High-speed atomic force microscope combined with single-molecule
fluorescence microscope
Shingo Fukuda, Takayuki Uchihashi, Ryota Iino, Yasutaka Okazaki, Masato Yoshida et al.
Citation: Rev. Sci. Instrum. 84, 073706 (2013); doi: 10.1063/1.4813280
View online: http://dx.doi.org/10.1063/1.4813280
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REVIEW OF SCIENTIFIC INSTRUMENTS 84, 073706 (2013)

High-speed atomic force microscope combined with single-molecule
fluorescence microscope
Shingo Fukuda,1 Takayuki Uchihashi,1,2 Ryota Iino,3 Yasutaka Okazaki,1 Masato Yoshida,1Kiyohiko Igarashi,4 and Toshio Ando1,2
1

Department of Physics, College of Science and Engineering, Kanazawa University, Ishikawa 920-1192, Japan
Bio-AFM Frontier Research Center, College of Science and Engineering, Kanazawa University, Ishikawa
920-1192, Japan3
Department of Applied Chemistry, School of Engineering, University of Tokyo, Tokyo 113-8656, Japan
4
Department of Biomaterial Sciences, Graduate School of Agricultural and Life Sciences, University of Tokyo,
Tokyo 113-8657, Japan
2(Received 22 April 2013; accepted 24 June 2013; published online 17 July 2013)
High-speed atomic force microscopy (HS-AFM) and total internal reflection fluorescence microscopy(TIRFM) have mutually complementary capabilities. Here, we report techniques to combine these microscopy systems so that both microscopy capabilities can be simultaneously used in the full extent.
To combine the two systems, we have developed a tip-scan type HS-AFM instrument equipped witha device by which the laser beam from the optical lever detector can track the cantilever motion in theX- and Y-directions. This stand-alone HS-AFM system is mounted on an inverted optical microscopestage with a wide-area scanner. The capability of this combined system is demonstrated by simultaneous HS-AFM/TIRFM imaging of chitinase A moving on a chitin crystalline fiber and myosin V
walking on an actin filament. © 2013 AIP Publishing LLC. [http://dx.doi.org/10.1063/1.4813280]
I. INTRODUCTION

To increase the imaging rate of atomic force microscopy (AFM), elementary devices and control techniques
were developed.1–6 Moreover, a new feedback control technique was also developed to make the high-speed and lowinvasive imaging capabilities compatible.7 Through thesedevelopments, high-speed AFM (HS-AFM) has recently
been established.8, 9 It permits direct imaging of biologicalmolecules in action at high spatiotemporal resolution, without disturbing their functions. In fact, dynamically functioning proteins have been successfully visualized in succession;
myosin V walking on an actin filament,10 rotational propagation of conformational changes over three β subunits of rotorless F1 -ATPase,11 bacteriorhodopsin responding to light,12cellulase moving unidirectionally during hydrolyzing a cellulose fiber,13 and others.14–17 The dynamic actions appearedin the molecular movies have provided great insights into thefunctional mechanisms of the proteins.9 However, in-liquid
AFM imaging, in general, has an intrinsic limitation as to theacquirable information. It is difficult to observe small chemicals such as ATP molecules. It is even impossible to visualize
their binding to and dissociation from proteins, meaning thateven when ligand-induce conformational changes of proteins
are observed with HS-AFM, it is hard to directly verify thatthe observed changes are really induced by the binding or dissociation of small ligand molecules. Moreover, AFM is poor
at identifying a specific target in a sample that contains multiple species of proteins, although efforts have been made to
overcome this poorness.18, 19
In contrast, fluorescence microscopy can easily detect fluorescence-emitting small molecules and identify one
species of molecules in a sample containing multi-species ofmolecules. When fluorophores with different colors are used,

