan high-speed atomic force
microscope
Shingo Fukuda, Takayuki Uchihashi, and Toshio Ando
Citation: Review of Scientific Instruments 86, 063703 (2015); doi: 10.1063/1.4922381
View online: http://dx.doi.org/10.1063/1.4922381
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REVIEW OF SCIENTIFIC INSTRUMENTS 86, 063703 (2015)

Method of mechanical holding of cantilever chip for tip-scan high-speed
atomic force microscope
Shingo Fukuda,1 Takayuki Uchihashi,1,2,3 and Toshio Ando1,2,3

1

Department of Physics, College of Science and Engineering, Kanazawa University,
Kakuma-machi, Kanazawa 920-1192, Japan
2
Bio-AFM Frontier Research Center, College of Science and Engineering, Kanazawa University,
Kakuma-machi, Kanazawa 920-1192, Japan
3
Core Research for Evolutional Science and Technology of the Japan Science and Technology Agency,
7 Goban-cho, Chiyoda-ku, Tokyo 102-0076, Japan(Received 23 February 2015; accepted 31 May 2015; published online 11 June 2015)
In tip-scan atomic force microscopy (AFM) that scans a cantilever chip in the three dimensions, thechip body is held on the Z-scanner with a holder. However, this holding is not easy for high-speed(HS) AFM because the holder that should have a small mass has to be able to clamp the cantileverchip firmly without deteriorating the Z-scanner’s fast performance, and because repeated exchange of
cantilever chips should not damage the Z-scanner. This is one of the reasons that tip-scan HS-AFM
has not been established, despite its advantages over sample stage-scan HS-AFM. Here, we present
a novel method of cantilever chip holding which meets all conditions required for tip-scan HS-AFM.
The superior performance of this novel chip holding mechanism is demonstrated by imaging of
the α3β3 subcomplex of F1-ATPase in dynamic action at ∼7 frames/s. C 2015 AIP Publishing
LLC. [http://dx.doi.org/10.1063/1.4922381]
I. INTRODUCTION

High-speed atomic force microscopy (HS-AFM) was established around 20081 through long-range efforts to increase
the response speed of cantilevers,2,3 the scanner,4–7 and electronics,1,2,8 as well as to make the high-speed performancecompatible with low-invasiveness to fragile samples9 (see reviews1,10,11). This established HS-AFM has mainly been used
to observe protein molecules in dynamic action at sub-100 ms
to subsecond time resolution. For example, bacteriorhodopsin
responding to light,12 myosin V walking on actin filaments,13
and rotor-less F1-ATPase (i.e., the α3β3 subcomplex of F1ATPase) with directional chemical-state rotation over thethree β subunits14 have been visualized (see reviews15,16).
These observations have provided greater insights than everbefore into how the proteins function, thus demonstrating
the innovative power of this microscopy. Recently, fast widearea scanners displaceable up to 23 × 23 µm2 or up to ∼50
× 50 µm2 have been developed17,18 together with a new vibration damping method for the wide-area scanners,18 expanding
the objects of HS-AFM observation to much larger ones. For
example, various dynamic processes occurring in live bacterial
and mammalian cells, such as endocytosis, membrane raffling,filopodia growth and bacteriolysis, were visualized withina few seconds.18,19 The HS-AFM instrument used in thesestudies employs the sample stage-scan mode. To achieve thehigh scan rate and minimize the hydrodynamic effect of fastsample-stage movement on the cantilever behavior, a small
sample stage (a glass rod with dimensions of 1–1.5 mm indiameter and 2 mm in height) is attached to the Z-scanner.
This small size limits the range of specimens to be placed on
the sample stage. Moreover, when the HS-AFM instrument iscombined with an optical microscope, the optical image of thesample oscillates during HS-AFM imaging. More seriously,

