doi:10.1016/j.jmb.2012.05.018

J. Mol. Biol. (2012) 422, 300–309
Contents lists available at www.sciencedirect.com

Journal of Molecular Biology
journal homepage: http://ees.elsevier.com.jmb

Single-Molecule Imaging on Living Bacterial Cell
Surface by High-Speed AFM
Hayato Yamashita 1 †, Azuma Taoka 2, 3 †, Takayuki Uchihashi 1, 3 ,Tomoya Asano 4 , Toshio Ando 1, 3 ‡ and Yoshihiro Fukumori 2, 3 ⁎‡1

School of Mathematics and Physics, College of Science and Engineering, Kanazawa University, Kakuma-machi,
Kanazawa 920-1192, Japan
2
School of Natural System, College of Science and Engineering, Kanazawa University, Kakuma-machi,
Kanazawa 920-1192, Japan
3
Bio-AFM Frontier Research Center, College of Science and Engineering, Kanazawa University, Kakuma-machi,
Kanazawa 920-1192, Japan
4
Division of Functional Genomics, Advanced Science Research Center, Kanazawa University, Kakuma-machi,
Kanazawa 920-1192, Japan
Received 12 April 2012;
accepted 14 May 2012
Available online
18 May 2012
Edited by
S. Kowalczykowski
Keywords:
scanning probe microscopy;
live cell imaging;
porin;
molecular resolution;
molecular dynamics

Advances in microscopy have contributed to many biologic discoveries.
Electron microscopic techniques such as cryo-electron tomography are
remarkable tools for imaging the interiors of bacterial cells in the near-native
state, whereas optical microscopic techniques such as ﬂuorescence imaging
are useful for following the dynamics of speciﬁc single molecules in living
cells. Neither technique, however, can be used to visualize the structuraldynamics of a single molecule at high resolution in living cells.
In the present study, we used high-speed atomic force microscopy(HS-AFM) to image the molecular dynamics of living bacterial cell surfaces.
HS-AFM visualizes the dynamic molecular processes of isolated proteins at
sub-molecular resolution without the need for complicated sample
preparation. In the present study, magnetotactic bacterial cells wereanchored in liquid medium on substrate modiﬁed by poly-L-lysine andglutaraldehyde. High-resolution HS-AFM images of live cell surfaces
showed that the bacterial outer membrane was covered with a net-likestructure comprising holes and the hole rims framing them. Furthermore,
HS-AFM captured the dynamic movement of the surface ultrastructure,
showing that the holes in the net-like structure slowly diffused in the cellsurface. Nano-dissection revealed that porin trimers constitute the net-likestructure. Here, we report for the ﬁrst time the direct observation of
dynamic molecular architectures on a live cell surface using HS-AFM.
© 2012 Elsevier Ltd. All rights reserved.

*Corresponding author. Department of Life Science, Graduate School of Natural Science and Technology, Kanazawa
University, Kakuma-machi, Kanazawa 920-1192, Japan. E-mail address: fukumor@staff.kanazawa-u.ac.jp.
† H.Y. and A.T. contributed equally to this work.
‡ T.A. and Y.F. contributed equally to this work.
Abbreviations used: AFM, atomic force microscopy; HS-AFM, high-speed atomic force microscopy; MALDI-TOF/
TOF, matrix-assisted laser desorption/ionization time-of-ﬂight tandem mass spectrometry; TEM, transmission electron
microscopy.
0022-2836/$ - see front matter © 2012 Elsevier Ltd. All rights reserved.