0034-6748/2013/84(7)/073706/8/$30.00

it is even possible to identify two or three species of molecules
in a sample with multiple components. Owing to these capabilities, fluorescence microscopy has been widely used inbiological studies.20–23 As a matter of course, fluorescencemicroscopy, however, cannot observe molecules that are not
emitting fluorescence. Moreover, unlike AFM, it cannot resolve the structure of proteins.
As seen above, AFM and fluorescence microscopy aremutually complementary. Therefore, numerous developmentsof AFM combined with fluorescence microscopy have beenmade.24–32 However, previous combined systems can perform
neither optical imaging at the single molecule level nor fast
AFM imaging. Here, we developed HS-AFM combined with
objective lens type of total internal reflection fluorescencemicroscopy (TIRFM) with a capability of single-molecule
imaging.33 For the HS-AFM unit, we employed the tip-scanmode,24 instead of the sample stage-scan mode, to makethe full and simultaneous use of TIRFM possible. The HSAFM unit is mounted on an inverted fluorescence microscope
equipped with a slow, wide-area XY scanner on which a sample cell (or a cover slip) is placed. Here, we report the detailsof this instrumentation and demonstrate the capability of the
combined system by simultaneous HS-AFM/TIRFM imagingof linear motions of proteins chitinase A and myosin V.

II. LASER-BEAM TRACKING

The current HS-AFM has employed the sample stagescan mode.8, 9 Its high-speed performance, however, imposes
restrictions on its application expansion. A large sample stagecannot be used, because it lowers the resonant frequencies ofthe scanner and hence the maximum possible imaging rate.
In addition, the HS-AFM system is inadequate for making

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simultaneous HS-AFM/fluorescence-microscopy observations possible, because the sample stage moves relative to theobjective lens equipped for fluorescence microscopy. To eliminate these restrictions, we here developed a new HS-AFM
system that operates in the tip-scan mode.
Designs for tip-scan AFM systems have beenreported.24, 34–37 A common technical issue in the systems is the optical lever detection of cantilever deflection.
The laser beam from the optical lever detector has to track
the cantilever that is moving in the X- and Y-directions.
HS-AFM uses small cantilevers and hence the size of a laser
spot to be focused on a cantilever should be as small as thewidth of small cantilevers (∼2 μm). Therefore, precise andfast laser-beam tracking is required for a tip-scan HS-AFM
system. In typical tip-scan AFM systems previously developed, for example, by Werf et al.,34 the laser diode andfocusing lens are positioned inside the piezo tube and thecantilever is attached to the end of the piezo tube. This design
is inadequate for HS-AFM, because an objective lens with ahigh numerical aperture (NA) has to be used to have a smalllaser spot on a small cantilever. Such an objective lens is tooheavy to be scanned quickly. In another design developed
by Nakano,35 two mirrors, each being attached to either endof the Y-scanner, guide a laser beam to the cantilever andthe laser beam reflected by the cantilever to the photodiodesensor. This method is only applicable to slow XY-scanners
and not precise enough to be used for small cantilevers. Here,
we use a laser beam tracking method that was previously used
for Sagnac interferometry.38 The tilt angles of a mirror placedin the optical path between the cantilever and the opticallever detector’s photodiode sensor are scanned in synchrony
with XY scanning of the cantilever. When the angle of thelaser beam incident onto the objective lens is altered, thelateral position of the focused spot is displaced, as shownin Fig. 1(a). Figures 1(b) and 1(c) show the orthogonal sideviews of the two-dimensional mirror tilter that we developed
here. The mirror tilter consists of two base plates, four piezo
actuators (AE0203D04, NEC-TOKIN Corp., Miyagi, Japan)
with the maximum displacement of ∼3 μm at 100 V, anda dichroic mirror (NT64-464, >90% transmission at 400–
690 nm, >95% reflection at 700–1150 nm, 12.5 × 12.5 mm2 ,Edmond Optics, Barrington, NJ, USA). The dichroic mirror
is supported by the X- and X -piezoactuators, each of whichis glued on the base plate 2 through a beveled block. This
whole unit is supported by the Y- and Y -piezoactuatorsthat are glued on the base plate 1. The respective pairs of
piezoactuators are displaced in the opposite directions, by

FIG. 1. Dichroic mirror tilter for laser-beam tracking of lateral cantilever
motion. (a) Schematic showing the working principle of laser-beam tracking by mirror-tilting method. (b) and (c) Side views of the two-dimensional
mirror tilter when viewed from two orthogonal directions.