various optical microscopy techniques are incompatible withthe configuration of mechanical and optical devices in the
HS-AFM instrument, restricting the functional expansion of
HS-AFM.
To overcome these limitations associated with the sample stage-scan mode, tip-scan HS-AFM has recently been
attempted to be developed. One of the common requirementsof tip-scan AFM that employs the widely used optical levermethod of detecting cantilever’s deflection is that the laser
beam focused onto the cantilever should be able to track thelateral motion of the cantilever. This tracking adoptable totip-scan HS-AFM was recently accomplished by laser beamscan in synchrony with the cantilever’s lateral motion using
either a mirror tilter20 or a focusing lens embedded in the
XY-scanner.21 The combined system of total internal reflection fluorescence microscopy (TIRFM) and tip-scan HS-AFM
equipped with the mirror tilter was demonstrated to be able
to capture topographic AFM and single-molecule fluorescentimages simultaneously at a few frames/s (fps).20 Thus, tip-scan
HS-AFM opened new opportunities to observe dynamic eventsin a large specimen placed on the stage of an inverted optical
microscope as well as to combine HS-AFM with various optical techniques, such as super-resolution fluorescence microscopy techniques22,23 and even optical tweezers.24
Nevertheless, an important issue, which is seemingly simple but practically difficult to resolve, has remained to beaccomplished. That is, how to attach a cantilever chip firmly
onto the fast, lightweight Z-scanner, without deteriorating thefast, precise response of the Z-scanner. As a matter of course,
one can firmly glue a cantilever chip onto the Z-scanner, as
has been done in the above tip-scan HS-AFM/TIRFM experiments. However, the chip has to be frequently replaced with a
new one, and therefore, this method is inadequate for routine
use. The use of thermoplastic adhesives is one possibility.

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Fukuda, Uchihashi, and AndoRev. Sci. Instrum. 86, 063703 (2015)

However, the adhesives cannot be removed completely but
partially remain after removal of the cantilever and gradually age by repeated heating, resulting in a rough surface of
the Z-scanner’s top face. Moreover, when the cantilever isimmersed in a buffer solution, such adhesives contaminate thesolution. The conditions required for the method of holding
a cantilever chip are as follows: (i) a cantilever chip bodyhas to be firmly clamped so that the chip does not generate
unwanted vibrations during fast scanning of the Z-scanner,(ii) the cantilever chip holder has to have a small mass and thefirst resonant frequency higher than that of the Z-scanner, (iii) a
cantilever chip has to be easily attached onto and removed
from the Z-scanner so that the scanner would not be damaged
during these operations, and (iv) the cantilever chip holder
should be durable without aging and wear so as to be used
repeatedly.
Magnetic or vacuum clamp may be a possible candidate
but magnetic clamp requires ferromagnetic coating of a cantilever chip, which is inadequate for routine use, while vacuum
clamp is too weak to hold a cantilever chip firmly enoughnot to generate unwanted vibrations. Therefore, mechanicalclamp seems to be a only candidate that would meet all therequired conditions. However, the entire body of the mechanical clamping system cannot be mounted directly on the Zscanner. This is partly because the mass effect of this mounting
would significantly reduce the resonant frequency of the Zscanner and primarily because repeated exchange of cantileverchips would damage the Z-scanner (e.g., the Z-piezoactuator is
unglued from the supporting base). In fact, this way of mounting was recently shown by Fukuma’s group to deteriorate thefrequency response of the Z-scanner even in the best caseamong various designs.25 Our group also has experienced thisproblem as well as the damaging problem. Therefore, we here
tested methods in which a cantilever chip clamping system
was indirectly mounted on the Z-scanner and reached a designthat did not affect the fast, precise response of the Z-scanner.
This excellent performance was demonstrated by imaging ofprotein molecules in dynamic action.
II. EXPERIMENTAL SETUP

