Ultramicroscopy 160 (2016) 182–196

Contents lists available at ScienceDirect

Ultramicroscopy
journal homepage: www.elsevier.com/locate/ultramic

Review article

Functional extension of high-speed AFM for wider biologicalapplications
Takayuki Uchihashi a,b,c, Hiroki Watanabe a, Shingo Fukuda a, Mikihiro Shibata a,
Toshio Ando a,b,c,naDepartment of Physics, Kanazawa University, Kanazawa 920-1192, Japan
Bio-AFM Frontier Research Center, Kanazawa University, Kanazawa 920-1192, Japanc
CREST/JST, Tokyo 102-0075, Japanb

art ic l e i nf o
Article history:
Received 6 March 2015
Received in revised form
25 September 2015
Accepted 12 October 2015
Available online 17 October 2015
Keywords:
AFM
High-speed AFM
Imaging
Biomolecules
Live cells

a b s t r a c t
High-speed atomic force microscopy (HS-AFM) has been established and used, which can visualize biomolecules in dynamic action at high spatiotemporal resolution without disturbing their function. Various studies
conducted in the past few years have demonstrated that the dynamic structure and action of biomolecules
revealed with HS-AFM can provide greater insights than ever before into how the molecules function. However, this microscopy has still limitations in some regards. Recently, efforts have been carried out to overcome
some of the limitations. As a result, it has now become possible to visualize dynamic processes occurring evenon live cells and perform simultaneous observations of topographic and ﬂuorescent images at a high rate. In
this review, we focus on technical developments for expanding the range of objects and phenomena observable
by HS-AFM as well as for granting multiple functionalities to HS-AFM.
& 2015 Elsevier B.V. All rights reserved.

Contents
1.
2.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182
Tip-scanning HS-AFM combined with TIRFM. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183
2.1.
Laser tracking method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183
2.2.
Combined Tip-scanning HS-AFM/TIRFM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 186
2.3.
Simultaneous HS-AFM/TIRFM imaging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187
2.4.
Potential of Tip-scanning HS-AFM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 188
3. Wide-area scanner for live cell imaging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 188
3.1.
Design of wide-area scanner. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 188
3.2.
Control techniques for wide-area scanner . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189
3.2.1.
Vibration damping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189
3.2.2.
Compensation for image distortion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189
3.2.3.
Z-scanner . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191
3.3.
Cantilever tip for cell imaging. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191
3.4.
Positioning of tip on cell surface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 192
3.5.
Live cell imaging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 192
4. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 193
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195

1. IntroductionnCorresponding author at: Department of Physics, Kanazawa University, Kanazawa 920-1192, Japan.
E-mail address: tando@staff.kanazawa-u.ac.jp (T. Ando).
http://dx.doi.org/10.1016/j.ultramic.2015.10.017
0304-3991/& 2015 Elsevier B.V. All rights reserved.

Since the invention of atomic force microscopy (AFM) [1], increasing its imaging rate has been one of the major technical

T. Uchihashi et al. / Ultramicroscopy 160 (2016) 182–196

challenges for enhancing the usefulness of AFM. The materialization of high-speed AFM (HS-AFM) required various developments
and breakthroughs, as described below, while it was expected tobring a great impact on various ﬁelds of science and technology.
Biological science in particular would most efﬁcaciously receive agreat deal of beneﬁt from the materialization. This is because thedirect observation of biological molecules in dynamic action athigh spatiotemporal resolution certainly facilitates our detailed
understanding of their functional mechanism. Driven by this motivation, Paul Hansma's group and Ando's group independently
embarked on the development of HS-AFM more than two decadesago. In the early stage, efforts were focused on the developmentsof a fast scanner [2–9], small cantilevers with a high resonant
frequency in water and a small spring constant [3–7], an opticalbeam deﬂection (OBD) detector that detects the deﬂection of asmall cantilever [3,5], and a fast amplitude detector that quickly
converts the deﬂection signal of the oscillating cantilever to its
amplitude signal [3,8]. By assembling these (or some of these)
devices, prototypic HS-AFM instruments employing the tapping
mode were built around 2000 and shown to be able to capture
images of protein molecules at a much higher rate than before[3,9]. However, it was evident that the feedback bandwidth was
still insufﬁcient and therefore the imaging rate was not high enough or the protein molecules were damaged when imaged too
fast. Then, Ando's group has further endeavored to enhance thecapacity of HS-AFM by developing various techniques such as anactive damping technique that suppresses the Z-scanner's unwanted vibrations as well as enhances its response speed [10], a
new scheme for the proportional-integral-derivative (PID) control(referred to dynamic PID control) that can make high-speed imaging compatible with low-invasiveness to fragile molecules andweak intermolecular interactions [11], a technique to compensate
for drift of the cantilever excitation power [11] and a fast phasedetector [12]. Moreover, Ando's group has developed smaller
cantilevers in collaboration with Olympus, a more robust fastscanner and a lower-noise, fast amplitude detector [13]. Finally,around 2008, HS-AFM of practical use was established that
achieved a feedback bandwidth of  100 kHz and therefore could
capture images of protein molecules at sub-100 ms temporal resolution, without disturbing their function [13].
Since the establishment of this HS-AFM, its excellent performance has been demonstrated by imaging studies on various
proteins in dynamic action. For example, conformational dynamics
of a photo-activated proton pump, bacteriorhodopsin [14–16]. anda rotary-motor protein, F1-ATPase [17], dynamic processes of linear-motor proteins, myosin V [18], cellulose [19–21] and chitinase[22], dynamic protein-protein interactions [23,24], dynamics occurring in the crystal lattices of a soluble protein and membraneprotein [25,26], and wiggling motion of intrinsically disordered
proteins [27,28] have been successfully visualized (see Reviews[29,30]). The exquisite dynamic images not only provided corroborative “visual evidence” for previous inferences but also solved
long-standing questions that had previously been difﬁcult or impossible to address by other approaches. Thus, HS-AFM has now
become holding an indisputable position as a new imaging tool inbiological science.
Nevertheless, the current HS-AFM that adopts the sample
stage-scanning mode has limitations in some regards. To achieve
the high 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 in diameterand 2 mm in height) attached to the Z-scanner has to be used [13].
This small size restricts the range of specimens to be placed on thesample stage. To remove this restriction, HS-AFM should adopt the
tip-scanning mode, in which the cantilever chip is scanned in thethree dimensions. Tip-scanning HS-AFM has another advantage