Rev. Sci. Instrum. 84, 073706 (2013)

which the respective tilt angles of the dichroic mirror arescanned. The base plates 1 and 2 have through-holes at theircenters through which the cantilever and the focused laser
spot can be viewed using a CCD camera, facilitating theadjustment of their relative position.
From the maximum displacement of the piezoactuators and the size of the dichroic mirror, the maximum tiltangle change ±θ max was estimated to be approximately ±4.8
× 10−4 rad for both tilters. The maximum displacement dmaxof the focused laser spot is a function of the focal length(f0 = 20 mm) of the ×20 objective lens (working distance,
24 mm; NA, 0.35; CFI L Plan EPI SLWD, Nikon, Tokyo,
Japan) used for laser beam focusing as well as ±θ max , which
is approximately given by dmax = 2fo × θ max . This relationship gives dmax = 19 μm, which approximately agrees withan experimentally measured value, 20 μm.
The mirror tilting shifts the position where the laser
beam reflected back by the cantilever is guided onto the twosegmented photodiode sensor. This shift produces a false cantilever deflection signal. However, this issue cannot be an impediment to tapping-mode HS-AFM which uses the cantilever
oscillation amplitude to detect tip-sample interactions. Thisis because the cantilever oscillation frequency is much higher
than the frequencies of mirror-tilter scanning, and because theamplitude signal is independent of the positional shift of thereflected laser beam at the photodiode sensor, which is muchsmaller than the diameter of the laser beam (∼2 mm).
To assess the dichroic mirror tilter’s dynamic performance, we have to directly measure the change in angle ordisplacement of a laser beam reflected back from the center of the dichroic mirror that is dynamically being tilted.
However, it is difficult to do so. Instead, we measured thesignal that is output from the optical lever sensor when thelaser beam was reflected by the dichroic mirror and then bya stationary mirror and finally reflected by the dichroic mirror again. As the stationary mirror, the supporting base ofa cantilever was used. Figure 2(a) shows frequency spectraof the signal that was output when the dichroic mirror wastilted by the X-and X -piezoactuators (i.e., X-tilt). The firstresonant peak appeared around 2.2 kHz, followed by a few
peaks at higher frequencies. When the X-tilter is driven by anisosceles triangle wave, this mechanical response deteriorates
the tracking and speed performance of the tilter, as shown in
Fig. 2(b). This issue was solved by an inverse compensation
method;39 an isosceles triangle wave was digitally filtered byan inverse transfer function constructed using the frequency
response shown in Fig. 2(a). Consequently, as shown by the
red line in Fig. 2(c), the X-tilter moves smoothly approximately in an isosceles triangle wave, even when driven at
1 kHz, which corresponds to an imaging rate of 10 frames/s(fps) for 100 scan lines in the Y-direction. The red line in
Fig. 2(c) is, however, slightly curved inwardly at the ascending regime and outwardly at the descending regime. This is
not due to an effect of hysteresis of the piezoactuators usedbecause such a nonlinear response does not appear when
the X-tilter is scanned slowly. It is likely due to forced deformation of the dichroic mirror. This effect was eliminatedby inverse compensation,39 as shown by the orange line in
Fig. 2(c).

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Rev. Sci. Instrum. 84, 073706 (2013)

FIG. 2. Dynamic response of X- and Y-tilters. (a) Frequency spectra of tilting motion of the X-tilter (black line, gain; blue line, phase). (b) Tilting motion of
X-tilter (red line) driven by 1 kHz isosceles triangle wave signal (black line). (c) Tilting motion of X-tilter (red line) driven by 1 kHz isosceles triangle signal
filtered through inverse compensation (black line). The orange line shows the tilting motion when the filtered driving signal is further modified for the eliminationof the curved response (blue line). (d) Frequency spectra of tilting motion of the Y-tilter (black line, gain; blue line, phase). (e) Tilting motion of Y-tilter (redline) driven by 10 Hz sawtooth wave signal (black line) whose precipitous downward part is slightly blunted.