The scanner used here is similar to that developed previously for tip-scan HS-AFM,20 except its modification for
cantilever chip holding. Briefly, for the Z-scanner, the stack
piezoactuator (AE0203D04F Tokin-NEC, Miyagi, Japan) isused that has dimensions 2 × 3 × 5 mm3, the measured firstresonant frequency in free oscillation 222 kHz (nominally 261
kHz), and the measured displacement efficiency 18.2 nm/V
(nominal maximum displacement, 4.6 µm at 150 V). This
Z-piezoactuator is glued onto the top of the supporting baseto be scanned in the X- and Y-directions. The same typeof a piezoactuator (Z′-piezoactuator) is also glued onto thebottom of the supporting base to counteract the impulsive force
produced by quick displacement of the Z-piezoactuator.3 The
gap space in the scanner is filled with an elastomer having ahigh loss factor to absorb the mechanical vibration energy.18
A wedge-shaped stage (hereafter referred to as “w-stage”) on
which a cantilever chip to be placed is glued onto the top of
the Z-scanner. This w-stage (32 mg) is made of silicon carbide with a high Young’s modulus-to-density ratio (Young’s

FIG. 1. Experimental setup for the characterization of Z-scanner’s frequency response. (i) Frequency response analyzer (FRA) simultaneously drives the Z- andZ′-piezoactuators with sinusoidal waves at various frequencies through a high-bandwidth piezodriver. (ii) The laser displacement sensor detects the displacementof target portions (i.e., the top face of the Z-scanner without both w-stage and cantilever chip holder, the top face of the w-stage, and the distal portion of thecantilever chip body). (iii) The FRA calculates the gain and phase of the signal from the sensor. The phase is relative to the driving signal.
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Fukuda, Uchihashi, and Andomodulus, ∼430 GPa; density, ∼3.2 g/cm3). The wedge angle
is 20◦, so that the mounted cantilever is tilted by this angle
with respect to the sample substrate. To facilitate guiding of
a cantilever chip onto the w-stage, a shallow groove slightly
wider than the cantilever chip is cut on the top face of the
w-stage. Another wedge-shaped stage (w′-stage) identical to
the w-stage is also glued on the top of the Z′-piezoactuator as
counterweight.
The experimental setup for analyzing the frequency responses described below is illustrated in Fig. 1. For the frequency response analysis, a frequency response analyzer(FRA5097, NF Corp., Kanagawa, Japan) was used. For the
analysis of the frequency response of the Z-scanner itself,
the w- and w′-stages were removed from the Z- and Z′piezoactuators, respectively. The Z- and Z′-piezoactuators
were simultaneously and sinusoidally oscillated in synchrony
with each other at amplitude of 6 nm and frequencies of
0–400 kHz through a high-bandwidth (∼1 MHz) piezodriver(M-2335, custom-made, Mess-Tek. Corp., Saitama, Japan).
A heterodyne laser displacement sensor (ST-3761, IWATSU,
Tokyo, Japan) was used to measure mechanical vibrations
occurring at target portions. Its laser beam was incident on
the Z-scanner without the w- and w′-stages, the w-stage glued
onto the Z-scanner (the w′-wage was also glued to the Z′piezoactuator) or the cantilever chip (BL-AC7DS-KU5,
custom-made, Olympus, Tokyo, Japan) clamped on the wstage with either of the two cantilever chip holding mechanisms described below (note that an identical cantilever chip
was glued to the w′-stage as counterweight). For the measurement of cantilever chip vibrations, the laser was incident on
the cantilever chip body at a distal position from which thecantilever extends. Note that for the vibration measurements,
we could not use the optical beam deflection sensor equippedin our tip-scan HS-AFM instrument because this sensor detects
an angle change of the target rather than its displacement. The
solid lines in Fig. 2 show the frequency response of the Zscanner itself, indicating the first resonant frequency of the
one-end-fixed Z-piezoactuator, 110 kHz, as expected fromthe measured resonant frequency of the piezoactuator in freeoscillation, 222 kHz. The attachment of the w-stage on the topof the Z-scanner only slightly changed the frequency responseof the Z-scanner, as shown with the dotted lines in Fig. 2.