183

that optical microscopy techniques can easily be incorporated tothe system, including ﬂuorescence microscopy techniques such asa total internal reﬂection ﬂuorescence microscopy (TIRFM) technique. AFM that visualizes the surface structure of objects lacks
the capability of identifying the target molecules when the molecules have no distinct structural features or exist in a crowded
system containing multiple species, whereas ﬂuorescence microscopy that detects ﬂuorescence emitted from ﬂuorescent dye-labeled molecules can identify the labeled molecules but can neither
reveal their structures nor detect the surrounding non-labeledmolecules and environment. As such, the combination of the two
microscopy techniques would redeem their weaknesses. Anotherrestriction of the current HS-AFM system is that the maximum
scan range is limited approximately to 2  4 μm2, which does notallow us to observe dynamic events occurring in large objects suchas live bacterial and mammalian cells. To remove this restriction,
we need to develop a wide-area scanner as well as an active
damping technique to suppress unwanted vibrations that are easily generated by fast scanning of such a large scanner having low
resonant frequencies.
To make HS-AFM more versatile for the use in more biologicalapplications, these new technical developments are essential. In
this review, we describe recent technical advances that have successfully removed the restrictions mentioned above [31,32]. Applications studies of this state-of-art HS-AFM have not yet been
explored as these advances were made very recently. However, we
also describe biological application studies and show their resultsthat have demonstrated the new capability, functionality and potential of the state-of-art HS-AFM.

2. Tip-scanning HS-AFM combined with TIRFM
Fluorescence microscopy is a proven technique to visualize
biological specimens over a wide range from single molecules tocells and tissues [33–38]. The most recent advance in super resolution ﬂuorescence microscopy has made it possible to resolve,
beyond the diffraction limit of light, the positions of individual
ﬂuorophores that are closely located (less than  20 nm apart) inbiological structures [39]. Fluorescence microscopy can detect
small molecules like ﬂuorescently labeled nucleotides, whereas
AFM cannot do so. Furthermore, it is even possible in ﬂuorescencemicroscopy to identify two or three species of molecules in asample containing multiple components by speciﬁc labeling of themolecules with differently colored ﬂuorescent dyes or by theconstruction of ﬂuorescent proteins with different colors [40,41].
However, in ﬂuorescence microscopy the structure of labeledmolecules themselves cannot be revealed even with super resolution since it only detects the ﬂuorescent light emitted from thelabeled molecules. Therefore, AFM and ﬂuorescence microscopy
are mutually complementary and hence their combined system
would be a valuable, versatile tool in biological studies. Thus far,various developments of AFM systems combined with ﬂuorescence microscopy have been reported [42–45]. Such a combined
system is also developed for HS-AFM [46,47]. However, in previous
combined systems, topographic AFM and single-molecule ﬂuorescence images cannot be captured simultaneously at high spatiotemporal resolution.
2.1. Laser tracking method
The major technical challenge in developing a HS-AFM system
combined with an optical microscope is that the tip-scanningmode has to be adopted, instead of the sample-scanning modethat has always been used for HS-AFM [3,5]. In the ﬁrst place, thisis because the sample stage must be stationary. Otherwise, the184

T. Uchihashi et al. / Ultramicroscopy 160 (2016) 182–196

optical image oscillates during HS-AFM imaging. Secondly, this isbecause the sample-stage scanner is incompatible with the objective lens for optical microscopy when the combined system isdesigned so that the cantilever does not interfere with opticalimaging. In tip-scanning AFM, the OBD detector has to scan its
laser beam to have the focused spot always on a given positon
within the cantilever under scan. As a matter of course, the OBD
detector itself can be scanned in synchrony with XY scanning of
the cantilever but such a heavy system cannot be scanned fast.
Mechanisms for this laser beam tracking have been designed andinstalled in conventional slow AFM systems, where the laser diodeand focusing lens are integrated into a piezo-tube scanner towhich the cantilever chip is ﬁxed [48], or mirrors and a focusinglens are integrated to the cantilever chip-scanner [49]. However,these designs cannot be used for HS-AFM. The piezo-tube scanner
is impossible to be used for HS-AFM; when its size is minimized toachieve high resonant frequencies, signiﬁcant cross-talk between
the three scan axes occurs. Also, multiple mirrors cannot be
equipped in a small-sized fast scanner. A tip-scanning HS-AFM
system is also reported that uses a focusing lens embedded in thefast XY-scanner to scan the laser beam in the X- and Y-directions[46]. The small single lens used therein has a high numericalaperture (NA) and a short working distance, and therefore, canfocus the laser beam to a small spot (2–4 μm in diameter), comparable to the width of small cantilevers (  2 μm). However, when
the Z-scanner is scanned more than  150 nm, the laser beam iscertainly defocused on the cantilever, resulting in a larger spot sizeand hence a signiﬁcant error in the amplitude detection.
Considering these issues, we adopted a two-dimensional

dichroic mirror

mirror tilter to scan the laser beam from the OBD detector in theX- and Y-directions [31]. The mirror is a dichroic type with 4 90%
transmission at 400–690 nm and 495% reﬂection at 700–
1150 nm. It reﬂects the incident laser beam of wavelength
4700 nm and guides the reﬂected laser beam onto an objective
lens with a long working distance, making a small focused spot (2–3 μm in diameter) on the small cantilever. The two directional
angles of the normal line to the mirror plane are changed insynchrony with XY-scanning of the cantilever, as depicted in Fig. 1(a). In addition to the dichroic mirror, the mirror tilter is composedof two base plates and four piezo actuators with the maximum
displacement of  3 μm at 100 V, as shown in Fig. 1(b) and (c). The
dichroic mirror is supported by the two X-piezoactuators glued on
the base plate 2 through a beveled block. This whole unit is further
supported by the two Y-piezoactuators glued on the base plate 1.
The respective pairs of piezoactuators are displaced in the opposite directions, by which the respective tilt angles of the dichroic
mirror are scanned. The maximum change of the tile angle wasestimated to be approximately 74.8  10  4 rad for X-directions.
This angle change corresponds to the maximum lateral displacements of the focused spot, approximately 19 μm, considering the
focal length of the objective lens, 20 mm. The actual maximum
displacement of the focused spot measured using a CCD camera
was about 20 μm, in good agreement with the estimation.
The laser beam reﬂected from the cantilever is collimated by
the objective lens, reﬂected by the dichroic mirror, and then guided onto the two-segmented Si PIN photodiode sensor throughthe λ/4 plate, polarization beam splitter and the focusing lens (seeFig. 3). Therefore, the incident position of the reﬂected laser on thedichroic mirror

beveled blocklaserbase plate 2

X’ piezo

X piezo
objective lensY piezobase plate1

Diriving Signal (a.u.)

