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High-speed AFM imaging
Toshio Ando1,2,3Proteins are dynamic in nature and function at the single
molecule level. To achieve a straightforward and in-depth
understanding of their underlying functional mechanism, weneed to directly observe protein molecules at work at highresolution, without the use of protein-attached markers. To
realize such objectives, high-speed atomic force microscopy(HS-AFM) has been developed and recently its capability has
been fully established. This approach opens a new avenue to
directly and closely observe individual molecules at
submolecular spatial resolution and sub-100 ms time
resolution. The captured molecular movies of proteins directly
report and provide great insights into how the proteins function.
Moreover, the very recent progress of HS-AFM technology has
extended its use to the observation of dynamic cellular
processes. In this article, I review imaging studies to show theinnovative power and potential of this new microscopy.
Addresses
1
Department of Physics, Kanazawa University, Kakuma-machi,
Kanazawa 920-1192, Japan
2
Bio-AFM Frontier Research Center, Kanazawa University, Kakumamachi, Kanazawa 920-1192, Japan
3
CREST, Japan Science and Technology Agency, 4-1-8 Honcho,
Kawaguchi 332-0012, Japan
Corresponding author: Ando, Toshio (tando@staff.kanazawa-u.ac.jp)

Current Opinion in Structural Biology 2014, 28:63–68
This review comes from a themed issue on Biophysical andmolecular biological methods
Edited by David Millar and Jill Trewhella

http://dx.doi.org/10.1016/j.sbi.2014.07.011
0959-440X/# 2014 Elsevier Ltd. All right reserved.

behavior of optical markers attached to protein molecules,
and hence, the protein molecules themselves are completely invisible, even with superresolution fluorescencemicroscopy bypassing the diffraction limit [4]. Due to thisindirectness of measurement, one has to infer how theprotein molecules are actually behaving behind therecorded data. Thus, there have been no means thatallow the simultaneous recording of structure anddynamics, preventing a straightforward understandingof how proteins function, except for proteins that serve
rather as scaffolds than as functional entities. Under thisadverse situation, a technique capable of observing both
structure and dynamics of single protein molecules haslong been desired, even with lower resolution than thoseof the conventional structural biology methods. To
meet this desire, high-speed atomic force microscopy(HS-AFM) has been developed since 1993 [5,6], which
was put into practical use around 2008 [7]. The imaging
rate is now remarkably increased to higher than 10 frames
per second (fps) and, importantly, the tip force acting on
the biological sample does not disturb its function, whilein spatial resolution HS-AFM is comparable to or slightly
better than conventional AFM [8,9]. This enhanced
performance has enabled the direct visualization of
dynamic structural changes and dynamic interactions
occurring in individual molecules, which is currently
not possible with other techniques. In fact, various applications of this microscopy have successfully revealed
dynamic molecular processes of proteins, yielding
remarkable findings and great insights into the functional
mechanisms of the proteins (see comprehensive reviews[9,10]). Moreover, this microscopy now allows theobservation of dynamic molecular processes occurringon the surfaces of live bacteria [11], specialized eukaryotic cells [12] and probably isolated intracellular organelles, as well as dynamic processes occurring ineukaryotic cells [13].

HS-AFM system
Introduction
The essential attributes of proteins, namely structure anddynamics, play central roles in their biological function.
Structural biology has been successful over the past 60
years in solving the detailed three-dimensional structuresof many proteins, by the use of X-ray crystallography,
electron microscopy, and nuclear magnetic resonancespectroscopy methods. And yet, these methods are virtually ineffective in solving the second important attribute. To study the dynamics of proteins, single-molecule
fluorescence microscopy [1,2] and optical trap nanometry[3] have been created and widely used over the last 20–30
years. However, these methods record the dynamic
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HS-AFM typically employs the tapping-mode, in which acantilever is oscillated in the Z-direction at or near its
resonant frequency so that the tip intermittently taps the
sample surface. In the HS-AFM system (Figure 1a),various devices, including cantilevers (Figure 1b),
circuits,thesample-stage
scanner
electronic(Figure 1c), and the cantilever deflection detection system, are optimized for achieving high-speed performance[7,9]. For example, the cantilevers are much more miniaturized (6–12 mm long) than conventional ones toachieve high resonant frequencies in water f c = 400 kHz
to 1.2 MHz and small spring constants kc = 0.1–0.2 N/m.
In addition, a new feedback control technique capable ofCurrent Opinion in Structural Biology 2014, 28:63–68

64 Biophysical and molecular biological methods

Figure 1(a)
Z-scanner
SampleStage(b)

