protocol

Guide to video recording of structure dynamics anddynamic processes of proteins by high-speed atomic
force microscopy
Takayuki Uchihashi1,2, Noriyuki Kodera2 & Toshio Ando1,2
Department of Physics, Kanazawa University, Kanazawa, Japan. 2Bio-AFM Frontier Research Center, Kanazawa University, Kanazawa, Japan. Correspondence should be
addressed to T.A. (tando@staff.kanazawa-u.ac.jp).
1© 2012 Nature America, Inc. All rights reserved.

Published online 24 May 2012; doi:10.1038/nprot.2012.047

High-speed atomic force microscopy (HS-AFM) allows direct visualization of dynamic structural changes and processes offunctioning biological molecules in physiological solutions, at subsecond to sub-100-ms temporal and submolecular spatial
resolution. Unlike fluorescence microscopy, wherein the subset of molecular events that you see is dependent on the site where
the probe is placed, dynamic molecular events unselectively appear in detail in an AFM movie, facilitating our understanding of
how biological molecules function. Here we present protocols for HS-AFM imaging of proteins in action, including preparationof cantilever tips, step-by-step procedures for HS-AFM imaging, and recycling of cantilevers and sample stages, together withprecautions and troubleshooting advice for successful imaging. The protocols are adaptable in general for imaging many proteinsand protein–nucleic acid complexes, and examples are described for looking at walking myosin, ATP-hydrolyzing rotorless F1-ATPaseand cellulose-hydrolyzing cellulase. The entire protocol takes 10–15 h, depending mainly on the substrate surface to be used.

INTRODUCTION
Proteins perform their functions through their dynamic structuralchanges and dynamic interactions with other molecules. Even cellular structures made of protein are highly dynamic. Therefore,
in the quest for better understanding of biological phenomena,acquiring details of structure dynamics and dynamic interplay
between subunits within a protein and between proteins is essential. Although the structure of proteins has been studied by X-ray
crystallography, electron microscopy, NMR and atomic force
microscopy (AFM), structures obtained are static snapshots. The
dynamic behavior of proteins has been studied by single-molecule
fluorescence microscopy and optical trap nanometry, but protein
molecules themselves are invisible in the observations. To understand how proteins perform their functions, inferences have to bemade from collected data with substantial resolution gaps. Directly
visualizing functioning protein molecules at high spatiotemporal
resolution has therefore been an important goal for structural
biology and biophysics.
AFM is now routinely used to directly acquire high-resolution
images of biological samples under physiological conditions, without sample staining1. To form an image, AFM acquires the sample
height information over many points on the sample. During theacquisition at each point, a sharp tip attached to the free end of a
cantilever is brought into contact with the sample. The mechanicalresponse of the cantilever upon this contact (change in, e.g., deflection, oscillation amplitude, oscillation phase or resonant frequency,
depending on the AFM operation mode to be used) is measured
and then the sample stage is moved in the z-direction to recover
the mechanical state of the cantilever back to a given state (i.e., set
point) through feedback control. Without this feedback control, theforce exerting from the cantilever tip to the sample cannot be kept
constant, resulting in serious damage or disruption of the sample.
The closed feedback loop, in which various devices are contained,
requires an amount of time for this recovery that depends mainlyon the slow response of the mechanical devices (i.e., cantilever andz-scanner). This series of operations is repeated many times for­different points on the sample during lateral scanning of the sample
stage, which means that it takes a relatively long time (at least 30 s)
to get an image.
Over the past 15 years, various efforts were carried out toincrease the imaging rate of AFM2–13, and HS-AFM has now
materialized14,15 (see recent reviews16–18). HS-AFM uses a smallcantilever with a high-resonant frequency (the first resonant frequency fc = 0.6 − 1.2 MHz in water) and a small spring constant(kc = 0.1–0.2 N m − 1). The HS-AFM instrument comprises a fastscanner, a feedback controller, fast electronics and an optical beam
deflection (OBD) detector for detecting deflection of this smallcantilever (Fig. 1). The system is operated in the tapping mode;a cantilever is oscillated at fc in the z-direction so that its tip isbrought intermittently into contact with the sample. It allows
imaging at a rate of 5–20 frames per second (fps) without disturbing the function of fragile proteins and delicate protein-protein
and DNA-protein interactions.
This high-speed and minimally invasive performance opens
up a new opportunity to directly visualize dynamic events ofproteins19–27, DNA-protein complexes28–30 and other molecularsystems31–33. In this protocol, we describe various preparations
and procedures to be done before the actual imaging experiments,
how to use the HS-AFM instrument to image dynamic eventsof proteins and cleanup steps for the next imaging experiments(Fig. 2). As designing substrate surfaces is particularly importantfor successful imaging, we provide, for example, a procedure forpreparing mica-SLBs (supported lipid bilayers; Steps 15–18).
Mica-SLBs containing biotin- or Ni-NTA–conjugated lipids areuseful for selective immobilization of proteins with either biotin
or His-tag, respectively34,35. Mica-SLBs can also be used for electrostatic immobilization of proteins under a controlled surface charge
density19,34,35. We describe three options for sample deposition:
direct sample deposition onto a bare surface of mica or HOPG

nature protocols | VOL.7 NO.6 | 2012 | 1193

protocol
z-scanner
Samplestage

x, y-piezo
driver

x
y

z

D/A

PC
A/D

Qcontroller

z-piezo
driverCantilever
Illuminator

Moreover, prototypical small cantilevers (kc = 0.15 N m − 1 and
fc = 1.2 MHz in air) are also available from NanoWorld.

x, y-scanners

Nikon
× 20

Cantilever
holderLaser diodeDynamic
PID
controller

Amplitude
detector

λ/4
Display

Signalconditioner

Dichroic
mirrorDigitalcameraPolarization
beam splitter

Quadrant
PIN diode© 2012 Nature America, Inc. All rights reserved.

Mirror
Lens

Figure 1 | Schematic of the HS-AFM system.

(highly ordered pyrolytic graphite; the example described is theone used for looking at cellulase-hydrolyzing cellulose23); covalentimmobilization of proteins onto a mica surface with glutaraldehyde (the example described is the one used for looking at rotary
catalysis of rotorless F1-ATPase22); and sample deposition onto
mica-SLBs (the example described is the one we used for looking at
walking myosin19).
We also provide videos to show procedures for imaging. We
have used this protocol in our previous studies on myosin V19,
bacteriorhodopsin (bR)20,21,24, rotorless F1-ATPase22 and cellulases23, and example results can be found in the ANTICIPATEDRESULTS section. The protocol can be adapted to many otherbiomolecular systems, mainly by optimizing the substrate surface
onto which the molecules are placed. More details on the preparation of substrate surfaces and considerations for their choice are
given elsewhere34–36.
Sources of supply of HS-AFM equipment and cantilevers
Currently, only a small number of laboratories are using HS-AFM
because the availability of the equipment has been limited until
recently. However, HS-AFM equipment, which is a copy of ourhomemade apparatus, is now available from a manufacturer, RIBM
(Tsukuba, Japan). This system is the fastest one, and other ­nominal
HS-AFM systems from different manufacturers are at least ten
times slower. Therefore, some aspects of the present protocol are
specific to the HS-AFM equipment manufactured by RIBM. Smallcantilevers made of silicon nitride (9–10 µm long, 2 µm wide and
130 nm thick; BL-AC10DS, Olympus, Tokyo) with fc = 1.5 MHz
in air and 0.6 MHz in water, kc = 0.1 N m − 1, and a quality factor
Q ~2 in water (Fig. 3) are available from Olympus, Atomic Force
F&E, Asylum Research and Bruker. Smaller cantilevers (BL-AC7DSKU2; 6–7 µm long, 2 µm wide and 90 nm thick) with fc = 3.5 MHz
in air and 1.2 MHz in water, kc = 0.2 N m − 1, and Q ~2 in water
can be custom-made by Olympus. We have been mostly using thelatter type. These cantilevers have bird beak–like tips, but electronbeam deposited (EBD) sharper tips are available as option.
1194 | VOL.7 NO.6 | 2012 | nature protocols