Diriving Signal (a.u.)cantilever

3.0
2.0
1.0
0.0

Tile Angle (a.u.)

Tile Angle (a.u.)

30
20
10
0
0

1

2
Time (ms)

3

4

3.0
2.0
1.0
0.0
20
15
10
5
0

0

1

2

3

4

Time (ms)Fig. 1. Dichroic mirror tilter for laser-beam tracking of lateral cantilever motion and its vibration damping. (a) Schematic showing the working principle of laser-beamtracking by mirror-tilting method. (b), (c) Perspective (b) and side (c) views of the two-dimensional mirror tilter. (d) 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).

T. Uchihashi et al. / Ultramicroscopy 160 (2016) 182–196

photodiode sensor is altered by scanning of the mirror tilter, andtherefore, the DC component of the differential signal that is
output from the photodiode sensor is also altered. However, its AC
component corresponding to the cantilever oscillation amplitude
is unaltered. In tapping-mode HS-AFM only the AC component isused for imaging, and therefore, this alteration of the DC component does not affect HS-AFM imaging.
The mirror tilter is relatively heavy and therefore has low resonant frequencies that would easily be excited by its fast scanning. When the X-tilter was scanned at various frequencies, thefrequency response of the tilting motion showed the ﬁrst resonantpeak around 2.2 kHz, which was followed by a few peaks at higher
frequencies. These resonant vibrations distort and delay the mechanical response of the X-tilter as shown in Fig. 1(d). This problem was solved by an inversion-based feed-forward damping[50]; an isosceles triangle wave as an original driving signal was
digitally ﬁltered, as follows. The isosceles triangle wave is characterized by amplitude X0 and fundamental angular frequency ω0.
The Fourier transform of its series of waves is given by
+∞
⎡1
⎤
2
1
F (ω) = 2πX 0 ⎢ δ (ω) − 2 ∑ 2 δ ( ω − kω 0 ) ⎥ (k: odd).
⎢⎣ 2⎥⎦
π k =−∞ k(1)

The compensated signal DX(t) to drive the X-tilter that isθωcharacterized by the transfer function G(iω)¼g(ω)  ei ( ) (corresponding to the frequency response; g, gain; θ, phase) is given by
the inverse Fourier transform of F(ω)/G(iω), which is expressed as

DX (t ) =+∞⎤1 ⎡⎢ X 0
4X
1
1
×− 20 ∑ 2cos ( kω 0 t ) ⎥ (k: odd),⎥⎦
α ⎢⎣ 2
ωGik
π k=1 k (0)(2)

where α (nm/V) is the extension coefﬁcient of the piezoactuators.
Practically, the sum of the ﬁrst 10 terms in the series of Eq. (2) is
sufﬁcient. When driven by this compensated signal of 1 kHz
shown with the black line in Fig. 1(e), the X-tilter moves smoothly
in an isosceles triangle wave, as shown by the red line in Fig. 1(e).
This driving frequency corresponds to an imaging rate of 10
frames/s (fps) for 100 scan lines in the Y-direction. The mechanicalresponses of the Y-tilter also showed the ﬁrst resonant peakaround 2.2 kHz and several larger peaks at higher frequencies. The
Y-tilter is driven by a saw-tooth wave containing a precipitous
downward regime for the tip being returned to the scan origin,which generates signiﬁcant vibrations. To avoid fatal vibration
generation, the Y-tilter scan was slowed down only in the downward regime. The Y-tilter moved smoothly by this driving. This
altered driving only slightly (  5%) lowers the imaging rate.
The performance of the laser-beam tracking system was assessed by monitoring the cantilever oscillation amplitude when
the oscillating cantilever was scanned laterally over a
650  650 nm2 area without feedback scan in the Z-direction,
while the tip was not in contact with the substrate surface. Fig. 2(a) shows an amplitude image acquired at 1 fps when the laser
beam tracking system was switched off. The signiﬁcant image
variation seen in Fig. 2(a) indicates that the optical lever sensitivity
varies due to positional shift of the cantilever relative to the stationary laser spot focused on it. The cross-section proﬁle along the
red line in the top of Fig. 2(a) indicates an apparent amplitudechange of  1.4 nmp–p, as shown in the bottom of Fig. 2(a). Whenthe laser-beam tracking system was switched on, this large error
almost disappeared as shown in Fig. 2(b). The cross-section along
the red line shown in the top of Fig. 2(b) shows an amplitude
variation of only  0.2 nm, as shown in the bottom of Fig. 2(b). It

2.4

Amplitude (nm p-p)

2.4

Amplitude (nm p-p)

185

2.2
2.0
1.8
1.6
1.4
1.2

2.2
2.0
1.8
1.6
1.4
1.2
1.0

1.0
0

200

400

600

Distance (nm)

800

200

400

600

800

Distance (nm)Fig. 2. Effect of laser-beam tracking of lateral cantilever motion on the OBD detection of cantilever oscillation amplitude. (a) Amplitude image captured at 1 fps for650  650 nm2 (256  256 pixels) without laser-beam tracking. 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 proﬁle of the image along the diagonal red line. For these imaging
experiments, the cantilever tip is not in contact with the substrate surface.

186

T. Uchihashi et al. / Ultramicroscopy 160 (2016) 182–196

two-segmented
photodiodeCCD camera
wave plate
mirror tilter

ZX

focusing lensY

IR lasercollimationlens
x20 objective
lens,polarization
beam splitterscannercantilever,

scample stage
large scanner

TIRFM

Fig. 3. Schematics and photograph showing the structure of tip-scan HS-AFM unit. (a) Conﬁguration of OBD detector with dichroic mirror tilter installed in the HS-AFM unit.
(b) Whole structure of the tip-scan HS-AFM unit. The numbers attached to respective parts correspond to the numbers shown in (a). (c) A photograph of the tip-scan HS-AFM
unit mounted on the optical stage in which the wide-area XY-scanner is installed.