Rev. Sci. Instrum. 86, 063703 (2015)

FIG. 2. Frequency responses of one-end-fixed Z-piezoactuator with andwithout w-stage. The solid lines (red line, gain; blue line, phase) indicate
the frequency response in the case without w-stage, while the dotted lines(red line, gain; blue line, phase) indicate the frequency response in the casewith the w-stage.

III. CANTILEVER CHIP HOLDING MECHANISMS

To avoid damaging the Z-scanner by repeated exchange of
cantilever chips, the entire chip holder should not be directly
mounted on the top of the Z-scanner. Otherwise, a pushingforce (or torque) exerted by an operator to the chip holder isdirectly applied onto the Z-scanner, often resulting in damageof the Z-scanner or ungluing of the holder from the Z-scanner.
The entire mounting is also inadequate in another regard;
it would result in that we have to clamp a cantilever chip
firmly with a small-sized lightweight mechanism. But, it isdifficult. When the holding mechanism is relatively heavy, itsignificantly impairs the Z-scanner’s resonant frequency; a Zpiezoactuator to be used for HS-AFM is small and its mass isapproximately in the range of 100–300 mg. As such, the chipholder should be supported on one prop or two props standingon the base block onto which the Z-piezoactuator is glued.
Moreover, the holder should have a simple structure so as toachieve high resonant frequencies.
Considering these factors, we first designed a holding
mechanism in which the clamping system is supported ona single prop, as shown in Fig. 3. The prop was monolithically fabricated on the base block mentioned above (stainlesssteel SUS303). The prop is positioned at the back of theZ-piezoactuator. The top face of the prop is slanted from thehorizontal plane at an angle the same as the wedge angle of

FIG. 3. Schematic diagram of the scanner with single-prop holding mechanism. (a) Side view, (b) top view, and (c) whole view.
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Fukuda, Uchihashi, and Ando

FIG. 4. Frequency responses of Z-scanner equipped with single-prop holding mechanism with and without elastic polymer sheet on the beam springhead. The solid lines (red line, gain; blue line, phase) indicate the frequency
response in the case with the polymer sheet. The dotted lines (green, gain;
purple, phase) indicate the frequency response in the case without the polymer sheet.

the w-stage, 20◦. The top part of the prop is internally threaded
perpendicularly to the slanted plane. One end of a beam spring(stainless steel SUS303) with the total length of 7.15 mm isclamped on the slanted top face of the prop with a screw so thatthe cantilever inserted between the beam spring head and the
w-stage is pushed down onto the w-stage. Note that the heightof the prop is adjusted so that clamping force can be applied
from the beam spring to the cantilever chip. That is, the top
face of the prop is positioned lower by 0.1 mm than the top
surface of the installed cantilever chip body. To keep the beamspring head from touching the sample of observation, its distaledge region is cut aslant at an angle of 20◦. The dotted lines
in Fig. 4 indicate the frequency response of the cantilever chip
held by this method, indicating appearance of an extra resonantpeak at ∼63 kHz, which is followed by a resonant peak at
105 kHz that originates from the w-stage-mounted Z-scanner
itself (compare the spectra to those shown with the dotted lines
in Fig. 2). The extra resonant vibrations at ∼63 kHz indicatethat the cantilever chip vibrates at the resonant frequency of thebeam spring that insufficiently clamps the chip. This insufficiency may result from incomplete contact between the surfaceof the beam spring head (0.8 × 1.9 mm2) and the cantileverchip body, which is very likely to be due to the deformationof the beam spring by clamping. To test this possibility andincrease the contact area, an elastic polymer sheet was attached