Overview of HS-AFM system
The HS-AFM system includes an original inverted optical
microscope (Fig. 1). An objective lens with a long working distanceof 24 mm (CFI L Plan EPI SLWD ×20, Nikon, Tokyo) that is used
for the optical microscope is a part of the OBD detector, and is also
used for viewing the cantilever and sample stage under the optical
microscope via a digital camera or a CCD camera4,15. A 0.5-mm
thick glass slide, to which a cantilever holder and a liquid cell areattached, is placed on the optical microscope stage. A cantileverchip is held in the holder so that its tip points upward (opposite to
the way in conventional AFM)4,15. A sample stage, attached to thez-scanner and facing downward, is placed over the cantilever. An
incident laser beam passing through the objective lens is focused
onto the small cantilever, and the light reflected back from thecantilever is collected and collimated by the same objective lensand guided to a quadrant-cell Si PIN photodiode4,15. The incident
and reflected laser beams are separated using a quarter-wavelength(λ/4) plate and a polarization beam splitter4,15.
Three types of scanners with different maximum scan ranges
are available. Type 1: x = 1 µm, y = 4 µm, z = 1 µm; Type 2:
x = 5 µm, y = 5 µm, z = 2 µm; and Type 3: x = 40 µm, y = 40 µm,
z = 6 µm. Type 1 is used for dynamic imaging of isolated proteins,
whereas the latter two types are used for dynamic imaging of larger
objects such as bacteria and eukaryotic cells. Q-control circuitswith mock z-scanners (LRC circuits) are implemented to theirz-scanners to damp mechanical vibrations6. In these scanners, thex-scan is made though inverse transfer function compensation
to damp unwanted vibrations15. The feedback controller (termed‘dynamic proportional-integral-derivative (PID) controller’) contains a circuit for suppressing or eliminating a tip ‘parachuting’effect8. In this context, parachuting means that the tip completely
detaches from the sample surface at inclined regions of the sample,
and time elapses until it lands on the surface again. In conventional
PID controllers, parachuting often occurs when the cantileveramplitude set point is set very close to the free oscillation amplitude
so that the tip lightly taps the sample surface8,15.
Spatial and time resolutions
In comparison with conventional large cantilevers, the small canti­
levers afford ~10–20 times higher sensitivity to the detection of
cantilever oscillation amplitude by the OBD detector and theamplitude detector. Even though the bandwidth for detection at every oscillation cycle (~0.83 µs period) is high, good(~0.1 nm) precision (hence, spatial resolution in the z-dimension)is achieved15. The spatial resolution in the x–y dimensions depends
on the radius of the tip apex. However, it is limited to 1–2 nm evenwhen a tip with an apex radius of ~0.5 nm is used22,37.
The time resolution T depends on the feedback bandwidth fB,
which is a function not only of the speed performance of devices
contained in the feedback loop but also of the ratio of the cantilever oscillation amplitude A0 to the maximum sample height h0(the amplitude set point generally affects fB, but in practice this is
not seen because the dynamic PID controller is used)8,15. However,for relatively small protein molecules, fB can be approximately
considered to be ~110 kHz. The time resolution T is also a functionof the scan range in the x-direction W, the number of scan lines N,

protocolac

For example, for biomolecules with
h0 ~5 nm, the force amounts to a maximum
Fabrication of
Steps 1–5
of ~100 pN when phase delay occurs bycantilever tip
Laser~20–30°. However, note that the mechanical
Steps 22 and 23
HS-AFMalignment
quantity that affects the sample (i.e., causes
imaginga momentum change) is not force itself but
Cleaning ofStep 24
Recycling ofz-piezo surf.
Step 46
impulse (force × force acting time). The
cantilever chip
Step 25
force acting time is very short (~100 ns
Option
C
(Step
28)
Which
options?
or less) when a cantilever oscillating at
b Making of mica
Steps 6 and 7
0.6–1.2 MHz is used. For relatively fragor HOPG disks
Option A or B
ile protein molecules, θm ≤~20°, whereas
Placing ofsample stage Steps 26 and 27
for relatively strong molecules θm can be
Prep. ofon z-piezo
Steps 10–14
Steps 8 and 9
sample stages
extended to 45° or further. For example,
Prep. of
Step 28
lipid vesicles
Option A
Which
under the imaging condition usedNO
Bare mica
options?
for rotorless F1-ATPase (W = 80 nm,
Steps 15–18
or HOPG?
Option B
Prep. of
N = 50 for a scan range of 40 nm in the
YES
Direct deposition
mica-SLBs
Covalent
Sample deposition
onto bare mica
y-direction, λ = 2 nm, and θm ~ 20°),
immobilization
onto
mica-SLBs
or HOPG(rotorless F1 ATPase)(myosin V)(cellulose + cellulase)
Equation (1) estimates that images can be
HS-AFM
captured at ~12.2 fps without damage toimaging
Scanner mounting
the sample. Consistent with this estimaSteps 29–38& approach
Recycling ofStep 45
tion, rotorless F1-ATPase was successfully
glass stage
imaged at 12.5 fps with A0 ~1 nm, without
Imaging
Steps 39–44
the function being disturbed22. Equation (1)Figure 2 | Flowcharts showing overview of procedures before and during HS-AFM imaging and cleanup
provides a guideline for the allowable
steps for the next imaging experiments. (a) Works for cantilevers. (b) Works for substrate surface
highest imaging rate under a given imagpreparation and sample deposition. (c) Procedures of high-speed AFM imaging.
ing condition. However, it should be determined by repeating test imaging on target
biological samples, because the feedback
the smallest sample surface corrugation to be observed λ and the phase delay cannot be measured during actual imaging. When thephase delay exceeds θm, the sample is damaged. For example, whensample fragility. The overall relationship can be expressed as
GroEL attached to a bare mica surface in the end-up orientation
T = pWN /(2l f Bq m ),(1) is imaged too fast, its top layer of the double ring is removed. As
GroEL is commercially available and easily deposited on the surface
where θm represents an sample fragility-dependent allowable maxi- of bare mica3,38, it is a good test sample that one can use to check
mum phase delay in the feedback control15,18. The meaning of the whether the HS-AFM setup itself works well and to examine thephase delay θ in the feedback control can be explained as follows; speed performance of other AFM systems.
supposing that the sample surface has a sinusoidal shape with theperiodicity λ and the maximum height h0 and that the sample stage Experimental design and control experimentis moved in the x-direction at velocity Vs, the sample height h right The experimental design depends on the target sample and dynamic
events to be visualized. For example, in the observation of myosin
under the cantilever tip changes with time t as
V walking along an actin filament19, we first placed actin filamentsh(t ) = (h0 / 2) × sin(2p ft ),(2) and myosin V on the surface of bare mica under buffer solutions
with various ionic strengths. Then, we chose an appropriate ionicwhere f is the feedback frequency at which the z-scanner is moved
and is given by f = Vs / λ. Because of feedback delay (delay time, τ0),
the sample stage is moved in the z-direction as© 2012 Nature America, Inc. All rights reserved.

Prep. of
Steps 19–21
AFM liquid cell

Z (t ) = − (h0 / 2) × sin[2p f (t − t 0 )].