was also conﬁrmed that the tracking system worked well even forfaster imaging rates up to 10 fps.
2.2. Combined Tip-scanning HS-AFM/TIRFM
Fig. 3(a) shows a schematic diagram of the OBD system for tipscanning HS-AFM. An infrared (IR) laser of wavelength 980 nm isused for the OBD system instead of a red laser used in the standard
HS-AFM system [51], because ﬂuorescent dyes frequently used in
ﬂuorescence imaging emit ﬂuorescence shorter than 800 nm. The
laser spot on the small cantilever can be monitored by the CCD
camera through the objective lens and the dichroic mirror, whichfacilitates the alignment of the laser beam and the cantilever. The
cantilever deﬂection is detected by the position-detection system
consisting of the two-segmented Si PIN photodiode, ampliﬁers,
and a signal conditioner with the bandwidth of 20 MHz. Other
optical components are optimized for the IR wavelength, including
the collimation lens, λ/4 plate, polarization beam splitter, and thefocusing lens placed at the front of the two-segmented PIN

photodiode. A smaller spot size of the focused laser on the photodiode has a higher position-detection sensitivity for a given
displacement of the laser spot. On the contrary, focusing into asmall spot reduces the displacement of the laser spot caused bycantilever deﬂection. Therefore, when the gap size (Δs) between
the photodiode segments is not considered, the spot size (D) has
no effect on the sensitivity of cantilever deﬂection detection as far
as the laser spot is within the effective area of the photodiode.
However, taking the gap size into an account, the sensitivity tocantilever deﬂection increases with decreasing spot size by a factor of Δs/D. Nonetheless, the laser spot has to be set at an appropriate size so that centering the laser spot on the photodiodesensor is easily performed. When the spot size is very small, thiscentering adjustment becomes difﬁcult. Considering these factors,
we adjust the spot size in the range of 0.5–1 mm in diameter withthe focusing lens.
Fig. 3(b) shows the side view of the tip-scanning HS-AFM unit.
The frame that supports the unit consists of the top and base parts.
All components in the OBD detector including the mirror tilter are

T. Uchihashi et al. / Ultramicroscopy 160 (2016) 182–196

assembled in the top frame and hung from its roof to ensure
mechanical stability. For coarse approach of the cantilever tip to
the sample surface, a stepper motor is installed in the base part.
The positions of the IR laser, the two-segmented photodiode, andthe scanner with the cantilever can be adjusted with precise
screws in the XZ-, YZ- and XY-planes, respectively. The wholeassembly is supported by a tripod consisting of two adjustablescrews and a screw connected to the revolving shaft of the stepper
motor.
As optical microscopy to be combined with the tip-scanning
HS-AFM, objective-lens type TIRFM was employed for single-molecule ﬂuorescence imaging. The tip-scanning HS-AFM unit issimply mounted on a modiﬁed stage of the inverted optical microscope as shown in Fig. 3(c). The stage of the inverted ﬂuorescence microscope (hereafter referred to “optical stage”) was
modiﬁed to hang the objective lens directly from the optical stage,as was done for our ﬁrst prototypic HS-AFM setup combined with
an optical microscope [3]. This hanging was necessary for thefollowing reason. An oil-immersion objective lens with a high NA
has to be used for TIRFM and therefore the front face of the objective lens is in contact with a cover slip through immersion oil.
In the standard conﬁguration of inverted optical microscopes, theoptical stage and the objective lens are mechanically separated;i.e., the objective lens is ﬁxed in the revolver, while the opticalstage is ﬁxed on the top of the supporting frame. In this conﬁguration, the objective lens and the optical stage vibrate nearly
independently of each other, and therefore, vibrations of the large
microscope body are easily transmitted to the cover slip mounted

187

on the optical stage through the objective lens [52]. When theobjective lens (100  , NA 1.49) is hung directly from the opticalstage, they vibrate together in phase, and hence, the vibrationtransmission is signiﬁcantly suppressed.
Another modiﬁcation of the optical stage was needed for precise assessment of the correlation between AFM and ﬂuorescence
images for the same sample. The ﬁeld of view of our TIRFM setup
is approximately 31  31 μm2. On the other hand, the lateral scan
range of the tip-scanning HS-AFM system is currently limited to800  800 nm2. This large difference of the ﬁeld of views complicates the correlation assessment. To ﬁll this large gap, an additional scanner that moves the whole tip-scanning HS-AFM unit up
to 15 μm in the X- and Y-directions was implemented in the optical stage. For wide-area AFM imaging, this XY-scanner was used
together with the Z-scanner of the tip-scanning HS-AFM system.
Although its imaging rate is much lower (4 3 min/frame) thantip-scanning HS-AFM, the wide-area scanner enables precise positioning of the cantilever tip on a region of interest within a preacquired TIRFM image.
2.3. Simultaneous HS-AFM/TIRFM imaging
Fig. 4(a) shows a TIRFM image of rhodamine-labeled actin ﬁlaments that are partially biotinylated and immobilized on a coverslip sparsely covered with avidin molecules, while Fig. 4(c) and(d) show HS-AFM images of the same sample captured for narrowareas located within the region shown with the rectangle in Fig. 4(a). It seems impossible to correlate the HS-AFM images with the

500 nm

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(d)

40 nm

40 nm

Fig. 4. Positional correlation between TIRFM, AFM, and HS-AFM images of partially biotinylated and fully rhodamine-labeled actin ﬁlaments placed on a sparsely avidinecoated cover slip. (a) TIRFM image. The white rectangle indicates the region for which (b) a wide-area AFM image was taken at 10 min/frame. (c) HS-AFM image captured at
an imaging rate of 8.3 fps with 100  45 pixels. The position of cantilever tip was set by reference to the TIRFM image. (d) HS-AFM image captured at an imaging rate of 2 fps
with 100  100 pixels. The position of cantilever tip was set by reference to the TIRFM image. (e) The overlaid TERM and AFM image. The imaged areas of (c) and (d) are thoseshown by the respective white rectangles.

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0s0.99 s1.98 s2.64 sHS-AFM
50 nm

TIRFM
500 nmFig. 5. Simultaneous HS-AFM/TIRFM imaging of proteins in action. (a) Cy3-labeled chitinase A on a chitin microﬁbril. The HS-AFM images (upper panel) captured at 3 fps
with 150  35 pixels show a single chitinase A molecule moving towards the top-right (arrows). The TIRFM images (lower panel) with 40  15 pixels also show the samemoving chitinase A molecule (arrows).