X, Y-scanners

ZXY

X, Y-Piezo
Driver

PC

D/A
A/D

Z-Piezo
DriverCantilever

QController

Cantilever
HolderDynafmic
PID
Controller

1 μm

1 μm

IIIuminator
Nikon
x 20

Laser Diode

Amplitude
Detector(c)

X-piezo

λ /4Balance Weight

Display

Sample Stage

SignalConditioner

Dichroic
Digital
Camera Mirror

Y-piezo

Z-piezo

Polarization Quadrant
Beam Splitter PIN Diode
MirrorLens
Current Opinion in Structural Biology

Schematic illustration showing HS-AFM system and devices contained therein. (a) Overall arrangement of all devices contained in the HS-AFM system(reprinted with permission from Ref. [14]. Copyright 2012 Nature Publishing Group). (b) Scanning electron micrograph of a small cantilever for HSAFM. The inset shows an electron beam deposited amorphous carbon tip grown on the original tip. (c) Structure of a HS-AFM scanner for narrow area
imaging (<1 mm  4 mm).

maintaining weak tip-sample interactions and active
damping techniques to suppress the scanner’s mechanicalvibrations have been implemented [7,9]. The speed
performance of an AFM system is expressed by the
bandwidth of feedback control f B, defined by the feedback frequency at which p/4 phase delay occurs in tiptracing of the sample surface. The most advanced HSAFM system has achieved f B = 110 kHz. The highest
possible imaging rate Rmax is a function of various
parameters, including f B, the scan range in the X-direction
W, the number of scan lines N, the spatial frequency ofthe sample surface corrugation to be imaged 1/l, and the
maximum possible phase delay in tracing the sample
surface um at which the resulting excessive force exerted
by the tip does not disturb the biological function of thesample. Rmax is expressed as Rmax = 2lumf B/(pNW)[8,14]. um depends on the sample fragility and is typically p/9 for proteins, according to previous imaging
studies [14]. For example, under the conditions
f B = 110 kHz, W = 200 nm, N = 100, um = p/9, andl = 10 nm, Rmax becomes 12 fps.

HS-AFM imaging of purified proteinsthird, diffusion and interactions in membranes; fourth,self-assembly processes; fifth, wiggling motion of andorder–disorder transitions in intrinsically disordered
proteins (IDPs); sixth, enzymatic reactions; and seventh,DNA–protein interactions. In some cases, molecular
structures are not well resolved. Nevertheless, the visualized dynamic events inaccessible with other approaches
have provided significant insights into the molecular
processes, as exemplified in the imaging study of cellulase
hydrolyzing crystalline cellulose fibers [36]. The visualization of interaction dynamics of membrane proteins
allows the assessment of interaction geometry and energy
in detail, as demonstrated [29,30]. The process of protein
self-assembly generally contains various events and structures: the formation of nucleation clusters, multiple intermediates, multiple sequential and parallel growing
pathways with different kinetics, and structural polarity
and anisotropy. HS-AFM can show most, if not all, of
these progressing events and growing structures in one
video recording [31,32]. The visualization of thin and very
flexible unstructured peptide chains allows the fast
identification and characterization of the disorderedregions in IDPs (Figure 2a) [33–35].

Thus far, various types of dynamic events occurring inprotein systems have been visualized, as listed in Table 1:
first, conformational changes; second, motor actions;Thus far, the most striking results have been obtainedin the observation of conformational changes ofCurrent Opinion in Structural Biology 2014, 28:63–68

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High-speed AFM imaging Ando 65