(3)

So, the phase delay θ is given by θ = 2πfτ0 = 2πVsτ0/λ.
Tip force versus imaging rate
The sum of h(t) and Z(t) is the feedback error E(t) and
expressed as
E(t ) = h0 × sin(q / 2) × cos(2p ft − q / 2).

(4)

Therefore, because of the phase delay, the force given by (kc / Q)× h0 × sin(θ /2) is excessively exerted at maximum from thetip to the sample, in addition to a constant force (~20–30 pN).

20 µm

Figure 3 | SEM micrograph of a conventional AFM cantilever (Olympus
OMCL-AC240TS) and a small cantilever (Olympus BL-AC10DS; encircled).
nature protocols | VOL.7 NO.6 | 2012 | 1195© 2012 Nature America, Inc. All rights reserved.

protocolstrength, in which actin filaments stably attached to the surface
while myosin V molecules were diffusing on the surface. Under
this condition, we could observe myosin V molecules walking ina hand-over-hand manner, but its success rate was low; moreover,
the walking velocity was much lower than that measured by fluorescence microscopy under the same buffer solution condition. We
then chose electrically neutral mica-SLBs containing a biotin lipid.
Partially biotinylated actin filaments were immobilized on the surface via streptavidin. Myosin V was never bound to the surface andonly interacted with the immobilized actin filaments to move unidirectionally. However, most of the molecules were moving, orienting
perpendicularly to the surface, so that we could not look at the characteristic sideways topography of the moving molecules. Next, weadded a positively charged lipid in the bilayer in various densities.
An appropriate density of the positively charged lipid was chosen
so that we could observe the sideways topography of the molecules moving at velocity similar to that measured by fluorescencemicroscopy. However, the molecular process occurring during a
step could not be resolved because it completes within a frame time(~143 ms). Finally, streptavidin molecules were further placed on
the substrate surface as moderate obstacles to the advance. This final
design allowed the visualization of detailed stepping processes.

It is important to check the possible effect of the tip-sample
interaction on the visualized molecular events. When a time constant for an observed dynamic event is previously known, it should
be compared with that obtained from the HS-AFM images. When
it is unknown, the imaging rate should be changed in a possible
range to examine whether the imaging rate (and hence the excessive
force determined by the rate) affects the time constant, as has been
done for the visualization of bR trimers that are binding to anddissociating from the edge region of two-dimensional crystal of bR
in the purple membrane24. This check is particularly crucial when
dynamic dissociation events of protein-protein or DNA-protein
are imaged, because their dissociation is forced to occur by overly
strong tip-sample interaction force.
One round of HS-AFM imaging of several molecules that appear
in a few different scan areas generates many frames, so that we
can thereby get statistically meaning results. As the same speciesof protein or DNA molecules have no individuality, quantitative
analysis of a few molecules is sufficient as far as many frames havebeen captured for them. However, note that bacteria, mammaliancells and intracellular organelles are likely to have individuality
within the same species, as has been indicated by a recent study of
HS-AFM imaging of E. coli cells39.

MATERIALS
REAGENTS CRITICAL All reagents should be of analytical grade.
• 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC; Avanti Polar Lipids)• 1,2-Dipalmitoyl-3-trimethylammonium-propane (DPTAP; Avanti
Polar Lipids)• 1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(cap biotinyl)(Biotin-cap-DPPE; Avanti Polar Lipids)• Streptavidin (Wako)• Chick brain myosin V-HMM40,41• Rabbit skeletal actin42• α(His6 at N terminus/C193S)3β(His3-Lys7 at N terminus)3 subcomplexof the F1-ATPase from thermophilic Bacillus sp. PS3 expressed in E. coli22• Crystalline cellulose from the cell wall of green alga Cladophora sp.43• Cellulase (Cel7A) from Tricoderma ressei44• Biotin-(AC5)2 Sulfo-OSu (Dojindo)• Phalloidin (Invitrogen)• 3-Aminopropyl-triethoxysilane (Shin-Etsu Chemical) ! CAUTION It isharmful to skin. Rubber gloves and safety glasses should be used. Store in alight-resistant bottle whose cap is sealed with Parafilm.
• Glutaraldehyde (25% (wt/vol); Wako) ! CAUTION It is harmful to skin. Rubber gloves and safety glasses should be used. Store in a light-resistant bottle
whose cap is sealed with Parafilm.
• Phenol (Nacalai Tesque) ! CAUTION It is harmful to the skin. Rubber gloves
and safety glasses should be used.
• Epoxy adhesive (high-quick or Super X black; Cemedine)• Colorless nail polish (available at supermarkets)• Acetone (Nacalai Tesque)• Water purified with Milli-Q (Millipore)• Sodium acetate• MgCl2• Tris-HCl• KCl• EGTA• Imidazole• DTT• Chloroform

1196 | VOL.7 NO.6 | 2012 | nature protocols• Methanol• HEPES• NaOH• MgSO4EQUIPMENT• Mica (natural Muscovite mica; The Nilaco Corporation)• HOPG (ZYH grade; Bruker)• Gel-Film (WF-55-X0-A; Gel-Pack)• Flat plastic container with Gel-Film glued onto the inner plane. This is used
for storing small cantilevers with sharpened EBD tips
• Auto-dry cabinet (Dry Keeper; Sanplatec) for storing scanners and cantilevers• Syringe-driven filter (0.22 µm, Millex GV; Millipore)• Precision wipes (Kimwipes; Kimberly-Clark)• Wipes for optics (Super cloth SA-25; Asahi Kasei)• Transparent container of cosmetic cream as a sealed container (volume,~10 ml; available at supermarkets) to be used for mica-SLB preparation• Glass rods as sample stages (custom-made, 1.5 or 2.0 mm in diameter and
2 mm in height; Japan Cell)• Container made of aluminum with a lid having through-holes of 0.1 mm indiameter (custom-made; see Fig. 4). This container is used for fabricating
EBD tips• Cantilever holder for growing EBD tips (custom-made; see Fig. 4)• Laboratory-built high-speed AFM (Nano Explore; recently commercialized
by RIBM)• Small cantilevers (BL-AC7DS-KU2; Olympus)• Bath sonicator (40 W; Elmasonic One; Elma)• Probe sonicator (10 W; UR-20P; Tomy Seiko)• Stereoscopic microscope (L-50; Hozan)• Rubber hand blower (Z-263; Hozan)• Peristaltic pump (TP-10SA; As One Corporation)• Field emission scanning electron microscope (FE-SEM; SUPRA40VP;
Zeiss)• Radiofrequency (RF) plasma etcher (PE-2000; South Bay Technology)• Puncher set for preparing mica and HOPG disks (constructed using,e.g., Mini straight punch, punch guide bush and button die; Misumi
Corporation)

protocol
Figure 4 | Pictures and schematics of a container for growing EBD tips with
SEM. (a) Top view of the container without the lid. Phenol power therein
is seen (arrow). (b) Cross-section of the container along the dotted line
drawn in a. (c) Top view of the cantilever holder placed on the container
lid. Fourteen cantilevers can be clamped to the holder with copper clamps.
The arrow indicates a clamped cantilever. (d) Cross-section of the cantilever
holder placed on the container along the dotted line drawn in c. The
through-holes are positioned so that each through-hole is close to the distal
end of each clamped cantilever.

ca© 2012 Nature America, Inc. All rights reserved.