TIRFM image due to the crowded actin ﬁlaments and the poor
resolution of the TIRFM image. Fig. 4(b) shows a wide-area AFM
image acquired as described above. By comparing the TRIFM andwide-area AFM images, we can easily ﬁnd the positional correlation between the TIRFM image and the HS-AFM images captured
for the narrow areas, as indicated with the small rectangles in theoverlaid image between the TIRFM image and the wide-area AFM
image, as shown in Fig. 4(e).
Chitinase A is an enzyme that degrades chitin microﬁbrils byhydrolysis and has been hypothesized to move processively andunidirectionally along the microﬁbrils [53]. This hypothesis wasrecently proven by HS-AFM imaging [22]. For ﬂuorescence imaging, chitinase A was labeled with Cy3. Crystalline chitin microﬁbrils suspended in water were immobilized onto a glass coverslip by spin coating. Fig. 5 shows successive images of chitinase A
moving along a chitin microﬁbril, which were simultaneously
captured by HS-AFM and TIRFM at an imaging rate of 3 fps. The
HS-AFM images (upper panel in Fig. 5) show binding of a single
chitinase A molecule to the chitin microﬁbril (0.99 s) and its
afterwards movement on the microﬁbril towards the upper-right(1.98 s and 2.64 s). A ﬂuorescent spot also appeared at 0.99 s in the
TIRFM image (lower panel in Fig. 5). The ﬂuorescent spot slightly
moved with time toward the right side, although the movement ismuch less discernible, because the ﬁeld of view in TIRFM is muchwider than that in HS-AFM.
2.4. Potential of Tip-scanning HS-AFM
In addition to imaging, AFM has also been used as a force
sensor to measure the strength of intra- and intermolecular bonds
at the single molecule level and the elasticity of biological objects[54–56]. It is of note that the speed of stretching biological molecules is now approaching to that of molecular dynamics simulations [56]. Along with this progress, theoretical descriptions forbond rupture under a wide range of loading rates have recently
been attempted [57]. The force measurements produce so-called
the force-distance curves, only from which useful information has
to be extracted. But the interpretation of the obtained curves is
difﬁcult unless the system under measurement is not well speciﬁed. Similarly, optical tweezers has been used in force and displacement measurements for single molecules and cells [58–60].
In the force measurements by these techniques, the objects undermeasurement cannot be visualized by AFM. It would be a breakthrough if we can visualize the objects under external force using
HS-AFM. Tip-scanning HS-AFM can accommodate optical tweezers
when it is combined with an optical microscope. Therefore, tipscanning HS-AFM has a potential to visualize biological molecules
under external force. It would provide a new opportunity to study
the effect of applied force on the functional action of the molecules

as well as detailed unfolding and refolding processes of themolecules.
Tip-enhanced ﬂuorescence microscopy operates based on thesigniﬁcant enhancement of electric ﬁeld of light in the vicinity of ametal or silicon tip that is illuminated with a laser beam [61]. It
has resolution less than 10 nm [62]. However, this microscopy
technique has not been of practical use, whereas other super-resolution ﬂuorescence microscopy techniques are now being disseminated [39,63,64]. Tip-enhanced ﬂuorescence microscopy canbe combined with tip-scanning HS-AFM. When combined, both
topological and ﬂuorescence images with identical pixel positions
would be acquired simultaneously at high spatiotemporal
resolution.

3. Wide-area scanner for live cell imaging
One of prospective applications of HS-AFM in biological scienceis high-resolution visualization of dynamic events occurring in live
cells, such as endocytosis, exocytosis, and reorganization of cytoskeletons which occur autonomically or in response to external
stimuli including mechanical and electrical stimuli, ligands forexternal membrane receptors and toxic substances.
Several techniques for fast, wide-area AFM imaging have been
proposed [65–69], and this type of imaging has also been demonstrated for hard (or relatively hard) materials such as Si
gratings [67–69] and some biological samples including driedcollagen ﬁbers [65], collagen ﬁbers on bone trabecula [70] and live
bacterial cells [66]. However, wide-area AFM imaging is difﬁcult
for soft biological samples such as live eukaryotic cells. To observeboth bacterial and eukaryotic cells, we have developed a wide-areascanner with the maximum scan range of  46  46 μm2 [32]. To
achieve precise, fast imaging using this scanner, we have also
developed several control techniques for damping mechanical vibrations, expanding the frequency bandwidth, minimizing thenonlinear displacement of piezoactuators and the cross-talk between the X- and Y-displacements. These techniques are essential
for a wide-area HS-AFM system but not necessarily requirsed of
the standard HS-AFM. In this section, we present the overviews of
the design of the wide-area scanner and these control techniques,and ﬁnally describe application studies of wide-area HS-AFM toshow its ability and potential.
3.1. Design of wide-area scanner
Large displacements of a scanner in the X- and Y-directions canbe achieved by the magniﬁcation of X- and Y-piezoactuators' displacements. For this magniﬁcation, we used the third-class leverage mechanism. Fig. 6(a) shows a schematic of the wide-area

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Zpiezo189

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YpiezoXpiezo10mm
Fig. 6. Design of wide-area scanner. (a) Structure of a newly designed wide-area scanner for sample stage-scanning high-speed AFM. (b) A photograph of the wide-areascanner.