Rev. Sci. Instrum. 86, 063703 (2015)

on the bottom surface of the beam spring head at its area to be incontact with the cantilever chip. This sheet (thickness ∼50 µm)
was prepared by thin coating of the beam spring head with anadhesive (Super-X, Cemedine, Tokyo, Japan) and then drying.
As shown with the solid red lines in Fig. 4, the large resonantpeak at ∼63 kHz disappeared by this treatment, suggesting alarger clamp force as well as an additional role of the polymer
sheet as a vibration damper; however, small resonant peaks
around 50 kHz remained.
To increase the clamping force, we then designed anotherchip holding mechanism in which a beam spring was bridged
between and supported by two props standing by the Zpiezoactuator, as shown in Fig. 5. As done for the single-prop
system, the height of the props is adjusted, the top face ofeach prop is slanted from the horizontal plane at an angleof 20◦, and the top part of each prop is internally threaded
perpendicularly to the slanted plane. The each end of a beamspring (stainless steel SUS303) that is 9.6 mm long, 3.5 mm
wide, and 0.3 mm thick was clamped on the corresponding
prop using a screw to push the inserted cantilever chip onto thew-stage. The distal edge region (width, 0.97 mm) of the beamspring is cut aslant at an angle of 20◦ (see Figs. 5(b) and 5(c).
As shown with the dotted lines in Fig. 6(a), even without elastic
polymer coating of the beam spring, the frequency responseof a cantilever chip held in this method was similar to thatof the w-stage-mounted Z-piezoactuator itself (see the green
line in Fig. 6(b) showing the difference amplitude spectrum
between those measured at the w-stage without the clamp
system and at the clamped cantilever chip body), suggesting
less deformation of the beam spring than the case of the singleprop system. When the beam spring was coated with an elastic
polymer film as above, the frequency response was improved,
as shown with the solid lines in Fig. 6(a), while the main
resonant frequency was only slightly lowered from 108 kHz
to 102 kHz. This vibration damping effect of the polymer
coating is more clearly shown with the red line in Fig. 6(b)that indicates the difference amplitude spectrum between thosemeasured at the w-stage without the clamp system and at thecantilever chip body that is clamped with the polymer-coatedbeam spring. Note that pushing forces exerted from an operator
are not directly applied to the cantilever chip (and hence tothe Z-scanner) but partially and indirectly exerted from the

FIG. 5. Schematic diagram of the scanner with two-prop holding mechanism. (a) Side view, (b) top view, and (c) whole view.
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Fukuda, Uchihashi, and AndoRev. Sci. Instrum. 86, 063703 (2015)

FIG. 6. Frequency responses of Z-scanner equipped with two-prop holding mechanism with and without elastic polymer sheet on the beam spring. (a) The
solid lines (red line, gain; blue line, phase) indicate the frequency response in the case with the polymer sheet. The dotted lines (green, gain; purple, phase)indicate the frequency response in the case without the polymer sheet. These responses were measured at the cantilever chip body held in this mechanism.
(b) Differential amplitude spectra between two cases: one is measured at the mounted cantilever chip body, whose data are the same as those shown in (a), andanother is measured at the w-stage without the cantilever holing mechanism, which is shown with the red dotted line in Fig. 2 (the former was subtracted fromthe latter). The green and red lines indicate the cases without and with an elastic polymer sheet on the beam spring, respectively.

beam spring. Moreover, the magnitude of the force applied
from the beam spring to the cantilever chip is restricted by the
spring constant of the beam spring (∼400 N/mm) as well as
by the height difference (0.1 mm) between the top surfaces ofthe props and the top surface of the mounted cantilever chip.
The upper limit of the force is further reduced by the polymer
film coated on the beam spring. The cantilever chip can beeasily removed from the holder by loosening of the screws.
As such, the Z-scanner would not be damaged by repeatedexchange of cantilever chips. Moreover, the displacement
efficiency of the Z-scanner with a cantilever chip held in thismethod was 15.2 nm/V, similar to that of the Z-scanner alone(18.2 nm/V). This slight reduction is consistent with the loaded
force (∼30 N) and the maximum force that can be generated
by the piezoactuator (∼200 N).
However, a minor drawback was that the base block to
be moved by the X- and Y-scanners became heavier because
the base block was needed to be larger to accommodate the