301

Single-Molecule Imaging by High-Speed AFM

Introduction
Imaging of single molecules within a cell isachieved using various techniques. Conventionaltransmission electron microscopy (TEM) techniquesreveal supramolecular structures at high resolution,
but these techniques require sample preparation
methods such as ﬁxation, staining, dehydration,embedding, and thin sectioning, all of which
potentially damage or alter the native supramolecular structure. Cryo-electron microscopy does not
require prior sample preparation and therefore
allows for visualization of molecular structures ina near-native, frozen hydrated state. 1,2 While thecryo-electron microscopy shows the static molecular
images, the molecular dynamics of the specimen
cannot be visualized because the specimen is in afrozen hydrated state. Optical microscopic methods,
such as ﬂuorescent video imaging, show molecular
dynamics in single living cells. 3,4 The optical
microscopic technique has been applied to imaging
of bacterial cells and provides insights aboutmolecular dynamics in vivo. For example, Kim etal. observed the dynamics of the single cytoskeletal
protein MreB in living bacterial cells using a totalinternal reﬂection ﬂuorescence microscope. 5 Thismethod visualizes ﬂuorescence signals of labeled
target molecules, and therefore only indirect molecular movement can be detected as a trajectory.
Moreover, the spatial resolution of optical microscopy is not sufﬁcient for observing the dynamic
structural change of intracellular molecules. Visualization of the structural dynamics in living cells isnecessary to gain more insight into the precise
function of biologic molecules.
Atomic force microscopy (AFM) is a complementary technique that has been used to visualize labelfree organic samples at sub-nano-order resolution
under physiologic conditions. Recent advances in
AFM provide new opportunities to study single cells
and single molecules in microbiology. 6,7 In particular, visualization of the architecture of a living cell
is a novel powerful approach to unveil the molecular organization in vivo. For example, the hexagonal S-layer of Corynebacterium glutamicum, 8 thepeptidoglycan organization of Lactococcus lactis, 9
and the germinating spores of Bacillus atrophaeus 10
have been directly visualized in living microbial
cells using AFM.
The time required to record a high-resolution
image with conventional AFM is much longer than
the timescale of most biologic processes. Therefore,
visualization of the molecular dynamics in living
cells using AFM has not been achieved. High-speed
atomic force microscopy (HS-AFM) was developed
to simultaneously capture the structure and dynamics of functioning biomolecules. 11–13 Large
improvements in its performance were recently
demonstrated in practical imaging studies of pro-teins in vitro. 14–16 Moreover, the dynamics ofmembrane proteins isolated from bacterial cells
can be visualized at high resolution with HSAFM. 17–19 HS-AFM will become even more usefulin biologic science if it can be used to observe themolecular dynamics of living cell membranes. A
large number of membrane proteins play importantroles in cell function (e.g., transport, signal transduction for extracellular stimuli, and maintenance of
cellular structure). Little is known, however, about
their dynamic molecular processes on living cells.
Here, we used HS-AFM to visualize the dynamics
of molecular complexes on living cell surfaces. In thepresent study, we used the magnetotactic spirillum
Magnetospirillum magneticum AMB-1 as a specimen
for observing the live cell surface, because previous
TEM analyses have provided data about the outermembrane structure of this bacterium. 20,21 Highresolution HS-AFM images of the live cell surface
showed that the bacterial outer membrane iscovered by a net-like structure, and the movies
captured by HS-AFM revealed dynamic movementsof outer membrane proteins.

Results
AFM observations of living cells on mica
M. magneticum AMB-1 cells were chemicallyimmobilized on mica to observe the detailed surface
architecture of living cells. First, bare mica wascoated with poly-L-lysine and then treated withglutaraldehyde. After the remaining unreacted
glutaraldehyde was completely removed by washing at least 10 times, the cell suspension was loaded
onto the treated mica and incubated for 1 h. Basedon light microscopic observations, the cells weredensely attached to the mica substrate (Fig. S1). The
viability of the immobilized cells was assessed using
a LIVE/DEAD BacLight Bacterial Viability Kit (Fig.
1a and b). Most of the cells remained alive on themica for at least 1 h after immobilization, which isenough time for HS-AFM observation. HS-AFM
images of cells immobilized on mica are shown in
Fig. 1c and d. The cell shape revealed by HS-AFM
was consistent with that observed by TEM (Fig. 1e).
The cells had a spiral shape, which is the characteristic architecture of M. magneticum AMB-1. The
height and width of the bacterial cells were
measured vertically along the long axis of the cells
to estimate the cell diameter (Fig. 1f). Based on AFM,
the cell was 542 ± 65 nm wide and 526 ± 78 nm high(n = 12), whereas based on TEM, the cell diameterwas 653 ± 74 nm ( n = 12). The larger diametermeasured on TEM images may be due to observation of the dried sample in the vacuum environment,
whereas AFM allows for visualization of the three-302

Single-Molecule Imaging by High-Speed AFM

(a)

(b)

(c)

(d)

600 nm

540 nmHeight0 nm(e)600 nm1100 nmHeight(f)

600 nmdimensional cellular structure in liquid environment. The magnetotactic bacterium M. magneticum
AMB-1 has unique bacterial organelles—‘magnetosomes’—that comprise the membrane vesicle andmagnetite crystal. TEM images revealed chain-likearrays of magnetosomes (Fig. 1e). AFM observation,
however, revealed no structural signature on the cellsurface at the predicted position of the magnetosomes. This ﬁnding suggests that intracellularmagnetosomes are not affected on the outer surface
structure.
Direct visualization of molecules on the living
cell surface
We recorded highly magniﬁed AFM images ofcells in liquid medium to visualize the molecules on
the cell surface. Figure 2a and b show the cell surface

0 nm

Fig. 1. Imaging of M. magneticum
AMB-1 cells using AFM. (a) LIVE/DEAD BacLight bacterial viability
stain of M. magneticum AMB-1 cells
in phosphate-buffered saline. (b)Viability of immobilized cells on
mica. Live bacteria with intact
membranes ﬂuoresce green, while
those with damaged membranes
ﬂuoresce red. (c) AFM observationof a living M. magneticum AMB-1
cell chemically immobilized onmica in medium. (d) AFM imageof M. magneticum AMB-1 cells
densely attached on mica. All cells
had a spiral shape and a smoothsurface. (e) TEM observation of M.
magneticum cells. Magnetosome
chains were observed in the centerof the cell (arrows). (f) Surface
proﬁle along the line indicated in(c). AFM images were recorded at
imaging rates of 5.0 s/frame with
200 × 200 pixels (c and d).