scanner. Its entire structure except the piezoactuators was madeby monolithic fabrication of a stainless steel block. The X- andY-piezoactuators are arranged symmetrically with respect to thecentral supporting base on which the Z-piezoactuator is attached.
One end of the lever with a length of 25 mm is ﬁxed to the frame,
which works as a fulcrum. The point of effort, to which a longstroke piezoactuator with a nominal unloaded displacement of
11.6 μm at 100 V and the ﬁrst resonant frequency of 69 kHz is
glued, is 5 mm distant from the fulcrum, i.e., the designed leverratio is ﬁve. This design achieved the actual maximum scan rangesof 46.7 μm for the X-direction and 45.7 μm for the Y-direction.
Fig. 6(b) shows a photograph of the fully assembled scanner. The
gap spaces in the scanner were ﬁlled with an elastomer consistingof polyurethane, which can passively damp low-frequency vibrations of the scanner.
3.2. Control techniques for wide-area scanner
3.2.1. Vibration damping
For scanning in the X-direction, a voltage signal with an isosceles triangle wave is usually applied to the X-scanner. However,high harmonic-frequency components are contained in the vertexes of the triangle wave, which easily excite the X-scanner withlow resonant frequencies, resulting in generation of unwanted
vibrations. The wide-area X-scanner developed above has the ﬁrst
resonant frequency around 2 kHz as shown in Fig. 7(a). The
X-scanner generated serious vibrations (red line) as shown in
Fig. 7(b), even though it was scanned by a triangle wave with thefundamental frequency of only 250 Hz (black line).
Instead of raster scanning, different two-dimensional scan
trajectories, such as sinusoidal [67], spiral [71], cycloid [72] andLissajous [73] curves, have been proposed. This is because in thesecases the driving signals for the XY-scanner do not contain highharmonic components, and therefore, can prevent generation of
unwanted vibrations. However, the procedures for producing thedriving signals, reconstructing the image, and compensating forhysteresis effects of piezoactuators are complicated. Moreover,these scan trajectories would result in the exertion of lateral forces
to the cantilever tip from various directions, and consequently thecantilever would be not only bent but also twisted by the tipsample interaction. Therefore, the interaction force is hardly
maintained constant. Here we used two methods and their combination to suppress the generation of unwanted vibrations; theinversion-based feed-forward damping [69] that is the same asthat described for the mirror tilter [31], simple rounding of thevertexes of the triangle wave [74] and combination of thesemethods. Fig. 7(c)shows the driving signal (black line) constructedby the inverse compensation method and the corresponding displacement of the X-scanner (red line). The construction of thedriving signal was made using the frequency response shown in
Fig. 7(a). The mechanical vibrations of the X scanner were signiﬁcantly suppressed by this method as seen in Fig. 7(c).
The rounding of the vertexes of the triangle wave is simple anduseful. There are several ways to generate rounded triangle waves
with reduced higher harmonics. Rather than using low-pass ﬁlters,
we generated a series of rounded triangle waves by removing
higher harmonics terms from the Fourier series given in Eq. (1)and then calculating the inverse Fourier transform of the resulting
Fourier series. Fig. 7(d) shows the rounded triangle wave constructed from harmonics up to the ninth order (black line) and thecorresponding displacement of the X-scanner (red line). The displacement did not show noticeable vibrations. The nonlinear scanregions that only appear around the vertexes of the scan wave areabout 10% of the full scan range, which would produce a nearly
negligible effect on AFM images to be acquired.
However, even with these methods, the X-scanner generated
unwanted vibrations when scanned at 1 kHz. Then, we combined
these methods. That is, the rounded triangle wave containing
harmonics up to the ninth-order was ﬁltered through the inversed
transfer function obtained from the measured frequency response.
As shown in Fig. 7(e), the X-scanner was successfully driven at
1 kHz, without generating unwanted vibrations and without asigniﬁcant deviation from linear trajectories in both trace and retrace regimes. Thus, the bandwidth of the X-scanner with the ﬁrst
resonant frequency of only 2 kHz was signiﬁcantly extended.
3.2.2. Compensation for image distortion
The hysteresis nonlinearity of piezoactuators is not negligible
for the case of wide-area AFM imaging. The positioning error due
to the hysteresis is generally 10–15% of the full scan range, causing
a signiﬁcant distortion in acquired AFM images [75]. To compensate for the nonlinearity, the closed-loop control that is based on
the actual displacements recorded by a displacement sensor isoften used in commercial AFM systems. However, this control
makes the scanner assembly complicated by the implementationof the displacement sensors. Moreover, the bandwidth of available
high-precision displacement sensors is too low to be used in thehigh-speed scanner. The open-loop method that is based on

T. Uchihashi et al. / Ultramicroscopy 160 (2016) 182–196

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Time (ms)Fig. 7. Vibration damping of the wide-area X-scanner. (a) Frequency spectra of mechanical response of the X-scanner (red line, amplitude; blue line, phase). (b) 256 Hz
driving signal of non-modiﬁed triangular wave (black line) and corresponding displacement of the X-scanner (red line). (c) 256 Hz driving signal of triangle wave modiﬁedby the inversed compensation method (black line) and corresponding displacement of the X-scanner (red line). (d) 85 Hz driving signal of rounded triangle wave containing
harmonics up to the 9the order (black line) and corresponding displacement of the X-scanner. (e) 1 kHz driving signal of triangle wave treated by both vertex rounding andinverse compensation (black line) and corresponding displacement of the X-scanner (red line).

feedforward compensation for the nonlinearity is rather simple[76]. For our wide-area scanner, we adopted the open-loop compensation method based on the pre-measured non-linear hysteresis curves of the X- and Y-scanners.
The nonlinearity of piezoactuators is independent of the displacement range. Therefore, we can construct a modiﬁed driving
signal that can compensate for the nonlinearity, using the following procedures. The numerical data of the measured hysteresis
curve of the displacement d as a function of the applied voltage V
is ﬁrst normalized by a given voltage V0 and the corresponding
displacement d0. From this normalized curve d¯ = d¯ (V¯ ), we obtain acurve of V¯ = V¯ (d¯ ), where d¯ ≡ d/d0 and V¯ ≡ V /V0 . This curve is ﬁtted
with a polynomial function. This polynomial function is used to
calculate the driving signal that displaces the scanner up to adesired range. Fig. 8(a) shows the HS-AFM image of a squaregrating with a pitch of 10 μm captured at 7 s/frame for a
33  33 μm2 area, without compensation for the nonlinearity. The
image shows grating pitches with large variations of approximately 7 20% and 715% in the X- and the Y-directions, respectively. The use of the open-loop feedforward compensation
method eliminated the nonlinearities, as shown in Fig. 8(b). The
variations of the grating pitch are improved to be less than 71%.
However, the image of the square grating is still distorted andappears rhombic, as shown in Fig. 8(c). This is due to displacement
cross-talk (coupling) between the X- and Y-scanners. This crosstalk results from the fact that the free ends of the levers used in
the displacement magniﬁcation mechanism cannot move straight.
The coupling ratio was measured to be independent of the scan
range. The ratio was 0.017 for the passive movement of the sample
stage in the Y-direction when the X-scanner was displaced, while

T. Uchihashi et al. / Ultramicroscopy 160 (2016) 182–196

8 μm

3 μm

191

8 μm

3 μm

Fig. 8. Compensation for nonlinearity of piezoactuators and cross-talk between X- and Y-scanners. (a) AFM image of a square-shaped grating sample with a pitch of 10 μm
obtained without compensation for nonlinearity (imaging rate, 7 s/frame; pixels, 256  256). (b) AFM image of the same grating sample obtained by compensation for
nonlinearity (imaging rate, 7 s/frame; pixels, 256  256). (c) Enlarged AFM image of the square-shaped grating sample obtained without compensation for cross-talkbetween X- and Y-scanners (imaging rate 7 s/frame, pixels, 256  256). (e) Enlarged AFM image of the square-shaped grating sample obtained with compensation for crosstalk between X- and Y-scanners (imaging rate, 7 s/frame; pixels, 256  256).