To measure the dynamic performance of the Y-tilter, thetwo-segmented photodiode equipped with the optical leversensor was rotated by 90◦ . The Y-tilter has the first resonantpeak at 2.2 kHz and several larger peaks at higher frequencies, as shown in Fig. 2(d). Ideally, the tip scanner is scannedin the Y-direction in a sawtooth wave containing a precipitous downward regime, meaning that the Y-tilter should also
be scanned in the same way. However, the precipitous scan
for returning to the scan origin easily generates vibrations inboth the tip-scanner and the dichroic-mirror tilter. We solved
this problem by simply slowing the scanning to the scan origin. Even when slowed down, the time that this scan spends
is much shorter than the time for which an image is captured.
By this slower scanning to the scan origin, it was unnecessary to use inverse compensation in order to move the Y-tiltersmoothly, as shown in Fig. 2(e).

Next, we evaluated the performance of the laser-beamtracking system by monitoring the amplitude of a small cantilever (BioLever fast BL-AC10DS-A2, Olympus; 9 μm long,
2 μm wide, and 130 nm thick; resonant frequencies 1.5 MHz
in air and 600 kHz in water; spring constant ∼0.1 N/m) which
was freely oscillating during its scanning in the X- and Ydirections, without its tip being in contact with the sample
surface. Figures 3(a) shows an amplitude image that was acquired at 1 fps without laser beam tracking when the cantilever tip oscillating in water at 600 kHz was scanned overa 650 × 650 nm2 lateral area (256 × 256 pixels). The image
contrast seen in Fig. 3(a) indicates that the optical lever sensitivity is changed by the positional shift of the cantilever relative to the laser spot focused on it. The height profile along
the red line shown in Fig. 3(a) indicates that the apparent
amplitude varies by ∼1.4 nmp-p , which is more than half the

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Rev. Sci. Instrum. 84, 073706 (2013)

FIG. 4. Schematics of fast tip-scanner. (a) Bottom view. (b) Side view.

III. SCANNER

FIG. 3. Effect of laser-beam tracking of lateral cantilever motion on the optical lever detection of cantilever oscillation amplitude. (a) Amplitude image
captured at 1 fps for 650 × 650 nm2 (256 × 256 pixels) without laser-beamtracking. The bottom shows a height profile of the image along the diagonal red line. (b) Amplitude image captured at 1 fps for 650 × 650 nm2 (256× 256 pixels) with laser-beam tracking. The bottom shows a height profileof the image along the diagonal red line. (c) Amplitude image captured at
10 fps for 250 × 250 nm2 (128 × 128 pixels) without laser-beam tracking.
The bottom shows a height profile of the image along the diagonal red line.
(d) Amplitude image captured at 10 fps for 250 × 250 nm2 (128 × 128 pixels) with laser-beam tracking. The bottom shows a height profile of the image
along the diagonal red line.

initial amplitude of 2.4 nmp-p . Using the laser-beam tracking,this large error disappeared as shown in Fig. 3(b). The heightprofile along the red line shows the amplitude variation of
only ∼0.2 nm, which mainly originates from thermal bendingof the cantilever. Even when the imaging rate was increased
to 10 fps for a smaller lateral area of 250 × 250 nm2 (128× 128 pixels), the tracking system worked well as shown in
Fig. 3(d). The height variation was only ∼0.2 nm, making
a large contrast with the large height variation that appeared
when the tracking system was switched off [Fig. 3(c)]. Thus,
the laser-beam tracking system can keep the optical lever’s
sensitivity of detecting cantilever oscillation amplitude constant, even when the cantilever tip is scanned at ∼1.3 kHz in
the X-direction.