morphology at ranges of 150 × 150 nm 2 and100 × 100 nm 2, respectively. The cell surface was
not ﬂat but had a slightly undulating topography(Fig. 2c: cross section of Fig. 2a) and small holes all
over. These holes produced a net-like structure (Fig.
2b) composed of the holes and hole rims (frame).
The cell surface was covered by the net-like hole
structures, which were irregularly arranged, suggesting that these structures are not packed tightly.
The diameter of the holes in the net-like structure
was 7.3 ± 1.4 nm (n = 210) and the depth was 0.85 ±0.52 nm (n = 210; Fig. 2e). Compactly arranged
particles were observed in the frame (Fig. 2b,arrows). Movie S1 shows the entire surface of awhole cell. When the imaging area was moved
along the long axis of the cell, these structures wereobserved on the entire cell surface. All of the cells
that were examined under high magniﬁcation had

303Single-Molecule Imaging by High-Speed AFM

(b)

(a)

30 nm

23.5 nm0 nm

Height20 nm

2.2 nm0 nm

Height(d)(c)(e) 6060

40

NumberNumber

N=211

20

N=211

40
20
0

0
0

5
10 15
Diameter (nm)

20

0

2

4

Depth (nm)these structures. We also observed the surface
structure of cells that were cultivated in low-ironmedium (1.8 μM Fe). The iron concentration,
however, did not affect the architecture or thenumber of hole structures (Fig. S2).
Dynamic molecular movement on living cells
To reveal the molecular dynamics on a living cellsurface, we recorded successive AFM images of theouter cell surface in liquid medium. Figure 3a shows
still images of a successive AFM movie (Movie S2)
with a frame time of 0.5 s. Many particles wereobserved on the rims of the holes in the net-likestructure (Fig. 3a, arrows). Because the particles
rapidly diffused in these structures (Movie S2), we
could not accurately follow their movements.
Representative trajectories of the holes in the netlike structure on the cell surface are shown in Fig. 3b
and c. A single hole in the net-like structure exhibits

6

Fig. 2. Highly magniﬁed AFM
observation of the cell surface. (a)AFM image of the cell surface at a
range of 150 × 150 nm 2. The cellsurface appeared undulated. (b)AFM image of cell surface at a
range of 100 × 100 nm 2. Small holes
were observed over the entire cellsurface. Compactly arranged particles were observed on the frame(the rims of these holes) in a net-likestructure (arrows). (c and d) Surface
proﬁle along the lines indicated in(a) and (b), respectively. (e) Histogram for widths and depths of netlike structure observed on the cellsurface. Curve represents the ﬁt to a
Gaussian distribution (diameter,
7.3 ± 1.4 nm; depth, 0.85 ± 0.52 nm).
AFM images were recorded at
imaging rates of (a) 2.0 s/frame,(b) 0.7 s/frame, and 200 × 200 pixels(a and b).

random movement, indicating that these movements are not the effect of a tip–sample interaction
force, but rather molecular diffusion on the living
cell surface. The diffusion speed was extracted fromeach single-structure trajectory (n = 23). The diffusion constants for individual holes are shown in Fig.
3d. Wade et al. reported that the diffusion constantsof molecules in the cell membrane follow a lognormal distribution. 22,23 In our study, the distribution of diffusion constants was ﬁtted with a lognormal distribution and the mean diffusion constantwas 3.2 ± 0.4 nm 2/s. This is the ﬁrst report of theobservation of molecular dynamics on a living cellsurface using HS-AFM. Because of the slow diffusion speed, the AFM scanning speed (0.5 s/frame)
used for the observation seems to have sufﬁcient
temporal resolution to detect the movement of theholes in the net-like structure (ca 7.3 nm indiameter). A faster scanning rate (0.2 s/frame) did
not affect the diffusion speed or the resolution of the304

Single-Molecule Imaging by High-Speed AFM

(a)

(b)

20 nm

20 nm

1.6 nm(c)

Height0 nm

1.6 nm0 nm

Height(d)
MSD (nm2)