FIG. 7. Effect of inverse feedforward compensation on the displacement of the X-scanner. (a) Frequency response of the X-scanner (red line, gain; blue line,
phase). (b) X-scanner displacement (red line) when driven by 3.45 kHz isosceles triangle wave signal (black line). (c) X-scanner displacement (red line) whendriven by 3.45 kHz isosceles triangle signal filtered through inverse compensation (black line).
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Fukuda, Uchihashi, and Ando

two props thereon, and therefore, the resonant frequency of
the X-scanner was lowered from 18 kHz to 10 kHz, while theresonant frequency of the Y-scanner was lowered from 1.5 kHz
to 1.3 kHz (the frequency response of the X-scanner is shownin Fig. 7(a)). Nonetheless, the lowered resonant frequency of
the X-scanner is within a range that the adverse responses of
the X-scanner can be removed by using an inverse feedforward
compensation method as described previously.18,26 In fact, thisremoval of adverse responses was proven, as follows. When
the X-scanner was driven with triangle signals of 3.45 kHz(black line in Fig. 7(b)), its displacement exhibited small vibrations (see slightly waved displacements shown with the redline in Fig. 7(b)). However, it was displaced smoothly (red line
in Fig. 7(c)) when driven with 3.45 kHz triangle wave signalsthat were modified by the inverse feedforward compensation
method (black line in Fig. 7(c)). The filter used in this signal
modification was constructed using the frequency response
spectra of the X-scanner (Fig. 7(a)). When 100 scan lines areused, this scan frequency corresponds to the imaging rates of∼34 fps. The Y-scanner is usually driven by a saw-tooth wave
containing a precipitous downward regime where the tip isreturned to the scan origin. When the Y-scanner is scanned fast,
it generates significant vibrations in this regime, which thendecay while ringing in the following slow upward regime. Thisvibration generation can be easily be suppressed by the scan
speed being lowered only in the precipitous downward regime,
as described previously.17 The time delay added by this slower
scan is negligible because it is much shorter than the frame
imaging time.
IV. IMAGING OF PROTEIN MOLECULES

Here, we demonstrate the superior performance of thecantilever chip holding method developed above (two-proptype) by imaging the α3β3 subcomplex of F1-ATPase in thepresence of 2 µM adenosine triphosphate (ATP), using our
tip-scan HS-AFM setup developed previously.20 The imaging
was carried out in amplitude modulation mode, with cantilever oscillation amplitude of ∼2 nm. The cantilever used was
BL-AC7DS-KU5 (Olympus, Tokyo, Japan) with resonant
frequencies of 3.5 MHz in air and 1.2 MHz in water and aspring constant of ∼0.2 N/m. The protein sample and the assay
system for HS-AFM observation were prepared as described
previously.14 Figure 8 shows successive AFM images captured
at 7.14 fps, which display conformational dynamics occurringin the α3β3 subcomplex during ATP hydrolysis. It is visible
in the images that the three β subunits undergo a conformational transition between the high (i.e., nucleotide-free) andlow (i.e., nucleotide-bound) states. In Fig. 8, a highest pixel
position in each frame is marked with a red closed circle.
Therefore, it can be seen that the position of the nucleotide-free
β subunit shifts counterclockwise. The rate constant of thisshift was comparable to the value obtained previously fromimages of the α3β3 subcomplex captured by sample stagescan HS-AFM at 12.5 fps.14 We attempted to image at thesame rate using the tip-scan HS-AFM system but could not
do so. This is solely because the resonant frequency of the
one-end-fixed Z-piezoactuator used in the tip-scan HS-AFM
apparatus (i.e., 110 kHz) is lower than that of the one-end-fixed

Rev. Sci. Instrum. 86, 063703 (2015)

FIG. 8. Successive AFM images of the α3β3 subcomplex of F1-ATPaseshowing counterclockwise propagation of conformational changes over thethree β subunits. Frame rate, 7.14 fps; scale bar, 2 nm. The highest pixel
position in each frame image is marked with a closed red circle.