We developed a fast scanner for the tip-scan HSAFM system, whose bottom and side views are shown in
Figs. 4(a) and 4(b), respectively. Its design is basically thesame as previously reported.40 The piezoactuators used
therein are AE0203D04 for X and AE0505D08 for Y,
both from NEC Tokin Corp. (Miyagi, Japan). The X- andY-scanners have the maximum displacements of 800 nm and1.5 μm at 100 V and resonant frequencies of 18 kHz and1.5 kHz, respectively. The mechanical vibrations producedby fast scanning of the X-scanner are suppressed by inverse
compensation.39 The Z-piezoactuator (AE0203D04) is glued
on the block to be scanned in the X- and Y-directions. The
electrodes protruding from the Z-piezoactuator are coveredby a waterproof film as it is immersed in a buffer solution
during AFM imaging. To maintain the Z-scanner’s resonant
frequency as high as possible, the cantilever holder to be attached to the Z-scanner is compact sized and made of siliconcarbide that has a higher Young’s modulus-to-density ratio
than stainless steel and aluminum. The cantilever holder has
a wedge shape with tip angle of 20◦ so that the cantilever heldby it is tilted by this angle with respect to the sample substrate.
The resonant frequency of the Z-scanner attached to the cantilever holder is 110 kHz and its maximum displacement is∼3 μm at 100 V. As a counterbalance, an additional piezoactuator (Z -piezoactuator) identical to the Z-piezoactuator is
attached to the opposite face of the Z-scanner’s supportingbase. A dummy cantilever holder with a dummy cantilever is
attached onto the Z -piezoactuator. By this counterbalancing,the impulsive force generated by the rapid displacementof the Z-piezoactuator is counteracted with the impulsive
force generated by the simultaneous displacement of theZ -piezoactuator with the same velocity in the opposite
direction.1
A piece of low-reflection glass (D263, Matsunami Glass
Co. Ltd., Osaka, Japan) is placed just above the cantileveras an optical window for the laser beam, which can reduce reflection of the laser beam at the air-water interface.
To acoustically excite the cantilever for tapping-mode operation, a thin piezoactuator (4.5Z4×4S-SYXC(C-82), Fuji
Ceramics Co. Ltd., Shizuoka, Japan) with a resonant frequency of 2 MHz is placed on the scanner in proximity to theZ-piezoactuator.

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IV. HS-AFM/TIRFM COMBINED SYSTEM

Figure 5(a) shows a schematic of the optical lever system for the tip-scan HS-AFM unit. Instead of a red laser
used in the standard HS-AFM system,8, 9 an infrared (IR)laser (L980P010, λ = 980 nm, Thorlab, Newton, NJ, USA) is
used because emission wavelengths of fluorescence dyes frequently used in fluorescence imaging are shorter than 800 nm.
To suppress the optical feedback noise often caused by thelaser beam that is reflected back or scattered back to thelaser window, a radio-frequency laser power modulation is
employed.41 An objective lens with a long working distance(×20, working distance 24 mm, NA 0.35, CFI L Plan EPISLWD, Nikon, Tokyo, Japan) is used to focus the infraredlaser onto the cantilever. The laser spot on the small cantilever can be observed with a CCD camera through the objective lens and the dichroic mirror, which facilitates the laseralignment. The cantilever deflection is detected by a position
detection system consisting of a two-segmented Si PIN photodiode (S3096-02, Hamamatsu Photonics, Shizuoka, Japan),
amplifiers, and a signal conditioner with total bandwidth ofRev. Sci. Instrum. 84, 073706 (2013)

20 MHz. Other optical components are optimized for the infrared wavelength: a collimation lens (LT110P-B, Thorlab,
Newton, NJ, USA), a λ/4 wave-plate (WPQ-9800-4M, Sigma
Koki, Tokyo, Japan), a polarization beam splitter (PBS103,
Thorlab, Newton, NJ, USA), and a focusing lens (LA1074-B,
Thorlab, Newton, NJ, USA) placed at the front of the twosegmented photodiode.
Figure 5(b) shows the side view of the newly designed
tip-scan HS-AFM unit. The supporting frame of the unit iscomposed of top and base parts. Materials used for the top
and base parts are aluminum and stainless steel, respectively.
The center of gravity of the AFM unit is lowered as much as
possible to make the unit robust to mechanical vibrations. All
optical components for the optical lever system including thetwo-dimensional dichroic mirror tilter are assembled in the
top frame. The fast scanner and a stepper motor (AM10202R-A-0.25-8-10+12/5 2050:1, Faulhaber GmbH, Weinheim,
Germany) for the coarse tip approach are installed on the base
part. The positions of the IR laser, the two-segmented photodiode, and the scanner with the cantilever can be adjustedwith precise screws (F3ESK1, Thorlab, Newton, NJ, USA)