200

100

0

10

Time (s)net-like structure. We also treated the cell surface
with glutaraldehyde and observed immobile netlike structures (Movie S3). Although movement of
the holes in the net-like structure ceased, moredetails of the structure could not be observed due tomolecular ﬂuctuations and vertical movement in the
non-supported membrane.
In vitro imaging of the isolated outer membrane
M. magneticum AMB-1 belongs to alpha-proteobacteria and is enveloped by two membrane layers:
the outer and cytoplasmic membranes. To elucidate
the composition of the net-like structure, we isolated
the outer membrane of M. magneticum AMB-1 andobserved it using HS-AFM. The puriﬁed outermembrane was ﬁrst negatively stained with uranyl
acetate and observed by TEM (Fig. 4a). The outermembrane formed a round-shaped patch that was
40 to 100 nm in diameter. TEM observation revealed
no distinct structural features in the patches. On theother hand, HS-AFM showed the net-like structureson the membrane patch (Fig. 4b). This structure was
the same as that observed on the living cell surface.
In addition to structural imaging, HS-AFM can beused as both a manipulator for the dissection ofindividual biologic samples and a recorder of thedissection process. 24,25 HS-AFM images of thedissection process of the net-like structure observed

20

Fig. 3. HS-AFM observation of
the molecular dynamics on a living
cell surface. (a) Highly magniﬁed
AFM image of cell surface at a rangeof 80 × 80 nm 2, showing a still imageof a successive AFM movie (Movie
S2). (b) AFM image with trajectories
of four net-like structures. These
trajectories were drawn by tracing
the movement of each hole. (c) Total
trajectories of the two-dimensional
diffusion for four net-like structures. The analyzed time spans
were 82 s for holes 1, 2, and 4, and47.5 s for hole 3. (d) Distribution ofdiffusion constants for individual
trajectories. The data follow a lognormal distribution characterized
by a mean D = (3.2 ± 0.4 nm 2 /s)(continuous line). Inset shows
mean square displacements of individual trajectories. The mean squaredisplacement increased linearlywith time, yielding diffusion constants. AFM images were recorded
at imaging rates of 0.5 s/frame with
200 × 200 pixels (a and b).

on the isolated outer membrane are shown in Fig. 4c
and Movie S4. While the net-like structures werebeing imaged, an additional tapping force wasapplied (3–7 frames). The net-like structures weredisassembled by the scanning stylus, which resulted
in the observation of several triangular molecules in
the membrane patch that were diffusing (13 framesin Fig. 4c). A cross section of membrane patches
before and after dissection is shown in Fig. 4d and e,
respectively. The height of the membrane remained
unchanged before and after dissecting, indicatingthat the structural alteration was not due to thedetachment of any component from the outermembrane. Subsequently, we recorded highly magniﬁed AFM images on the membrane patch after
dissecting. Figure 4f shows that most of themolecules in the membrane patch formed a triangle
shape very similar to the porin trimer structure. 26
Together, the ﬁndings from the dissection experiment provided direct evidence that the triangularmolecule is one of the components constituting thenet-like structure.
Identification of the molecular components inthe net-like structure
An SDS-PAGE proﬁle of the outer membrane
fraction isolated from M. magneticum is shown in
Fig. 4g. Approximately 15 bands were observed.

305

Single-Molecule Imaging by High-Speed AFM

(b)

(a)

Height14.3 nm

20 nm

50 nm

0 nm

(c)

0

4

6

13

9.4 nmHeight

20 nm

0 nm

(d)

(e)

1 2

(g)

(f)

kDa

1.0 nm250
150

Height

100
75
50
37

1 0nm
0 nm

25
20
15
10

Fig. 4. In vitro imaging and nanodissection of isolated outer membrane. (a) TEM image of isolated
outer membrane from M. magneticum AMB-1. (b) AFM image ofisolated outer membrane adsorbedon mica. Net-like structure wasobserved in a membrane patch. (c)HS-AFM observation of dissociation process of a net-like structure
in an outer membrane patch. Numbers indicate frame number. Notethat triangular molecules appeared
after dissection of the net-like structure. (d and e) Surface proﬁle along
the lines indicated in (c). (d) and (e)show the surface proﬁle before(frame number 0) and after (frame
number 13) the dissection process,respectively. (f) Highly magniﬁed
AFM image of molecules observed
after dissociation. (g) SDS-PAGE gel
proﬁle of extracted proteins fromthe isolated outer membrane (lane2). The protein band indicated by
the arrow was identiﬁed as porin.
The molecular masses of the standards (Precision Plus protein standards; Bio-Rad) are indicated on theleft sides of the lanes. AFM images
were recorded at imaging rates of
1.0 s/frame with 100 × 100 pixels (band c) and 160 × 160 pixels (f).