it was 0.023 for the passive movement of the sample stage in the
X-direction when the Y-scanner was displaced. To eliminate thiscoupling, an appropriate fraction of the voltage applied to one
piezoactuator (X- or Y-piezoactuator) was subtracted from thedriving signal for the other piezoactuator. This simple method
gave a nearly distortion-free image, as shown in Fig. 8(d).
3.2.3. Z-scanner
The Z-piezoactuator is one of the key devices that determine
the feedback bandwidth [10,13]. Its resonant frequency should behigh for HS-AFM but is restricted by the required maximum scan
range. In general, the resonant frequency of a piezoactuatorchanges in inverse proportion to its maximum stroke. This conﬂict
relationship has to be compromised especially for the observationof large-sized specimens. For the Z-scanner of the wide-areascanner, we chose a stack piezoactuator with a displacement of
2.5 μm at 100 V and the ﬁrst resonant frequency of 222 kHz in
free oscillation. This piezoactuator was glued on the top of thecentral supporting base to be moved in the X- and Y-directions.
The same type of piezoactuator (Z'-piezoactuator) was also glued
to the bottom of the supporting base to counteract the impulsive
force exerted to the supporting base by quick displacement of theZ-piezoactuator [3,10]. Moreover, the mechanical resonances ofboth Z- and Z’-piezoactuators were suppressed by an active
damping method based on the quality factor control techniquethat uses an LRC circuit, as a mock scanner, having a frequency

response similar to those of the Z- and Z’-piezoactuators attachedto the supporting base [10].
3.3. Cantilever tip for cell imaging
The small cantilevers (BL-AC7DS-KU2 and BL-AC10DS-A2,
Olympus, Tokyo) that we have been being used for HS-AFM have abird beak-like tip end. But the tip is not sharp enough to gain highresolution images. To have a stylus-shaped sharp tip, we deposit
amorphous carbon on the very end of the original tip by electron
beam deposition using the spot mode of scanning electron microscopy (SEM), and then sharpen the grown tip by RF-plasma
etching under argon environment. The tip end radius results in 3–
5 nm. The length of the carbon tip used for imaging of biologicalmolecules is usually  1 μm. Since the growth rate of carbon tip isapproximately 600 nm/min, the focused electron beam is usually
irradiated for 1–2 min. However, the tip with length of 1 μm is
unsuited for the observation of live cells of higher than a few μm
as the cantilever itself would directly contact with the cell surface.
To obtain a longer tip, the electron beam deposition for 1 min was
repeated 5–7 times [77]. For each time of deposition, the focus
position of the electron beam was readjusted to compensate formechanical drift of the SEM. This procedure produces a tip withlength of 3 μm. Its treatment by RF-plasma etching was brieﬂy
performed (for  10 s) to prevent shortening of the tip, resulting in
the tip end radius of 5–8 nm.

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z

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FilterSplit PIN Photodiode

Half Mirror
CCD

Fig. 9. Simple ﬂuorescence microscopy system installed in the sample stage-scanning HS-AFM setup. (a) A schematic illustration of the system. An arc lamp for ﬂuorescence
excitation and a CCD camera to capture bright-ﬁeld and ﬂuorescence images are installed in the bottom of the HS-AFM system. (b) An overlaid image of cantilever andﬂuorescence images. The white dotted line displays the outline of the cantilever's shadow. The white square with the arrow indicates the area size that the wide-area scanner
can cover (  40  40 mm2).

3.4. Positioning of tip on cell surface
The maximum scan range of the wide-area scanner,
46  46 μm2, is usually still too small to observe a whole eukaryote cell. A central region of a eukaryotic cell without underlying stiff structures is easily deformed by the loading force fromthe cantilever tip and therefore hard to be imaged. Therefore, we
needed to position the cantilever tip onto ﬂat peripheral regions ofa cell supported by the stiff substrate. In our HS-AFM setup, thesmall cantilever can be monitored using a CCD camera, allowing us
to align the positions of the cantilever, the sample stage and thelaser spot focused onto the cantilever. However, the cells on thesample stage cannot be observed by this optical system, andtherefore, tip positioning at a desired location of the cell sample
cannot be conducted. To solve this problem, we added a simple
ﬂuorescent microscopy system to the standard HS-AFM setup, as

0s

shown in Fig. 9(a) [47,77]; the excitation lamp, dichroic ﬁlters andthe CCD camera are installed in the bottom part of the HS-AFM
setup. By using this system and cells transfected with green
ﬂuorescent protein (GFP), the cantilever tip can be positioned at anarbitrary region of the cell sample (Fig. 9(b)).
3.5. Live cell imaging
First, the wide-area scanner was used for the observation of thebacteriolysis process of rod-shaped Bacillus (B.) subtilis subjected
to lysozyme. B. subtilis is susceptible to antibacterial action of lysozyme that hydrolyzes 1,4-beta-linkages between N-acetyl glucosamine and N-acetyl muramic acid, leading to degradation of
peptidoglycan on the cell wall [78]. We monitored morphologicalchanges of the cell surface caused by this degradation to demonstrate the capability of wide-area HS-AFM [32]. The bacterial cells

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Fig. 10. HS-AFM images of B. subtilis captured using the wide-area scanner. (a) Topographic image of B. subtilis in a culture medium solution before the addition of lysozyme(imaging rate, 15 s/frame, pixels, 200  200). (b) Successive HS-AFM images showing bacteriolysis process of B. subtilis subjected to lysozyme. Lysozyme was injected at
t¼ 240 s (imaging rate, 20 s/frame; pixels, 256  256).