FIG. 5. Schematics showing the structure of tip-scan HS-AFM unit. (a) Optical lever detector with dichroic mirror tilter for laser-beam tracking. (b) Whole
structure of the tip-scan HS-AFM unit. The numbers attached to respective parts correspond to the numbers shown in (a). (c) Optical microscope stage withwide-area XY-scanner.

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in the XZ-, YZ-, and XY-planes, respectively, as shown in
Fig. 5(b). The tilt angle of the dichroic mirror is also adjustable using three screws. The objective lens is also movable along its long axis for the adjustment of its focal point at
the cantilever. The whole assembly is supported by a tripod
consisting of two adjustable screws and a screw connected tothe stepper motor. This tip-scan AFM unit is more compact
than the standard HS-AFM head, facilitating its mounting on
an optical microscope stage.
As an optical microscope to be combined with the
tip-scan HS-AFM system, we used an inverted microscope(ECLIPSE Ti, Nikon, Tokyo, Japan) with modifications. In
objective lens-based TIRFM, a high NA objective lens with ashort working distance has to be used. The front face of theobjective lens is usually in contact with a cover slip throughimmersion oil. In the standard configuration of inverted optical microscopes, the sample stage and objective lens are separately held within the microscope body. In this configuration,
vibrations of the large microscope body are easily transmitted to the cover slip through the objective lens.32 To minimize
the mechanical coupling between the microscope body andthe cover slip, the oil-immersion objective lens (Apo TIRF
100×, NA 1.49, Nikon, Tokyo, Japan) is directly hung from acustom-made microscope stage (Chukousya, Tokyo, Japan),instead of using the originally equipped objective lens revolver. In this microscope stage, the focus of the objective
lens can be adjusted.
For video imaging by TIRFM, a relay lens (VM Lens
C2.5X, Nikon, Tokyo, Japan) is connected to an EM CCD
camera (Ixon3, Andor Technology, Belfast, Northern Ireland).
The IR laser used in the HS-AFM’s optical lever system is cutat the front of the CCD camera using an optical low-pass filter with 91% transmission at 400-810 nm and less than 0.01%
transmission at 850–1190 nm (NT86-105, Edmond Optics,
Barrington, NJ, USA).
The TIRFM setup has a wide field of view of ∼31
× 31 μm2 , whereas the maximum lateral scan range of the
HS-AFM system is limited to 800 nm × 1.5 μm. This large
difference makes it difficult to correlate acquired AFM images to TIRFM images. To fill this large gap, a wide-area XYscanner is constructed in the optical microscope stage, using
long-stroke piezoactuators (AE1010D44H40F, NEC Tokin
Corp., Miyagi, Japan), as shown in Fig. 5(c). This wide-areascanner provides AFM images over a 15 × 15 μm2 area, enabling precise positioning of the cantilever tip on the region ofinterest visualized by TIRFM. The Z-scanner in the HS-AFM
unit is also used for slow AFM imaging performed using thewide-area XY-scanner.