306
The trypsin-digested gel slices containing thesebands were analyzed by MALDI-TOF/TOF (matrix-assisted laser desorption/ionization time-ofﬂight tandem mass spectrometry). The mass spectrafrom the tandem mass spectrometry were analyzed
against the M. magneticum AMB-1 whole genome
database 27 (Fig. S3). The result corresponded to theprevious proteomic analysis of M. magneticum AMB1 membrane fractions. 28 The most abundant proteinin the outer membrane fraction is a 40-kDa protein(Fig. 4g) that was identiﬁed as a porin homolog(amb0025: Msp1). The amino acid sequence of
amb0025 was similar to porin 41 of Rhodospirillum
rubrum (30% identity) and the major outer membrane protein OmaA of Azospirillum brasilense Sp245(26% identity). Tanaka et al. reported the highexpression level of this outer membrane protein,which was used as an anchoring molecule for a cellsurface display system. 29
The crystal structures of a number of bacterial
porins have been deposited in the Protein Data Bank
database. In the present study, AFM images wereconstructed from the crystal structural data by
simulation software SPM simulator (Fig. S4). Extracellular and periplasmic faces of simulated porins
were determined by referring to general bacterial
porin structures reviewed in Refs. 30 and 31. The
simulated AFM images of the extracellular sides arewell reproduced in the observed image (Fig. S4). In
the construct, the three ring structures corresponding to the periplasmic side were never observed in
the HS-AFM images of the outer membrane. The
periplasmic side of the outer membrane might be
attached selectively on the mica. The simulated
porin image is well ﬁtted to the observed triangularstructure. These results suggest that the net-likestructure on the outer cell surface comprises porin
molecules.

Discussion
Recent dramatic improvements in AFM haveopened up new avenues for analyzing cellular
structures, such as isolated membranes 26 and cellenvelopes, 8,9,32 and for probing their spatial organization, interaction, and elasticity at a molecularresolution under physiologic conditions. The temporal resolution of conventional AFM, however, is
not sufﬁcient to analyze the molecular dynamics on
living cell membranes. Direct observation of the
movement of single molecules on living single cells is
one of the most challenging techniques in cellbiology. Our study presents, for the ﬁrst time, direct
observation of dynamic movements of outer membrane proteins on living cells at single molecularresolution using a laboratory-built HS-AFM. We
improved the spatial resolution to capture thedynamics of membrane proteins at a high resolution

Single-Molecule Imaging by High-Speed AFM

and succeeded in visualizing lateral movement andstructural changes of proteins in isolated biologic
membranes. 17–19 HS-AFM is, at present, capable ofimaging soft biologic molecules non-invasively
without damage, suggesting that it has the potential
to capture the molecular dynamics of the surface of aliving cell. The bacterial cell, however, has asubstantially higher topography than isolated molecules. In the present study, we used a longer AFM
stylus, which seemed suitable for cell imaging,
because a normal stylus disturbs imaging when thecells come into contact with the cantilever. We wereable to discern the movement of surface structures at
a lateral resolution of about 2 nm. Although thisresolution is insufﬁcient to determine the precise
molecular organization, the lateral resolution
obtained in this study is the best achieved so far forthe dynamics of living cell surface molecules. The
low resolution may be due to the spiral andundulating topography of the specimen or to thesoftness of the cell surface.
Our AFM observations revealed that the cellsurface is completely covered with a net-likestructure in the liquid medium. The AFM movies
showed many small particles in the net-like structure. Moreover, according to the nano-dissection
experiment and proteomics analysis, we suggestthat outer membrane porin constitutes the net-likestructure. The small particles visualized in the netlike structure may correspond to porin molecules.
The prokaryotic surface layer (S-layer) resembles a
net-like structure. 8,33 While a variety of S-layers
have been observed in archaea and Gram-positivebacteria, only a few Gram-negative bacteria possess
the S-layer. 34,35 We detected no homologs of the Slayer subunit in our proteomic analysis of the M.
magneticum AMB-1 outer membrane fraction. Also,
there is no typical S-layer protein subunit in the
annotated genome sequence of M. magneticum
AMB-1. Additionally, after dissociation of the netlike structure by dissection with the AFM stylus, theheight of the outer membrane patch was notchanged. If the single layer of the S-layer subunit
array is bound to the surface of the outer membrane,
the height of the outer membrane patch should bedecreased. Moreover, the holes and small particles
in the net-like structure randomly moved in themembrane. These ﬁndings are not consistent with acrystalline monomolecular lattice, which could nothave such dynamics.
In this study, we successfully produced highresolution images of cell surface structures onbacterial cells using HS-AFM. Surface structures on
various types of cells were visualized using conventional AFM. High-resolution images were achieved
mainly on microbial cells, 8–10 while high-resolution
images of animal cells remain challenging. 36 Thisdifference may be due to the softness of animal cells.
Actually, Young's modulus values (indicating the307

Single-Molecule Imaging by High-Speed AFM

softness of a sample surface) of living microbial cells(in the 0.1 MPa-to-0.1 GPa range) 37–39 are signiﬁcantly larger than those obtained on animal cells (inthe 100 Pa to 0.1 MPa range), 40–42 which wasestimated by nano-indentation measurements
using AFM. In the next step, further improvementsof our HS-AFM will lead to elucidate the structures
on soft cell surfaces in high resolution.
Our live cell AFM images provide novel visualization of the bacterial cell outer membrane. The netlike arrayed structure might contribute to the highpermeability of the outer membrane. Our observations are the ﬁrst to show the advantage of HS-AFM
for recording dynamic changes on the surfaces ofliving single cells. This report demonstrates theenormous potential of HS-AFM imaging for microbiology and cellular biology. The HS-AFM technique can be applied to other cells such as plant,
animal, protozoan, and yeast cells, and even to
eukaryotic cell organelles such as mitochondria andchloroplasts.