Table 1
Various types of dynamic events of proteins visualized by HS-AFM
Conformational changes- Bacteriorhodopsin responding to light [15–17]- Rotary propagation of conformational changes over the b subunits of F1-ATPase [18]- ATP-induced channel pore dilation of P2X4 receptor [19]- ATP-induced height change of Ca2+ pump [20]- Agonist-induced height change of NMDA receptor [21]- Height change of acid-sensing ion channel in response to acidification [22]- Straight-to-curving structural change of FtsZ polymers [23]- ATP-dependent rotation of AAA+ chaperone p97 [24]Motor action- Walking myosin V on actin filaments [25]Diffusion and interactions in membranes- Interaction between aquaporin-0 tetramers in reconstituted eye lens fiber cell membranes [26]- Diffusion of and interaction between bacterial outer membrane protein OmpF molecules in reconstituted membranes [27]- Diffusion and fusion of vacancy defects in streptavidin 2D crystals formed on supported lipid bilayers [28]- Membrane-mediated interaction between ATP-synthase c-rings [29]- Interaction of bR trimers with bR crystal edges in purple membranes [30]Self-assembly processes- Amyloid-like fibril formation by lithostathine [31]- 2D crystallization of sphingomyelin-specific pore-forming toxin on planar lipid membranes [32]Dynamics occurring in intrinsically disordered proteins- Wiggling and shortening/extension motion of disordered regions in FACT protein [33]- Order–disorder transition of a disordered region in FACT protein and effect of phosphorylation [34]- Wiggling and shortening/extension motion of a disordered region in CENP-T [35]Enzymatic reactions- Processive movement and traffic congestion of cellulase hydrolyzing crystalline cellulose fibers [36]DNA–protein interactions- One-step and two-step dissociation of ssDNA binding protein oligomer from ssDNA [37]- Cleavage of DNA by type IIF restriction enzyme [38]- Nicking enzyme-mediated unidirectional movement of DNA motor along DNA stator track [39]- ATP-independent unwrapping of mono-nucleosomes [40]Diffusion and interactions on live cell surfaces- Diffusion of crowded porin molecules on live bacterial outer surface [11]- Water channel aquaporin-0 binding to and dissociating from junctional microdomains on the lens cell surface [12]bacteriorhodopsin (bR) [15–17] and F1-ATPase [18] andmotor actions of myosin V [25]. The dynamic behaviors ofthese proteins were visualized in unprecedented detail,leading to significant findings. This is partly because theirdynamic actions are closely related to their biological
functions, and therefore, the functions themselves are
directly displayed on screen. A molecular movie of myosin V walking on actin filaments showed its forward
movement with a 36 nm stride in a hand-over-handmanner, rotational motion of the detached trailing headaround the forwardly biased neck-neck junction(Figure 2b), foot stomp in the leading head
(Figure 2c), and the lever-arm swing of the leading headspontaneously following the trailing head detachmentfrom actin (Figure 2b). The foot stomp and the swinging
lever-arm motion were observed for the first time by HSAFM. HS-AFM movies of actin-bound myosin V captured in the presence of ADP or under nucleotide-free
condition revealed other dynamic actions, which led tothe clarification of the hand-over-hand walking mechanism and an important discovery concerning the usage of
ATP energy. As exemplified in this HS-AFM study, HSAFM imaging has the unique feature that multiple
dynamic events can be detected simultaneously in one
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video recording. This feature makes a stark contrast withprevious single-molecule techniques wherein only a subset of events can be detected in a specifically designed
assay. Furthermore, unlike previous single-molecule
techniques, HS-AFM allows us to know how individual
molecules are really situated under assay conditions
because of the visibility of both molecules and their local
environments. Owing to these excellent features of HSAFM imaging, the observed molecular events can bestraightforwardly interpreted, leading to definitive conclusions; hypotheses and data interpretations play a muchsmaller role in deriving conclusions.
The high-resolution power of HS-AFM, capable of distinguishing individual domains and subunits within aprotein molecule, is important, especially in identifying
a local portion undergoing structural changes and finding
interplay between subunits. For example, in the study of
the a3b3 subcomplex of F1-ATPase (i.e., rotor-less F1ATPase) in the presence of ATP [18], the individual
subunits were clearly distinguished. Therefore, conformational changes occurring in a defined rotary sequenceamong the subunits were observed (Figure 2d), which led
to a discovery that the intrinsic cooperativity responsible
Current Opinion in Structural Biology 2014, 28:63–68

66 Biophysical and molecular biological methods

Figure 2

(a)

0s
(b)

(c)

0.47 s0.74 s1.74 s2.75 s0s0.14 s0.43 s0s0.14 s0.43 s6.51 s

7.38 s

7.65 s

(d)

0s

1.6 s

0.72 s0.32 s

2.0 s

2.8 s

(e)

0s

1s

5s

9s
Current Opinion in Structural Biology

HS-AFM images showing various dynamic events of proteins. (a) Wiggling motion of and order–disorder transitions observed in an intrinsicallydisordered region of facilitates chromatin transcription (FACT) protein. A shorter ID region belonging to the SPT16 subunit is deleted. Imaging rate,
15 fps; scan area, 100 nm  100 nm. (b and c) Stepping behavior (b) and foot stomp (brief detachment from and re-binding to actin) at the leadinghead (c) of myosin V. The arrow in (c) indicates detachment of the leading head from actin. Imaging rate, 7 fps; scan area, 150 nm  75 nm. (d)Counterclockwise rotary propagation of conformational changes of the a3b3 subcomplex of F1-ATPase in an ATP-containing solution (each
15 nm  15 nm image was clipped from an 80 nm  80 nm area image). The red dots indicate the highest pixel positions. Frame rate, 12.5 fps. (e)Light-induced conformational changes of D96N bR in the purple membrane. The illumination of green light was switched on and off at the beginningand the end of the second image, respectively. The white and light-blue triangles indicate a trimer and a trefoil, respectively. Imaging rate, 1 fps; scanarea, 25 nm  25 nm.