10 mm

REAGENT SETUP
Buffer solutions Filter all buffer solutions using 0.22-µm syringe-driven
filters and stock them in well-cleaned glass bottles.  CRITICAL It is important to stock the buffer solutions in cleaned glass bottles (not in plastics
bottles). Otherwise, many particles that may hamper the intended imaging
will appear in AFM images.
Imaging buffer for cellulose and cellulase (buffer A) Imaging buffer issodium acetate (20 mM, pH 5.0). It can be stored at 4 °C for a few months.
Imaging buffer for rotorless F1-ATPase (buffer B) Combine 2 mM MgCl2,
10 mM Tris-HCl (pH 8.0), and various concentrations of nucleotides. It canbe stored at 4 °C for a few months.
Imaging buffer for actomyosin V (buffer C) Combine 25 mM KCl, 2 mM
MgCl2, 1 mM EGTA, 20 mM imidazole/HCl (pH 7.6), 5 mM DTT, andvarious concentrations of nucleotides. It can be stored at 4 °C for a few
months.
Nail polish Dilute colorless nail polish three times with acetone.
 CRITICAL This dilution is very effective for quickly gluing a sample stage
glass rod onto the z-piezo (z-scanner) and for fixing it firmly.
EQUIPMENT SETUP
High-speed AFM instrument The imaging rate of HS-AFM is much higher
than that of conventional slow AFM. Therefore, HS-AFM imaging is not­hampered by slow mechanical and electrical drifts, meaning that we do not

b

d

Screw
Clamp

Phenol powder

10 mm

Rod forSEM holder

Cantilever
Through-hole(Φ 0.1)

Evaporated phenol
Phenol powder

have to wait at all until the drifts cease. Moreover, effects on the image
quality of varying imaging parameters, such as the amplitude set point and
the PID controller gains, appear instantly on the acquired images.
This excellent performance facilitates the tuning of imaging parameters
for attaining high-quality images.
Small cantilevers As already mentioned, we use the small cantilevers
custom-made by Olympus (BL-AC7DS-KU2). The apex of the beak-like tipof the cantilevers is not small enough (25–100 nm) to acquire high-resolution images. Therefore, we grow an EBD tip at the top end of the original
beak-like tip using the FE-SEM. The EBD tip can be sharpened by argon or
oxygen plasma etching. Note that the EBD tip can be completely removed byoxygen plasma etching over 1 d, so that we can reuse a small cantilever chip
several times.

PROCEDURE
 CRITICAL Note that stages of the procedure that need to be modified for different sample types (myosin V, rotorless
F1-ATPase and cellulose + cellulase) are indicated by the word “modify” in parenthesis.
Fabrication of cantilever tips ● TIMING 3–5 h (depending on the number of cantilevers)
1| Place a small amount of phenol powder (0.1–0.5 g) into the small container (volume, ~1 ml; Fig. 4a,b) with small
through-holes (~0.1 mm diameter) in the lid15.
2| Place small cantilevers into the cantilever holder (Fig. 4c,d). To reduce mechanical drift, the cantilevers should betightly fixed in the holder using mechanical clamps. Do not use double-sided adhesive tape to fix cantilevers onto thecontainer lid. Adjust the position of the holder relative to the lid so that each cantilever is positioned near a through-hole
drilled in the lid. Note that it is not necessary to coat the small cantilevers with osmium to make them conductive if you use
the low vacuum mode available in an SEM.
3| Place the container in the FE-SEM chamber and wait for 1–2 h until mechanical drift ceases.
4| Focus a spot-mode electron beam onto each original tip (Fig. 5a). It produces a stylus composed of amorphous carbon
at a growth rate of ~17 nm s − 1 under low-vacuum conditions. The focused electron beam is usually irradiated onto theoriginal tip for ~1 min, which results in growth of a ~1 µm long EBD tip (Fig. 5b). We can fabricate an EBD tip every 5 min.
 CRITICAL STEP Search for an appropriate SEM condition for growing sharp EBD tips. Typically, small working distance(3–4 mm), standard aperture size (for example, 30 µm), high acceleration voltage (higher than 20 kV), small spot size,
well-turned focus and good stigma adjustment produce sharper EBD tips. Furthermore, the spot-mode electron beam should
be applied at an angle relative to the original beak-tip so that in HS-AFM imaging the EBD tip touches the substrate surface
perpendicularly (Fig. 5b). Thus, the cantilevers (hence, the container) should be set in the SEM chamber with a certain tilt
angle (typically 10°).
nature protocols | VOL.7 NO.6 | 2012 | 1197

protocol
Figure 5 | SEM photographs of small cantilevers with and without EBD tip.
(a) An original beak-like tip. (b) Approximately 1-µm-long EBD tip grown on
the original tip. The EBD tip makes an angle of ~10° relative to the normalline of the lever plane so that in HS-AFM imaging the tip taps the substratesurface perpendicularly. The inset shows a top view of the cantilever end
with the EBD tip.

a

b

~10°

1 µm

© 2012 Nature America, Inc. All rights reserved.

5| After fabricating EBD tips, remove the container fromthe SEM chamber while holding the cantilever holder. Attach
the cantilevers to a glass slide using double-sided adhesive tape and put the glass slide into the RF-plasma etcher chamber.
Apply argon or oxygen plasma etching to sharpen the EBD tips for ~8 min. Typical power of the RF plasma is about 18 W andthe gas pressure is ~180 mTorr. It can reduce the apex radius from 15–25 to 4–5 nm. In the best case, it can be reduced to
0.5 nm, although the yield for tips of this radius is ~0.1 (refs. 20,22).
 PAUSE POINT For convenience and time saving, fabricate many cantilever tips at once. After fabrication, put the smallcantilevers with sharpened EBD tips in a plastic flat container with a Gel-Film onto which the cantilevers can be immobilizedbased on surface tension (plastic containers supplied from cantilever manufacturers can also be used). Then, store thecontainer in the auto-dry cabinet. The shelf life is practically not limited (1–2 years).
Preparation of mica and HOPG disks (modify) ● TIMING ~30 min
 CRITICAL If you wish to use mica disks, go to Step 6. If you wish to use HOPG disks, go directly to Step 7. In general,mica substrate is used for depositing hydrophilic molecules, whereas HOPG substrate is used for hydrophobic samples such as
crystalline cellulose.
6| Prepare a mica sheet (~10 × 100 mm2) and cleave it to a thickness of less than 0.05 mm. Next, punch the thinly cleaved
sheet using the puncher set to produce mica disks with a diameter of 1.5 mm. Go to Step 8.
 CRITICAL STEP The use of a thin mica sheet to produce disks is very important for achieving successful imaging, because
disks punched out from a thicker mica sheet often have burrs at the edges and loosely packed layers. Such mica disks hamper
imaging because of both low mechanical stability and contaminations.
7| Prepare HOPG sheets (~5 × 5 mm2, < 0.1 mm thick) by using a sharp knife. Punch them using the puncher to produce
disks with a diameter of 2.0 mm. Much thinner HOPG disks are spontaneously produced from the punched HOPG disks andcan be easily separated with a sewing needle.
 CRITICAL STEP Because HOPG is softer than mica, it is hard to make regularly shaped disks of diameters smaller than
2.0 mm. Again, the use of thin HOPG sheets for fabricating disks is very important in order to avoid low mechanical stability
and contaminations.
Preparation of sample stages ● TIMING more than 3 h (working time ~1 h)
8| We use a glass rod (1.5 or 2.0 mm in diameter and 2 mm in height) as a sample stage4,45. Note that a larger diameter
sample stage will produce a higher hydrodynamic pressure when it is moved fast, which perturbs the response of cantilever
oscillation amplitude to tip-sample interaction45,46. Before use, clean glass rods with Kimwipes wetted with acetone, rinsethem with Milli-Q water, place them on Kimwipes for drying, and then store them in a clean plastic box.
9| Under a stereoscopic microscope, glue either a mica disk or an HOPG disk onto the top of a sample stage using an epoxy
adhesive. Then, wait for more than 3 h for drying.
 CRITICAL STEP The reverse side of a disk should be fully glued to the top surface of a sample stage to accomplish firm
attachment.
 PAUSE POINT After preparation, the sample stages with mica or HOPG disks can be stored in a clean plastic case. For
convenience and time saving, prepare many sample stages with the disks at once.
Preparation of lipid vesicles and mica-SLB (modify) ● TIMING ~3 h for vesicle preparation; variable for mica-SLB preparation
 CRITICAL If you do not wish to use mica-supported planar lipid bilayers (mica-SLBs), directly go to Step 19. If you dowish to use mica-SLBs for specific or electrostatic immobilization of proteins, follow or adapt the following procedures(Steps 10–18). See reference 47.
10| Preparation of lipid vesicles. Dissolve each lipid compound in chloroform or in a mixture of chloroform, methanol andwater in a glass tube (follow the instructions provided by the manufacturer). We used chloroform (100%) for dissolving DPPC,DPTAP and biotin-cap-DPPE. Store the lipid solutions at −20 °C.
1198 | VOL.7 NO.6 | 2012 | nature protocols