Materials and Methods
Microorganism and culture
M. magneticum AMB-1 (ATCC 700264) was cultured inMS-1 liquid media under an O2 (1%)–N2 (99%) atmosphere at 25 °C in the dark. 43 For AFM analyses, bacterial
cells were collected by centrifugation at 8000 g for 10 min
at room temperature from 45 ml of late exponentiallygrowing cultures (optical density at 600 nm of ca 0.1). The
bacterial pellet was resuspended in 100 μl of MS-1 liquidmedium.
LIVE/DEAD staining
The viability of immobilized M. magneticum AMB-1 on
mica was assessed using the BacLight LIVE/DEAD
staining system according to the manufacturer's protocol(Molecular Probes, Carlsbad, CA). Cells that retained thegreen ﬂuorescence color were live, whereas red ﬂuorescent cells were considered dead. Fluorescence microscopy
images were observed using Eclipse E600 (Nikon, Tokyo,
Japan). Images were acquired using a DS-Qi1Mc monochrome quantitative digital camera (Nikon) and processedwith NIS-Elements software (Nikon) and/or Adobe
Photoshop CS2 (Adobe, San Jose, CA).
HS-AFM measurements
Imaging was performed with a laboratory-built HSAFM, an extensively improved version of the previously
reported AFM. 11,12 The HS-AFM was equipped withsmall cantilevers (k = 0.1–0.2 N/m, f = 800–1200 kHz inwater) and operated in tapping mode. The AFM styli weregrown on each cantilever by electron beam deposition. 44
The previous deposition time was 90 s, whereas thedeposition time in this study was increased to 180 s to

generate a longer stylus, which was adjusted for cell
imaging in this study. The substrate for observation of thecell surface was prepared as follows. Freshly preparedmica was treated by depositing 0.1% poly-L-lysine solution
and left to dry at room temperature. The resulting mica
was then treated with 0.1% glutaraldehyde for 15 min. The
M. magneticum cell suspension was loaded on the treatedmica in liquid medium. After 60 min, the sample wasrinsed with fresh medium and measured in liquidmedium. The puriﬁed outer membrane was adsorbed on
freshly cleaved mica and observed in 300 mM KCl and
10 mM phosphate buffer (pH 7).
Simulation of AFM image
We used software (SPM simulator, Advanced Algorithm Systems Co., Tokyo, Japan) to simulate AFM imagesof the porin attached to a substrate surface at either the
extracellular or the periplasmic side. The simulation was
performed with a simple hard-sphere model. The cantilever tip was modeled as a circular cone (apex angle, 5°) witha small sphere (radius, 0.5 nm) at the apex. The crystal
structures of the porin trimer were used as the samples.
Each atom in the protein was modeled as a hard spherewith a corresponding van der Waals radius. We simulated
AFM images using various radii for the tip–apex sphereand found that a radius of 0.5 nm produced the images
most similar to the actual AFM images. The simulated
images were processed using a low-pass ﬁlter with a cutoff
wavelength of 2 nm, because the spatial resolution of the
AFM image was approximately 1 nm based on 2D Fouriertransformation of the actual AFM images.
Tandem mass spectrometry analyses and protein
identification
In-gel digestion of protein bands and MALDI-TOF/
TOF analysis were performed as described previously. 45
To identify proteins, we used the 4800Plus MALDI-TOF/
TOF™ Analyzer (Applied Bioscience, Carlsbad, CA), andresults were analyzed using Protein Pilot TM software.
Preparation of cellular component
Outer membrane fractions were prepared using themethod reported by Mizushima and Yamada. 46
Specimen preparation for TEM
Formvar- and carbon-coated grids were put on a dropof the cell suspension or puriﬁed outer membrane forabout 1 min to prepare the specimen for TEM observation.
Some of the grids were negatively stained with 4% uranyl
acetate or 2% sodium tungstate for several seconds. The
specimens were studied with a JEOL JEM 2000EX TEM
operating at 120 kV or JEOL JEM 2010FEF TEM operating
at 200 kV in bright-ﬁeld mode.
Physical and chemical measurements
The protein content was determined using the bicinchoninic acid method (BCA Protein Assay Kit, Pierce

308
Chemical) with bovine serum albumin as the standard.
SDS-PAGE was performed according to the method ofLaemmli. 47 Immunoblotting analysis was performed as
described previously. 48

Acknowledgements
This work was supported by a Grant-in-Aid for
Scientiﬁc Research, Basic Research (S) (20221006); a
Grant-in-Aid for Young Scientists (B) (22780063);
and a Grant-in-Aid for Scientiﬁc Research on
Innovative Areas (23111508) from the Japan Society
for the Promotion of Science; and by the Knowledge
Cluster Initiative from the Ministry of Education,
Culture, Sports, Science, and Technology.