Z-piezoactuator (PL033.30, Physik Instrumente (PI) GmbH,
Karlsruhe, Germany) used in the sample stage-scan HS-AFM
apparatus (∼180 kHz).
V. CONCLUSION

In this study, we solved one of the difficult issues in establishing tip-scan HS-AFM, i.e., how to hold a cantilever chip on
the Z-scanner. The holding mechanism developed in this study
only slightly lowered the resonant frequency and displacement
efficiency of the Z-piezoactuator (<10% and ∼16%, respectively) and lowered the resonant frequencies of the X- andY-scanners by ∼44% and ∼13%, respectively. The relatively
large reduction of the X-scanner’s resonant frequency is due
mainly to the increased size (by ∼400%) of the base block to
be displaced by the X-scanner. However, the resulted resonant
frequency of the X-scanner (10 kHz) is not the main factor
that limits the imaging rate, as demonstrated above using theinverse feedforward compensation method. Even for the widearea scanner displaceable up to ∼50 × 50 µm2 in the X- andY-directions, this compensation method permits its X-scannerwith the first resonant frequency of ∼2 kHz to be displaced at
1 kHz approximately in an isosceles triangle form as a functionof time, without producing unwanted vibrations.18 Rather, theZ-scanner’s resonant frequency severely limits the imaging
rate because the feedforward compensation technique cannot
be applied to the Z-scanner.
By the combination of the chip holding mechanism developed here together with the mirror tilter system for laser
beam tracking of the lateral motion of the cantilever, tipscan HS-AFM is now established. Note that other devices
for HS-AFM have already been established in the courseof the development of sample stage-scan HS-AFM. By thisestablishment of tip-scan HS-AFM, we can now apply large
and/or heavy specimens to HS-AFM imaging, such as live
cells in a culture dish and a semiconductor wafer. Moreover,various optical techniques can be combined with tip-scan HSAFM, such as optical tweezers and super-resolution and tipenhanced27,28 fluorescence microscopy techniques. This large
expansion of the applicable samples and the functionality will
significantly increase the usefulness of tip-scan HS-AFM not
only in life science but also in industrial inspection, testing, andmeasurements.
However, for the use of tip-scan HS-AFM in liquid environment, one minor issue remaining to be resolved is to makethe Z-piezoactuator waterproof because it has to be locatedThis article is copyrighted as indicated in the article. Reuse of AIP content is subject to the terms at: http://scitationnew.aip.org/termsconditions. Downloaded to IP:
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Fukuda, Uchihashi, and Andocloser to the cantilever chip covered with a liquid than in thecase of sample stage-scan HS-AFM. This is the reason that
we used the waterproof piezoactuator AE0203D04F (TokinNEC, Miyagi, Japan) despite its lower resonant frequency
than the non-waterproof piezoactuator PL033.30 (PI GmbH,
Karlsruhe, Germany).

Rev. Sci. Instrum. 86, 063703 (2015)
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ACKNOWLEDGMENTS

We thank Dr. R. Iino for providing us with the sample
of puried α3β3 subcomplex. This work was supported by the
CREST program of the Japan Science and Technology Agency(JST) (to T.A.), the JST program on Development of Systems
and Technology for Advanced Measurement and Analysis (toT.A.), KAKENHI for Basic Research (No. 24227005 to T.A.
and Nos. 23115008, 24241048, 26104514, and 26102515 toT.U.), and KAKENHI for Scientific Research on Innovative
Areas: Research in a Proposed Research Area (No. 26119003
to T.A.) and Grant-in-Aid for Research Fellows (to S.F.) fromthe Japan Society for the Promotion of Science.

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