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200 nmFig. 11. HS-AFM images of live COS-7 cells in response to insulin application. (a) Fluorescence image of a COS-7 cell transfected with mEGFP. The white dotted line indicatesthe outline of the cantilever's shadow. The white square indicates the area for HS-AFM imaging. (b), (c) HS-AFM topographical images of the same COS-7 cell as shown in (a)(b) before and (c) after the addition of insulin with the ﬁnal concentration of 20 mg/ml into the observation solution (imaging rate, 10 s/frame; pixels, 200  200). The cell was
starved with serum-free medium for  1hour before the experiments. (d), (e) HS-AFM images taken at the indicated times (d) before and (e) after the insulin addition. These
images are overlaid with the corresponding images at 0 s. The areas shown in green and magenta indicate newly appeared and disappeared structures in the entire imaged
region, respectively, while the ﬁlled and open white arrows indicate newly appeared and disappeared structures at the leading edge of the cell, respectively.

were immobilized on a glass stage coated with poly-L-lysine(0.1 mg/ml) and then imaged in a culture medium (Fig. 10(a)).
Fig. 10 (b) shows clipped images of the bacterium before and after
the addition of lysozyme, captured at 20 s/frame (256  256 pixels). At t¼0 s, the bacterial cell surface appeared smooth. At
t¼ 240 s, lysozyme was added to the observation solution at theﬁnal concentration of 80 μM. After t¼ 320 s, the surface morphology gradually varied and wrinkle structures perpendicular tothe long cell axis appeared, while the whole cell gradually swelled(t ¼1060 s, 1460 s and 1580 s). Finally, at t¼ 1720 s, the cell burst
presumably due to an excessive osmotic pressure from the insideof the cell.
Next, we observed eukaryotic COS-7 and Hela cells that aremuch softer than bacterial cells [77]. Here, we only show the results with COS-7. The COS-7 cells were transfected with monomeric enhanced GFP (mEGFP). The cantilever tip was positioned ata peripheral region of one of the cells under the simple ﬂuorescence microscope (Fig. 11 (a)). The membrane dynamics and its
activation induced by insulin were observed in real time. Fig. 11(b) and (c) show topographic images captured for the peripheral
region of the same COS-7 cell before and after the addition of
insulin, respectively. Fig. 11(d) and (e) show HS-AFM images taken
at the indicated times (d) before and (e) after the insulin application. These images are overlaid with the corresponding images
captured at t ¼0 s (i.e., images (b) and (c)). The areas shown ingreen and magenta indicate newly appeared and disappeared
structures in the entire imaged region, respectively, while the ﬁlled and open white arrows indicate newly appeared and disappeared structures at the leading edge of the cell, respectively. It
can be seen that the membrane rufﬂing at the leading edge of thecell is activated in response to insulin application.
We also observed morphological changes of live neurons.
Hippocampal neurons from newborn rats were planted at low
density on a top layer of glia cells placed on a glass stage andcultured for 9 days [79]. Under the ﬂuorescence microscope, theAFM tip was positioned near thin dendrites of the neuron transfected by mEGFP, as shown in Fig. 12(a). As seen in Fig. 12(b), rapid
ﬁlopodia extension and retraction were observed. Furthermore, athin and sheet-like rufﬂing structure on top of glia cells arounddendritic shafts was also observed (data not shown). This structure
would be difﬁcult to image under an optical microscope due to its
small width (150–500 nm) and thickness (  70 nm), which arebeyond the resolution of conventional optical microscopes.

4. Summary
The maturity of HS-AFM has enabled us to directly visualize
various dynamic processes occurring in biological molecules inreal time, and consequently, gain more detailed and deeper

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Fig. 12. HS-AFM images of live hippocampal neurons cultured for 9 days in vitro. (a) Fluorescence image of a hippocampal neuron transfected with mEGFP. The white dottedlines show the outline of the cantilever's shadow. The white square indicates the area for HS-AFM imaging. (b) A sequence of HS-AFM topographical images of a dendrite
captured for the area indicated in (a) (imaging rate, 5 s/frame; pixels, 200  200). The white arrows indicate the growth and retraction of ﬁlopodia. The size of the dendrite is 400 nm in width and  200 nm in height.

insights into their functional mechanisms. Combination of HSAFM with other powerful techniques that can provide complimentary information should make HS-AFM more powerful andversatile in biological science. In this review, we ﬁrst describedvery recent technical developments of tip-scanning HS-AFM thatcan be easily combined with ﬂuorescence microscopy. This combination allows us to acquire the information of both conformational dynamics and chemical states of individual protein molecules. This will open an opportunity to precisely identify conformational changes of a protein molecule triggered by, for example, nucleotide binding, hydrolysis, and product release.
Moreover, the identiﬁcation of molecular species by ﬂuorescencemicroscopy during HS-AFM imaging makes it easier to interpret
AFM images of complex systems consisting of several species ofmolecules. However, for this task, the combined system we developed is not powerful enough, as the spatial resolution differs
largely between the two microscopies. And yet, this will soon be
resolved because in principle the tip-scanning HS-AFM can be
combined with confocal microscopy and super-resolution ﬂuorescence microscopy including stimulated emission depletion(STED) microscopy [39], stochastic optical reconstruction microscopy (STORM) [64], and photo-activated localization microscopy(PALM) [63]. Such ultra-hybrid microscopy systems will bring amajor impact on single-molecule biophysics of isolated biologicalmolecules and a less impact on cell biology as brieﬂy described
below. Optical tweezers can also be incorporated to tip-scanning
HS-AFM, which will open a new opportunity to visualize proteins
molecules under external force. Tip-scanning HS-AFM can also
serve for non-biological or industrial applications, as it can accommodate large and heavy specimens, such as semiconductor
wafers.
The wide-area scanner is also an important technical breakthrough that extends the application rage of HS-AFM from single
molecules to live cells. We described the mechanical design of thewide-area scanner as well as several control techniques to suppress unwanted vibrations, nonlinear displacement of piezoactuators and cross-talk between two lateral scan axes. Now, more
complex systems including live cells and organisms have becometargets of high-speed AFM. Although we have not yet completed,the implementation of the wide-area scanner in the tip-scanning
HS-AFM system combined with an optical microscope will largely
expand the capability and functionality of HS-AFM. As AFM only
visualizes a surface, its use with ﬂuorescence microscopy must be
also useful for studying dynamic molecular events occurring on
the surfaces (not in interiors) of live cells or isolated organelles.
However, its targets are limited to small and hence relatively rigid

T. Uchihashi et al. / Ultramicroscopy 160 (2016) 182–196

structures like neuronal spines, small mitochondria and nuclei.
Nevertheless, in-situ visualization of dynamic molecular eventsoccurring on these higher-order structures will bring impact oncell biology, as dynamic molecular phenomena occurring there aremostly unclear.

Acknowledgments
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 (to T.A.), Grant-in-Aids for Basic Research(#24227005 to T.A., and #23115008, #24241048, #26104514 and#26102515 to T.U.), Grant-in-Aid for Research on Innovative Area(#26119003 to T.A.), Human Frontier Research Program (to T.A.)
and Grant-in-Aid for Research Fellows (#14J05613 to S.F.) from theJapan Society for the Promotion of Science.

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