Supplementary Data
Supplementary data related to this article can befoundonline at doi:10.1016/j.jmb.2012.05.018.

References
1. Milne, J. L. S. & Subramaniam, S. (2009). Cryo-electron
tomography of bacteria: progress, challenges andfuture prospects. Nat. Rev., Microbiol. 7, 666–675.
2. Tocheva, E. I., Li, Z. & Jensen, G. J. (2010). Electron
cryotomography. Cold Spring Harbor Perspect. Biol. 2,
a003442, http://dx.doi.org/10.1101/cshperspect.
a003442.
3. Li, G. W. & Xie, X. S. (2011). Central dogma at thesingle-molecule level in living cells. Nature, 475,
308–315.
4. Miyanaga, Y., Matsuoka, S. & Ueda, M. (2009). Singlemolecule imaging techniques to visualize chemotactic
signaling events on the membrane of living Dictyostelium cells. Methods Mol. Biol. 571, 417–435.
5. Kim, S. Y., Gitai, Z., Kinkhabwala, A., Shapiro, L. &Moerner, W. E. (2006). Single molecules of thebacterial actin MreB undergo directed treadmilling
motion in Caulobacter crescentus. Proc. Natl Acad. Sci.
USA, 103, 10929–10934.
6. Dufrêne, Y. F. (2008). Towards nanomicrobiology
using atomic force microscopy. Nat. Rev., Microbiol.
6, 674–680.
7. Scheuring, S. & Dufrêne, Y. F. (2010). Atomic force
microscopy: probing the spatial organization, interactions and elasticity of microbial cell envelopes atmolecular resolution. Mol. Microbiol. 75, 1327–1336.
8. Dupres, V., Alsteens, D., Pauwels, K. & Dufrêne, Y. F.
(2009). In vivo imaging of S-layer nanoarrays on
Corynebacterium glutamicum. Langmuir, 25, 9653–9655.
9. Andre, G., Kulakauskas, S., Chapot-Chartier, M. P.,
Navet, B., Deghorain, M., Bernard, E. et al. (2010).
Imaging the nanoscale organization of peptidoglycan
in living Lactococcus lactis cells. Nat. Commun. 1, 27.

Single-Molecule Imaging by High-Speed AFM

10. Plomp, M., Leighton, T. J., Wheeler, K. E., Hill, H. D. &Malkin, A. J. (2007). In vitro high-resolution structuraldynamics of single germinating bacterial spores. Proc.
Natl Acad. Sci. USA, 104, 9644–9649.
11. Ando, T., Kodera, N., Takai, E., Maruyama, D., Saito,
K. & Toda, A. (2001). A high-speed atomic force
microscope for studying biological macromolecules.
Proc. Natl Acad. Sci. USA, 98, 12468–12472.
12. Ando, T., Uchihashi, T. & Fukuma, T. (2008). Highspeed atomic force microscopy for nano-visualizationof dynamic biomolecular processes. Prog. Surf. Sci. 83,
337–437.
13. Ando, T., Uchihashi, T., Kodera, N., Yamamoto, D.,
Miyagi, A., Taniguchi, M. & Yamashita, H. (2008).
High-speed AFM and nano-visualization of biomolecular processes. Pflugers Arch. 456, 211–225.
14. Igarashi, K., Uchihashi, T., Koivula, A., Wada, M.,
Kimura, S., Okamoto, T. et al. (2011). Traffic jams
reduce hydrolytic efficiency of cellulase on cellulose
surface. Science, 333, 1279–1282.
15. Uchihashi, T., Iino, R., Ando, T. & Noji, H. (2011).
High-speed atomic force microscopy reveals rotary
catalysis of rotorless F1-ATPase. Science, 333, 755–758.
16. Kodera, N., Yamamoto, D., Ishikawa, R. & Ando, T.
(2010). Video imaging of walking myosin V by highspeed atomic force microscopy. Nature, 468, 72–76.
17. Shibata, M., Yamashita, H., Uchihashi, T., Kandori, H.
& Ando, T. (2010). High-speed atomic force microscopy shows dynamic molecular processes in photoactivated bacteriorhodopsin. Nat. Nanotechnol. 5,
208–212.
18. Shibata, M., Uchihashi, T., Yamashita, H., Kandori, H.
& Ando, T. (2011). Structural changes in bacteriorhodopsin in response to alternate illumination observedby high-speed atomic force microscopy. Angew.
Chem., Int. Ed. Engl. 50, 4410–4413.
19. Yamashita, H., Voïtchovsky, K., Uchihashi, T., Contera, S. A., Ryan, J. F. & Ando, T. (2009). Dynamics ofbacteriorhodopsin 2D crystal observed by high-speed
atomic force microscopy. J. Struct. Biol. 167, 153–158.
20. Martins, J. L., Keim, C. N., Farina, M., Kachar, B. &Lins, U. (2007). Deep-etching electron microscopy ofcells of Magnetospirillum magnetotacticum: evidence forfilamentous structures connecting the magnetosome
chain to the cell surface. Curr. Microbiol. 54, 1–4.
21. Balkwill, D. L., Maratea, D. & Blakemore, R. P. (1980).
Ultrastructure of a magnetotactic spirillum. J. Bacteriol.
141, 1399–1408.
22. Wade, W. F., Freed, J. H. & Edidin, M. (1989).
Translational diffusion of class II major histocompatibility complex molecules is constrained by theircytoplasmic domains. J. Cell Biol. 109, 3325–3331.
23. Saxton, M. J. (1997). Single-particle tracking: thedistribution of diffusion coefficients. Biophys. J. 72,
1744–1753.
24. Hansma, H. G., Vesenka, J., Siegerist, C., Kelderman,
G., Morrett, H., Sinsheimer, R. L. et al. (1992).
Reproducible imaging and dissection of plasmid
DNA under liquid with the atomic force microscope.
Science, 256, 1180–1184.
25. Hoh, J. H., Sosinsky, G. E., Revel, J. P. & Hansma, P. K.
(1993). Structure of the extracellular surface of the gap
junction by atomic force microscopy. Biophys. J. 65,
149–163.