protocol
11| Mix lipid solutions at a desired ratio in a glass tube. In the case of observing myosin V walking on actin filaments, atypical lipid composition is DPPC, DPTAP and biotin-cap-DPPE in a weight ratio of 0.85:0.05:0.1 (lipid composition A)19.
12| Dry the organic solvent under a stream of N2 gas, while warming up the glass tube by gripping it with your hand to
facilitate solvent evaporation. To ensure full evaporation, leave the glass tube in a vacuum desiccator for at least 30 min.
13| Add a buffer solution to the glass tube (typical final concentration of lipids, 1 mg ml − 1), vortex it for ~30 s and sonicate
it for ~3 min using the bath sonicator to disperse the lipids in the medium. At this stage, multilamellar lipid vesicles areformed. In the case of lipid composition A, we use Milli-Q water instead of a buffer solution.
14| Divide the lipid suspension into small aliquots (10 µl each in a 0.5-ml sample tube).
 PAUSE POINT After preparation, the aliquots containing lipid vesicles can be stored at − 80 °C before use.

© 2012 Nature America, Inc. All rights reserved.

15| Preparation of mica-SLBs. Thaw the multilamellar lipid vesicles stored at −80 °C by gripping the tube with your hand.
Dilute the solution to 0.05–0.1 mg ml − 1 by adding a buffer solution. In the case of lipid composition A, we add a 10 mM
MgCl2 solution for the dilution.
16| To obtain small unilamellar vesicles, sonicate the multilamellar vesicle suspension (typically 100–150 µl) with the probesonicator at intervals of 1 s with a duty ratio of ~0.5 until the suspension becomes transparent (typically no more than 30 cycles).
17| Deposit a drop (~2 µl) of the small unilamellar vesicle solution on freshly cleaved mica glued on a sample stage(prepared in Steps 8 and 9). Usually, we prepare 3–5 vesicle-deposited sample stages at once.
18| Put the vesicle-deposited sample stages in the sealed container, in the inside of which a Kimwipe wetted with Milli-Q
water is stuffed. Incubate the vesicle-deposited sample stages until the mica surfaces are fully coated by lipid bilayers(Fig. 6). In the case of lipid composition A, the incubation time is more than 3 h (typically overnight) after the deposition.
The container can maintain high humidity to avoid sample drying. To keep the sample stages from falling, place them on
the Gel-Film glued on a glass slide (Fig. 6c). The sealed container should be used upside down so that the sample stages
are easily handled with tweezers. The details of other useful lipid compositions, practical methods to prepare mica-SLBs andtheir application to several samples are well described elsewhere35.
 PAUSE POINT After preparation, mica-SLBs on the sample stages can be stored in the sealed container for up to 3 d at room
temperature (25 °C).
Preparation and mounting of the AFM liquid cell ● TIMING ~10 min
19| Clean the AFM liquid cell equipped with the cantilever holder by using a piece of Super cloth containing mild detergent,and then rinse it abundantly with tap water. Sonicate it for 3 min in a beaker filled with tap water and further rinse it repeatedly with Milli-Q water. Then, place it on a Kimwipe and dry it using a rubber hand blower.
20| Clamp a small cantilever to the cantilever holder as shown in Figure 7a.
 CRITICAL STEP The cantilever should be tightly clamped into the holder, otherwise unwanted vibrations and insufficient
feedback control will result (Fig. 7)46.
21| Mount the AFM liquid cell on the high-speed AFM apparatus. Gently wash the liquid cell 2–3 times using an imaging
buffer (~60 µl) to remove dirt, while sucking the buffer using a peristaltic pump. Next, fill the liquid cell with the imaging
buffer (~60 µl).
! CAUTION Do not fill the liquid cell with more than 60 µl of imaging buffer. Otherwise, the z-piezo to be mounted later will
get wet and result in a short circuit and thus in fatal damage when high voltage is applied.
 CRITICAL STEP After this step and until the end of AFM measurement, keep the cantilever from drying. Dirt contaminants
tend to be accumulated on the cantilever during drying by water evaporation.
Laser alignment ● TIMING ~3 min
22| Align the laser focusing position relative to the cantilever by moving both lateral and vertical positions of the cantilever
holder so that the intensity of light reflected back from the cantilever is maximized (Fig. 8 and Supplementary Video 1).
23| Adjust the photodetector position so that the reflected laser guided to the photodetector makes a spot at its center(Supplementary Video 1).
nature protocols | VOL.7 NO.6 | 2012 | 1199

protocolaWetted Kimwipesb

Sealed container(bottle cap)
Sample stage

Sealed container (bottle)
Sample stage (inside)c

Sealed container (bottle)
Wetted Kimwipes
Droplet containing
lipid vesicles
Mica
or
HOPG

Glass rodsample stage

Gel-Film
WettedKimwipes
Gel-Film
glued on glas slide

Sealed container(bottle)Glass slide

WettedKimwipes(inside)1 Euro coin

Wetted Kimwipes

Sealed container (bottle cap)dWetted Kimwipes(inside)e

Sealed container (bottle cap)

Humid hood

© 2012 Nature America, Inc. All rights reserved.

Humid hoodx-piezo (x-scanner)(inside of elastomer)

Glass rodsample stage

Monolithically madehigh-speed scanner
metal part

Wetted Kimwipes

StopperDroplet containing
samples
Mica or HOPG

z-piezo(z-scanner)

Dummy weightfor counterbalancing
y-piezo(y-scanner)

Glass rodsample stage

Sealing ringz-piezo(z-scanner)

Metal part of high-speed scanner

Figure 6 | Storage tools used to preserve a solution droplet on the sample stage without drying it. (a) Photograph showing an opened container containing
sample stages (note that the sample stages are placed in the bottle cap). (b) Photograph showing the sealed container. (c) Schematic showing the sealedcontainer. (d) Photograph showing a humid hood used during incubation of solution on a sample stage glued onto the z-piezo. (e) Schematic showing thehumid hood covered over the sample stage glued onto the z-piezo.