Rev. Sci. Instrum. 84, 073706 (2013)

FIG. 6. Successive AFM images showing processive movement of M5HMM along actin filament. The images were captured at 5 fps for an areaof 150 × 75 nm2 (100 × 40 pixels).

was coated with planar lipid bilayers using procedures
similar to those used previously.10, 42 The lipid compositionis 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC),
1,2-dipalmitoyl-3-trimethylammonium-propane (DPTAP),
and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N(cap biotinyl) (biotin-cap-DPPE) in a weight ratio of
0.85:0.05:0.1. Partially biotinylated actin filaments were
immobilized on the lipid bilayer surface through streptavidin
with a low surface density. M5-HMM was injected to the
2 μM-ATP-containing buffer solution on the cover slip.
Then, HS-AFM imaging was carried out at 5 fps for an
area of 150 × 75 nm2 (100 × 40 pixels). Figure 6 shows
successive HS-AFM images of M5-HMM unidirectionallymoving along an actin filament, from right to left. Thus,
it was demonstrated that the tip-scan HS-AFM systemdeveloped here has an ability to capture protein molecules inaction without disturbing their activity. However, the speed
performance is somewhat inferior to the standard HS-AFM
system because a piezoactuator used for the Z-scanner of the
tip-scan HS-AFM has a longer working stroke (∼3 μm) thanthat used for the Z-scanner of the standard HS-AFM system(∼1 μm).
Figure 7(a) shows a TIRFM image of rhodamine-labeledactin filaments immobilized on a cover slip through avidin.
After taking this image, positioning of the cantilever tip was
made by reference to the TIRFM image, and then, HS-AFM

V. SIMULTANEOUS HS-AFM/TIRFM IMAGINGFirst, we tested the performance of the tip-scan HS-AFM
system by imaging tail-truncated myosin V (M5-HMM)
molecules moving on actin filaments. As a sample substrate,
cover slips (C030401, Matsunami Glass Co. Ltd., Osaka,
Japan) were used. A circular area (7 mm in diameter) ona cover slip was surrounded by a Teflon sheet (965383,
Niraco Co. Ltd, Tokyo. Japan) to avoid spreading of a buffer
solution on the cover slip. Then, the cover slip surface

FIG. 7. Position-correlated TIRFM, AFM, and HS-AFM images of
rhodamine-labeled actin filaments placed on a cover slip via avidin. (a)TIRFM image. The inset shows a wide-area AFM image captured at
10 min/frame for an area shown by the white rectangle in the TIRFM image.
(b) HS-AFM image captured at 8.3 fps with 100 × 45 pixels. The position of
cantilever tip was set by reference to the TIRFM image. (c) HS-AFM image
captured at 2 fps with 100 × 100 pixels. The position of cantilever tip wasset by reference to the TIRFM image. (d) The overlaid TIRFM/AFM images.
The imaged areas of (b) and (c) are those shown by the black rectangles.

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FIG. 8. Simultaneous HS-AFM/TIRFM imaging of proteins in action. (a)Cy3-labeled chitinase A on a chitin microfibril. The HS-AFM images (left)
captured at 3 fps with 150 × 35 pixels show a single chitinase A moleculemoving toward the top-right (arrows). The TIRFM images (right) with40 × 15 pixels also show the same moving chitinase A molecule (arrows).
(b) Cy3-labeled M5-HMM on an actin filament. The HS-AFM images (left)
captured at 3 fps with 100 × 36 pixels2 show a single M5-HMM moleculemoving toward the bottom-left. The TIRFM images (right) with 17 × 9 pixels shows corresponding correlated fluorescent spots (white arrows) slightly
moving from right to left.

Rev. Sci. Instrum. 84, 073706 (2013)