for torque generation to rotate the g subunit is elicitedthrough the b–b interplay alone, while the g subunit ispassively subjected to the torque. The high-resolution
power also allowed the detection of small outwards movement (0.8 nm) of the E–F helix loop of each bR molecule from the trimer center under light illumination(Figure 2e) [15]. The outwards movement results inthe contact between three bR molecules (a triad designated as ‘trefoil’), each belonging to an adjacent trimer(second frame in Figure 2e). This observation led to thediscovery of positive and negative cooperativity effects ofthe bR–bR contact within a trefoil (not within a trimer) on
the decay kinetics of the photo-activated bR; the kinetics
is decelerated for a molecule early activated within atrefoil, whereas accelerated for a molecule activated last
within a trefoil. It is hard to imagine how other approaches
Current Opinion in Structural Biology 2014, 28:63–68

would allow the finding of these interplays between
subunits or neighboring molecules which underlie thefunction or functional modulation of these proteins. As
such, HS-AFM imaging will disclose many intriguing
phenomena occurring in proteins, which other approaches
may fail to notice, even for protein systems that havepreviously been scrutinized.

Future challenges in HS-AFM imaging
One of the greatest challenges in HS-AFM imaging is toobserve the dynamic action of individual protein molecules on the surfaces of live cells and isolated organelles.
This is because the surfaces are very soft and deformed by
the contact with the AFM tip. For the relatively rigid
surfaces of live bacterial and specialized eukaryotic cells,
HS-AFM can visualize individual protein molecules andwww.sciencedirect.com

High-speed AFM imaging Ando 67

their dynamics, as demonstrated in the imaging of densely packed porin (Msp1) molecules diffusing on theouter surface of the magnetotactic spirillum Magnetospirillum magneticum AMB-1 [11] and of water channel aquaporin-0 binding to and dissociating from junctional
microdomains on the lens cell surface [12]. The surface
rigidity depends on its size. Therefore, it will probably
become possible to observe the dynamic molecular processes occurring on the surfaces of, for example, neuronal
synapses and relatively small mitochondria and Golgi
apparatuses (0.2–0.5 mm).
The direct visualization of morphological changes occurring in live cells is also a challenge in HS-AFM, because alarge area has to be scanned at a high velocity. The recent
progress of high-speed/wide-area scanners [13,41] andactive vibration damping techniques [13] has made thisvisualization possible, as demonstrated in the visualization of various dynamic events occurring in live bacterial
and eukaryotic cells; bacteriolysis caused by lysozyme[13] and an antimicrobial peptide [42], the process of
endocytosis from the initial pit formation to budding of
protrusions around the pits and their disappearance [13],and actin retrograde flow [13]. The resolution is higher
than optical microscopy but the information acquirablewith the HS-AFM imaging is limited and therefore cannot provide significant findings. To expand the usefulness, HS-AFM should be combined with fluorescencemicroscopy to make it possible to correlate the morphological changes with underlying molecular processes thatproceed in the cell interior.
For this combination, HS-AFM needs to employ the tipscan mode rather than the sample-stage scan mode. Veryrecently, a tip-scan HS-AFM system has been developed,
allowing simultaneous capture of topographic and totalinternal reflection fluorescence microscopy images, as
demonstrated [43]. Various optical techniques can be
implemented with this new HS-AFM system. For
example, combining the system with the optical tweezers
will allow the visualization of a single protein molecule
under external force. Combining with tip-enhanced fluorescence microscopy [44,45] will materialize high-speed
superresolution fluorescence microscopy, which allows
simultaneous dynamic recording of high-resolution topographic and fluorescence images in the same field of view.
Thus, HS-AFM technology has more potential to evolveand hence will become an even more useful tool in the
biological sciences.

Conclusion
HS-AFM has recently opened new opportunities todirectly observe biological molecules in action at highresolution, which allows more comprehensive understanding of how they function, even for those previously
scrutinized, as demonstrated in the recent studies
reviewed here. Because of this capability, HS-AFM will
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become more common and indispensable in biologicalsciences in the near future. The HS-AFM technology iswell established, but is still evolving toward its use for awider range of biological samples and phenomena. It is
hard to imagine what will become possible once all of thetechnological capabilities of HS-AFM imaging have been
fully realized.

Acknowledgements
This work was supported by Human Frontier Science Program Grant (no.
RGP0069) and Grant-in-Aid for Basic Research from the MEXT, Japan(nos. 20221006 and 24227005).

References and recommended reading
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