Cleaning of the z-piezo surface ● TIMING ~1 min
24| Clean the surface of the z-piezo (z-scanner) with a piece of Super cloth wetted with acetone to remove any dust and to
confirm the flatness of the z-piezo surface.
Sample deposition on the substrate (modify) ● TIMING ~15–30 min
25| Procedures for sample deposition onto a substrate surface depend on the surface to be used. For sample deposition
onto mica-SLBs with biotin (for example, acto-myosin V) go directly to Step 28C. For other types of sample deposition(for example, rotorless F1-ATPase and cellulose + cellulase), perform Steps 26 and 27 first, and then choose the appropriate
option in Step 28.
26| Place a drop of nail polish on the top of z-piezo. Immediately after that, place a sample stage with a glued mica or
HOPG disk on the top of the z-piezo using tweezers and wait for 10 min until the sample stage is firmly fixed on the z-piezo.
 CRITICAL STEP To avoid contamination, do not touch the sample stage except when placing the z-piezo.
27| Cleave a few top layers of the mica or HOPG using adhesive tape (e.g., Scotch tape) to have a clean surface. If you cannot cleave the surface anymore, remove the glass stage from the z-piezo and go to the recycling process (Step 45), and thenrepeat Steps 24–27.
28| For direct sample deposition onto a bare surface of mica or HOPG, go to option A. For covalent immobilization of proteins
onto a mica surface with glutaraldehyde, go to option B. To observe walking myosin V, go to option C (proceed directlyfrom Step 25).
 CRITICAL STEP Carefully inspect that the mica or HOPG surface shows a clean surface without burrs after the cleavage.
1200 | VOL.7 NO.6 | 2012 | nature protocols

protocolac30

z-piezo(z-scanner)

Side view

20
10
Buffer solution

Cantilever baseGain (dB)

Sample stage

0
–10

Mica

Liquid cell
(cantilever holder)

Small cantilever–20

Glass slide
d1

–30

d2
Cantilever baseTop view

360

Mica

0

Small cantileverb–720–1,080–1,440Side view–1,8001

d1
Top view

d2

Signal (V)d10Frequency (kHz)

100

1,000

2.5

25

2.0

20

1.5

15

1.0

10

0.5

5

0

Signal (nm)© 2012 Nature America, Inc. All rights reserved.

Liquid cell
(cantilever holder)

Phase (deg)

–360

0
0

20

40

60

80

100

Time (µs)

Figure 7 | Importance of firmly mounting the cantilever base and selecting the imaging region for successful imaging. (a) Schematic showing appropriate
imaging region selection and holding of the cantilever base. The cantilever base is deeply inserted into the cantilever holder. The imaging region locates at anouter area of the mica disk. The distances of d1 and d2 shown should be less than 1 mm and 0.2–0.3 mm, respectively. (b) Schematic showing inappropriate
imaging region selection and holding of the cantilever base. (c) Frequency responses of the sinusoidally driven z-scanner displacement detected by deflectionof a small cantilever whose tip is in contact with the sample stage. The red and navy blue curves show the responses detected in the appropriate setting
shown in a and in the inappropriate setting shown in b, respectively. The peaks at 171 kHz indicate the first resonant frequency of the z-scanner. Note thatthe z-scanner is actually moving in the same way for both a and b, but in the setting shown in b the cantilever as a sensor cannot precisely detect thez-scanner movement, because the cantilever chip itself is moved by a hydrodynamic pressure produced by z-scanner movement. (d) Amplitude responses ofan oscillating cantilever to z-piezo movement. The cantilever tip is in contact with the sample stage. Black curve shows a signal input to the z-piezo; the red
curve shows the response in setting a; the navy-blue curve shows the response in the setting b (considerable delay occurs in the response).

(A) Deposition directly onto bare mica or HOPG surface ● TIMING ~5 min(i) Deposit a drop of sample solution onto the freshly cleaved surface, cover the sample stage with a humid hood(Fig. 6d,e), and wait for a while (typically 3 min). After that, rinse the deposited sample with an appropriate buffer
solution to remove any excess of molecules (Supplementary Video 2). Go to HS-AFM imaging (Step 29). In the caseof observations of cellulase on cellulose on an HOPG substrate23, 2 µl of cellulose suspension in water (0.1–0.5%(vol/vol)) was dropped on the freshly cleaved HOPG surface and incubated for 5–10 min. Then the solution was rinsed
with 18 µl of buffer A before AFM imaging (cellulase was added while imaging was going on).
 CRITICAL STEP Avoid drying of the sample solution on the substrate disk in order to prevent degeneration of thebiomolecules attached to the substrate surface.

nature protocols | VOL.7 NO.6 | 2012 | 1201

protocol© 2012 Nature America, Inc. All rights reserved.

Figure 8 | Optical microscopic views of a smallcantilever. (a,b) Magnified views of a smallcantilever without incident laser (a) and withincident laser (b). (c,d) Low-magnified views of
the cantilever when the mica surface is far fromthe cantilever (c) and when the surface is close
to the cantilever (d).

acdMica surface

20 µm

Small cantilever

Edge ofglass stage

Cantilever baseb(B) Covalent immobilization of
200 µm200 µm
20 µm
proteins onto a mica surface withglutaraldehyde ● TIMING ~12 min
 CRITICAL STEP This immobilization method has been applied to observe rotary catalysis of the α3β3 subcomplex of
F1-ATPase with Lys-tag at the N terminus22. Therefore, we here describe the procedure to immobilize this protein. However,the procedure should be adaptable for many other proteins with amino groups on their outer surfaces, although theorientation of the immobilized molecules is mainly determined by the position of the most reactive amino group.
(i) Deposit a drop (~2 µl) of 3-aminopropyl-triethoxysilane (0.05–0.1% (vol/vol) in Milli-Q water) on a freshly cleaved
mica surface, cover the sample stage with a humid hood (Fig. 6d,e), wait for 3 min, and rinse the surface(Supplementary Video 2) with Milli-Q water.
 CRITICAL STEP The preparation of a diluted 3-aminopropyl-triethoxysilane solution should be done just before
deposition because the aminosilane quickly reacts with water.
(ii) Deposit a drop (~2 µl) of glutaraldehyde (diluted to 0.1–0.25% (wt/vol) with Milli-Q water), cover the sample stage
with a humid hood (Fig. 6d,e), wait for 3 min, and rinse the surface (Supplementary Video 2) with a buffer solution
containing no primary amine (we used 10 mM HEPES/NaOH (pH 7.4), 10 mM KCl, 5 mM MgSO4).
(iii) Deposit a drop (~2 µl) of protein solution (1–10 nM, in a buffer containing no primary amine), cover the sample stage
with a humid hood (Fig. 6d,e), wait for 5 min, and then rinse the surface (Supplementary Video 2) with a buffer
solution containing a reagent with primary amine (we used 10 mM Tris-HCl (pH 8.0)) to terminate residual active
glutaraldehyde on the surface.
(iv) After that, we can use any buffer solutions to observe the protein molecules immobilized on the surface (we usedbuffer B for observing the α3β3 subcomplex of F1-ATPase). Proceed to HS-AFM imaging (Step 29).
 CRITICAL STEP Avoid drying of the sample solution on the substrate disk in order to prevent degeneration of thebiomolecules attached to the substrate surface.
(C) Deposition for observing walking myosin V ● TIMING ~20 min(i) From the sealed container, take a sample stage with mica on the top of which SLBs are formed (Fig. 6a–c).
(ii) Place a drop of nail polish on the top of the z-piezo. Immediately after that, place the sample stage on the top of thez-piezo by using tweezers. After that, immediately cover the humid hood over the sample stage as shown in Figure 6d,e.
 CRITICAL STEP Do not dry the planar lipid bilayers on mica, or most of the bilayers will certainly be damaged.
These procedures should therefore be performed quickly (ideally, within 1 min).
(iii) Rinse the sample surface (Supplementary Video 2) with Milli-Q water dropwise (20 µl × 5) and replace the solution
with buffer C. Then, deposit a drop (2 µl) of streptavidin (1 µg ml − 1) in buffer C on the bilayer surface, cover the
sample stage with a humid hood (Fig. 6d,e), and then wait for 3 min.
 CRITICAL STEP Rinse the sample thoroughly for removing the residual vesicles.
(iv) After rinsing with buffer C (20 µl × 3), deposit a drop (2 µl) of partially biotinylated actin filaments that are stabilizedwith phalloidin (~1 µM in buffer C) on the bilayer surface, cover the sample stage with a humid hood (Fig. 6d,e),
and then wait for 10 min.
 CRITICAL STEP Rinse the sample thoroughly to remove the residual streptavidin.
(v) After rinsing with buffer C (20 µl), deposit a drop (2 µl) of myosin V-HMM solution (0.1–1 nM) on the bilayer surface,cover the sample stage with a humid hood (Fig. 6d,e), and wait for 3 min. At this point, the sample stage becomes
firmly fixed on the z-piezo. Go to HS-AFM imaging (Step 29).
 CRITICAL STEP In order to avoid contamination, do not touch the sample surface. By using the humid hood (Fig. 6d,e),maintain the sample surface covered with a buffer solution so as not to degenerate the molecules on the surface.
? TROUBLESHOOTING
High-speed AFM imaging (approach) ● TIMING ~5 min
29| Mount the scanner, with the z-scanner attached to the sample stage with a sample to observe, over the liquid cell
containing a buffer solution (Supplementary Video 3).
! CAUTION Make sure that the top of the sample stage is sufficiently far away from the cantilever to avoid crashing. If it
crashes, the cantilever is often damaged and all previous procedures have to be repeated.
1202 | VOL.7 NO.6 | 2012 | nature protocols