HS-AFM and the exposure time was adjusted to be slightly
shorter than the frame time of HS-AFM. As test samples, weused chitinase A and M5-HMM, which are linear motor proteins. Chitinase A is known as an enzyme which degrades
chitin microfibrils by hydrolysis and is considered to moveprocessively and unidirectionally along the microfibrils.43 For
the fluorescence imaging, chitinase A was labeled with Cy3.
Crystalline chitin microfibrils suspended in water were attached to a glass cover slip by spin coating. Figure 8(a) shows
successive images of chitinase A moving along a chitin microfibril simultaneously observed by HS-AFM and TIRFM
at an imaging rate of 3 fps. HS-AFM images (left) clearlyshows binding of a single chitinase A molecule to the chitin
microfibril at 0.99 s, as indicated by the white arrow. Then,the molecule moved with time on the microfibril toward theupper-right. A fluorescent spot appeared also at 0.99 s in the
TIRFM images (right), as indicated by the white arrow. The
fluorescent spot slightly moved with time toward the rightside, although the movement is less conspicuous, because thefield of view in the TIRFM is much wider than that of the
HS-AFM.
Figure 8(b) shows simultaneously recorded HS-AFM and
TIRFM images of a Cy3-labeled M5-HMM molecule boundto an actin filament in the presence of 3 μM ATP. Here,the glass surface was coated with lipid bilayers, as describedabove. The AFM images (left) show hand-over-hand movement of M5-HMM toward the bottom-left of the images.
On the other hand, several fluorescent spots appeared in the
TIRFM images (right). This is again because of the wide
field of view in the TIRFM image. However, a fluorescentspot and a M5-HMM molecule appeared simultaneously in
the TIRFM and HS-AFM images at 0.99 s, as indicated by
the arrows. Thus, it was demonstrated here that the combined
HS-AFM/TIRFM system can simultaneously capture respective images of the same biological molecules in action.

VI. CONCLUSION

imaging was carried out. The acquired HS-AFM images areshown in Figs. 7(b) and 7(c). However, it is difficult to findprecise positional correlation between the TIRFM image andthe HS-AFM images because individual actin filaments are
not well resolved in the TIRFM image, particularly in thecrowded area. However, wide-area AFM imaging with the useof the wide-area XY-scanner can solve this problem. The inset in Fig. 7(a) shows a wide-area AFM image taken by reference to the TIRFM image. This AFM image facilitates precise
positional correlation between the TIRFM image and the HSAFM images, as indicated in the overlaid TIRFM and AFM
image shown in Figs. 7(d).
Finally, we tested the ability of our combined HSAFM/TIRFM system to capture simultaneously the topographic and fluorescence images of single protein moleculesin action. For the simultaneous recording, the synchronization between the two types of video recordings is required.
For this synchronization, the exposure of the CCD camera
was triggered at the beginning of each frame imaging by

We developed the combined HS-AFM/TIRFM system
which can perform simultaneous topographic and fluorescence imaging at the single molecule level. The compact design of the HS-AFM unit with a laser-beam tracking capability allows its stand-alone operation and mounting on the
inverted optical microscope stage. The slow but wide-area
XY-scanner incorporated in the optical microscope stage fills
the large gap in the field of view between HS-AFM and
TIRFM, permitting precise finding of the positional correlation between their images. The tip-scan AFM can be operated at 5 fps, without disturbing the function of biological molecules. This speed performance is much better thanconventional AFM systems but lower than the standard HSAFM system. However, the speed performance can be improved when water-proof piezoactuators with a shorter working stroke are available. Moreover, since a fast wide-areascanner has recently been materialized,44 the relatively narrow field of view in the tip-scan HS-AFM system can be
improved in the near future, which will significantly expand
the scope of HS-AFM/TIRFM system’s application from

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Fukuda et al.

imaging of isolated biological molecules to imaging of dynamic events occurring in live cells.
The stand-alone configuration of the tip-scan HS-AFM
unit permits its flexible use. We can combine the unit withdifferent types of inverted optical microscopes: confocal microscopes and super-resolution fluorescence microscopes including stimulated emission depletion,45 stochastic opticalreconstruction,46 and photo-activated localization47 microscopes. Such ultra-hybrid microscopy systems will become
powerful and innovative tools for biological science.

ACKNOWLEDGMENTS

This work was supported by Grant-in-Aid for Basic
Research (Project ID 24227005 to T.A., 23115008 and
30326300 to T.U.), a JST’s program on Development of Systems and Technology for Advanced Measurement and Analysis (to T.A.), HFSP (to T.A.), the Knowledge Cluster Initiative Project from the Ministry of Education, Culture, Sports,
Science, and Technology Japan (to T.A.), and JST/ALCA(to T.U.).
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