protocol
30| Adjust the scanner position so that the cantilever tip is positioned near the substrate disk (Fig. 8 and Supplementary
Video 3).
 CRITICAL STEP Positioning of the lever portion of the cantilever to a region near the substrate disk edge is crucial to avoid
disturbance of the amplitude measurement by low frequency vibrations of the lever and cantilever chip itself, which are produced
by a hydrodynamic pressure45,46.
31| Execute a coarse approach while carefully watching the cantilever and the substrate disk surface on the digital camera(or CCD camera) display. Once the substrate disk surface is seen on the monitor, stop the approach.
 CRITICAL STEP To avert a crash, this careful watching and quick halting of the coarse approach is very important. It isalways better to halt the coarse approach before a clear view of the substrate disk surface appears.
32| Measure a thermal fluctuation spectrum of the cantilever using the fast Fourier transform (FFT) analyzer (in the HS-AFM
software) to find the first resonant frequency of the cantilever.

© 2012 Nature America, Inc. All rights reserved.

33| Apply sinusoidal AC voltage to a piezo actuator fixed in the cantilever holder to excite the cantilever at the measured
resonant frequency.
34| Adjust the cantilever oscillation amplitude to 0.1–0.5 V while looking at the oscillation signal on an oscilloscope screen(typical amplitude: 1–5 nm; the amplitude in nm cannot be exactly known before calibrating the optical lever sensitivity.
See Step 43).
35| Adjust the amplitude detector gain to have its output of 0.1–0.5 V.
36| Adjust the set point voltage of the PID controller to ~60–70% of the amplitude detector output voltage, and thenswitch on the PID controller. Upon switching on, the z-piezo becomes fully extended because the cantilever tip is not yet incontact with the sample surface.
37| While carefully checking both PID output and cantilever amplitude signal, execute slow approach until the computer
automatically stops the approach upon detecting tip-sample contact (a software program is always monitoring both theamplitude and PID output).
 CRITICAL STEP When the cantilever is approaching the sample surface, the oscillation amplitude usually increases first(due to an unspecified effect). Readjust the set point voltage of the PID controller to ~60–70% of the amplitude detector
output voltage.
? TROUBLESHOOTING
38| After the approach is automatically halted, adjust the z-piezo position by changing the bias DC voltage of the z-piezo
driver so that the PID output voltage becomes around 0 V. Slowly increase the integral gain of the PID controller until the
feedback signal starts to oscillate. Then slightly decrease the gain so that the oscillation ceases. Do likewise for the
proportional gain and the derivative gain.
High-speed AFM imaging (imaging) ● TIMING usually 2–5 h
39| Before starting imaging, increase the set point voltage of the PID controller to ~95% of the free oscillation amplitude.
40| Start imaging. At first you will obtain noisy images like the one shown in Figure 9 (0.15 s) and Supplementary Video 4.
Slowly decrease the set point voltage of the PID controller until clearer images appear (Fig. 9, 2.94–3.24 s). Owing to ‘tip
parachuting’ (i.e., complete detachment of the cantilever tip from the sample surface at the downhill regions of the sample),noisy lines extending to the plus x-direction (imaging direction) appear in the images. In this stage, you can choose oneof the following two operations to obtain clear images like those shown in Figure 9 (6.33–11.92 s) and Supplementary
Video 4. Further decrease the set point voltage or switch the PID controller from the conventional mode to the dynamic PID
control mode8 and then adjust the gain that is especially equipped for this mode in addition to the PID gains. In this mode,
the PID gain is unchanged for the uphill regions of the sample but is increased for the downhill regions8. The effect ofthe dynamic PID control mode is shown in Supplementary Video 5. The choice depends on the fragility of your sample.
For samples with higher fragility, the latter operation should be chosen.
41| When no improvement is attained in the image quality, change the cantilever chip to a new one, because noisy
images are usually due to tip smear. When the image quality is good enough, move to the next steps.
? TROUBLESHOOTING
nature protocols | VOL.7 NO.6 | 2012 | 1203

protocol© 2012 Nature America, Inc. All rights reserved.

Figure 9 | Clipped high-speed AFM images showing relationship between
applying tapping force and image quality. From 0.15 s to 3.24 s, tapping
force was gradually increased. In other words, the set point voltage of the
PID controller was gradually decreased to increase the tapping force. Next,
appropriate objects, depending on the particular experiment (in this case,actin filaments), are searched by laterally moving the sample stage bychanging the offset DC voltages of the piezo-driver for the x- and y-scanners.
In this model experiment, myosin V–HMM movement (from bottom left to top
right) along an actin filament was observed (see the arrows). Imaging rate,
6.8 fps; scan size, 150 × 75 nm2; number of pixels, 80 × 40; z-scale, 10 nm.

0.15 s6.33 s11.18 s2.94 s10.15 s3.09 s10.74 s11.77 s3.24 s

10.89 s11.92 s+ End

11.62 s42| Find target molecules by scanning different areas(change the offset DC voltages of the x- and y-piezo
30 nm
drivers) and readjust imaging parameters (cantilever
oscillation amplitude, excitation frequency and set point
voltage) until high-resolution images appear (Figs. 10 and 11; Supplementary Video 4).
 CRITICAL STEP The AFM tip often picks up contaminants, which considerably deteriorates the image quality. When thisoccurs, repeatedly vary the set-point voltage within a certain range while imaging. By this operation, the cantilever tip
interacts with the sample strongly and weakly. This procedure can sometimes remove attached contaminants and improves
the image quality (Fig. 11). It is advisable to scan over a wider area and to use a faster frame rate to increase the tipsample interaction, which might be effective for removing attached contaminations from the AFM tip. After capturing
enough images, switch off both the PID controller and the wave generator used for exciting the cantilever.
43| Measure force curves to calibrate the OBD detection sensitivity48.
 CRITICAL STEP To know the cantilever oscillation amplitude not in voltage but in nm, calibration should have been done
before starting imaging experiments. However, because force curve measurements are apt to damage the cantilever tip, we
often make this calibration after completing imaging. The OBD detection sensitivity does not change much as long as thesame type of cantilever is used.
44| Release the sample surface from the cantilever tip using the stepper motor used for the tip approach.
Recycling of glass stages ● TIMING ~1 d
45| After finishing the imaging experiments, remove the sample stage from the z-piezo using acetone. Then immerse theused sample stages in nondiluted sulfuric acid to remove adhesives and clean the glass rods.
! CAUTION Never detach the glass rod by applying force, or the z-piezo will be damaged.
 CRITICAL STEP Put drops of acetone around the interface between the z-piezo and the glass rod and wait until the glassrod naturally detaches from the z-piezo.
Recycling of cantilevers ● TIMING ~1 d
46| After use, rinse the cantilevers with Milli-Q water. To remove the EBD tip and contaminants from the cantilevers, apply
RF argon/oxygen plasma etching to the cantilever over 1 d. Typical power of the RF plasma is about 20 W and the gaspressure is ~180 mTorr.

a 147 ms

30 nm

b

c 0s

+
293 ms0.75 s*d –ATP

e

0s

15 nm
5 nm

*

+ATP

30 nm
5.44 s

734 ms
5 nm

Figure 10 | High-speed AFM images of proteins in action. (a) Swinging lever arm motion at theleading head of myosin V–HMM on an actin filament19. The ‘+’ symbol indicates the plus end of theactin filament. (b) bR in response to light (black and green bars indicate dark and light illumination
conditions, respectively)20. (c) Alternate binding of GroES (marked with the asterisks) to two rings of
GroEL34. (d) ATP-induced conformational change of β-subunits in rotorless F1-ATPase (α3β3 subcomplex)22.
(e) Cellulase moving left while hydrolyzing cellulose fibers23. Each arrow (yellow or blue) indicates the
position of a cellulose molecule.
1204 | VOL.7 NO.6 | 2012 | nature protocols? TROUBLESHOOTING
Sample preparation (Step 28C)
When you are not yet used to thesample deposition, you should
confirm whether or not each step for
sample preparation (e.g., lipid-bilayer
preparation, streptavidin deposition,actin deposition) is successful by
imaging the sample surface with
HS-AFM after each step is done.
Tip approach (Step 37)
You may have a difficulty in executing
the slow approach because of oscillation of the PID controller or reduction

protocol
Figure 11 | Effect of AFM tip smear and its removal on image quality.
Top, schematics illustrate the speculated respective situations from therespective AFM images. Improvement of the image quality of double-stranded
DNA (dsDNA) is demonstrated. The z-positions of a substrate disk areindicated with broken lines drawn nearby. At 2.73 s, the AFM tip picked up
contaminants, resulting in a blurred image. By varying the set point voltageof the PID controller, the image quality drastically improved (rightmost
image). Imaging rate, 9.9 fps; scan size, 600 × 600 nm2; z-scale, 3.5 nm.

Mean shape ofoscillating cantilever
Dirt

dsDNA

Mica
7.68 s2.73 s

7.58 s34.07 s200 nm© 2012 Nature America, Inc. All rights reserved.

of the reflected laser power. When this problem is encountered, you should check the fixing of the glass stage on thez-piezo, inspect the mica disk or readjust the sample position relative to the cantilever tip (Fig. 7).
Imaging (Step 41)
When no improvement is attained in the image quality even after changing the cantilever once or twice, at least one step inprevious procedures has not been done appropriately. It is difficult to find a cause for noisy images. The best troubleshooting is to conduct each step as described in this protocol, particularly with respect to the indicated critical steps. For samples
that we have imaged many times (such as myosin V and rotorless F1-ATPase), we can obtain fairly good images with asuccess rate of over 80%. However, whether or not we can capture high-quality images largely depends on the tip condition.
Accordingly, we often change the cantilever chips one after another until we can acquire high-quality images.
● TIMING
Steps 1–5, fabrication of cantilever tips: 3–5 h (depending on the number of cantilevers)
Steps 6 and 7, preparation of mica and HOPG disks: ~30 min
Steps 8 and 9, preparation of sample stages: more than 3 h (working time ~1 h)
Steps 10–14, preparation of lipid vesicles: ~3 h
Steps 15–18, preparation of mica-SLBs: variable, depending on lipid compositions (typical working time, ~15 min)
Steps 19–21, preparation and mounting of the AFM liquid cell: ~10 min
Steps 22 and 23, laser alignment: ~3 min
Step 24, cleaning of the z-piezo surface: ~1 min
Steps 25–28, sample deposition on the substrate: ~15–30 min
Steps 29–38, high-speed AFM imaging (approach): ~5 min
Steps 39–44, high-speed AFM imaging (imaging): usually 2–5 hStep 45, recycling of glass stages: ~1 d
Step 46, recycling of cantilevers: ~1 d
ANTICIPATED RESULTS
HS-AFM is a new method for directly obtaining visual information on dynamics of functioning proteins. For instance,
the following dynamics have thus far been successfully captured on video: the myosin V lever arm swing at the leadinghead after trailing head detachment from actin (Fig. 10a)19, the structural change of bR upon green light illumination(Fig. 10b)20,21, alternate binding of GroES to the two rings of GroEL (Fig. 10c)34, the structural change of rotorless
F1-ATPase during ATP hydrolysis (Fig. 10d)22, the processive run of cellulase on crystalline cellulose fibers (Fig. 10e)23,
the dynamics of bR 2D crystals24, amyloid-like fibril formation from lithostathine25, the wiggling motion of intrinsicallydisordered regions of FACT protein26, the membrane-mediated interaction between c-rings of ATP synthase27, and the
dynamics of nucleosomes28. Although it is only imaged from the top direction, the whole topography of a protein
molecule and its minute temporal changes can be visualized with HS-AFM. Unlike fluorescence microscopy, various
dynamic events of molecules appear in an AFM video without planned selection, meaning that both expected andunexpected dynamic molecular behaviors can appear simultaneously. Therefore, we have a great chance of learning thedetails of several facts of molecular action in one imaging experiment. Moreover, the molecular action that appears onvideo can often be interpreted in a straightforward manner without sophisticated analysis, thereby helping to obtainconvincing conclusions. These excellent general features of HS-AFM imaging markedly facilitate and accelerate ourunderstanding of the functional mechanisms of proteins.

nature protocols | VOL.7 NO.6 | 2012 | 1205

protocol
Note: Supplementary information is available in the online version of the paper.
Acknowledgments We thank D. Yamamoto for technical assistance. This work
was supported by the Core Research for Evolutionary Science and Technology(CREST) program of the Japan Science and Technology Agency (JST); a Grant-inAid for Basic Research (S) from the Japan Society for the Promotion of Science(JSPS) (no. 20221006); a Grant-in-Aid for Scientific Research on Innovative
Areas (Research in a proposed Research Area) from the Ministry of Education,
Culture, Science, Sports and Technology (MEXT)-Japan; and the Knowledge
Cluster Initiative/MEXT-Japan.
AUTHOR CONTRIBUTIONS  All the authors designed and discussed the experiments.
T.U. and N.K. equally contributed to this work, conducted the experiments, prepared
all figures and movies, and drafted the MATERIALS and PROCEDURE sections.
T.A. wrote the introductory part of manuscript and edited the whole manuscript.
COMPETING FINANCIAL INTERESTS  The authors declare no competing financial
interests.

© 2012 Nature America, Inc. All rights reserved.

Published online at http://www.nature.com/doifinder/10.1038/nprot.2012.047.
Reprints and permissions information is available online at http://www.nature.
com/reprints/index.html.
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