Single-Molecule Imaging by High-Speed AFM

26. Jaroslawski, S., Duquesne, K., Sturgis, J. N. &Scheuring, S. (2009). High-resolution architectureof the outer membrane of the Gram-negative
bacteria Roseobacter denitrificans. Mol. Microbiol. 74,
1211–1222.
27. Matsunaga, T., Okamura, Y., Fukuda, Y., Wahyudi,
A. T., Murase, Y. & Takeyama, H. (2005). Complete
genome sequence of the facultative anaerobic magnetotactic bacterium Magnetospirillum sp. strain AMB-1.
DNA Res. 12, 157–166.
28. Tanaka, M., Okamura, Y., Arakaki, A., Tanaka, T.,
Takeyama, H. & Matsunaga, T. (2006). Origin ofmagnetosome membrane: proteomic analysis of magnetosome membrane and comparison with cytoplasmic membrane. Proteomics, 6, 5234–5247.
29. Tanaka, M., Nakata, Y., Mori, T., Okamura, Y.,
Miyasaka, H., Takeyama, H. & Matsunaga, T.
(2008). Development of a cell surface display
system in a magnetotactic bacterium, “Magnetospirillum magneticum” AMB-1. Appl. Environ. Microbiol. 74,
3342–3348.
30. Schulz, G. E. (2002). T0068e structure of bacterial outermembrane proteins. Biochim. Biophys. Acta, 1565,
308–317.
31. Koebnik, R., Locher, K. P. & Van Gelder, P. (2000).
Structure and function of bacterial outer membraneproteins: barrels in a nutshell. Mol. Microbiol. 37,
239–253.
32. Garcia, R., Magerle, R. & Perez, R. (2007). Nanoscale
compositional mapping with gentle forces. Nat. Mater.
6, 405–411.
33. Messner, P., Steiner, K., Zarschler, K. & Schaffer, C.
(2008). S-layer nanoglycobiology of bacteria. Carbohydr. Res. 343, 1934–1951.
34. Lee, S. W., Sabet, M., Um, H. S., Yang, J., Kim, H. C. &Zhu, W. (2006). Identification and characterization of
the genes encoding a unique surface (S-) layer ofTannerella forsythia. Gene, 371, 102–111.
35. Fletcher, C. M., Coyne, M. J., Bentley, D. L., Villa, O. F.
& Comstock, L. E. (2007). Phase-variable expression ofa family of glycoproteins imparts a dynamic surface toa symbiont in its human intestinal ecosystem. Proc.
Natl Acad. Sci. USA, 104, 2413–2418.
36. Muller, D. J. & Dufrêne, Y. F. (2011). Atomic force
microscopy: a nanoscopic window on the cell surface.
Trends Cell Biol. 21, 461–469.

309
37. Touhami, A., Nysten, B. & Dufrêne, Y. F. (2003).
Nanoscale mapping of the elasticity of microbial cellsby atomic force microscopy. Langmuir, 19, 4539–4543.
38. Gaboriaud, F. & Dufrêne, Y. F. (2007). Atomic force
microscopy of microbial cells: application to nanomechanical properties, surface forces and molecular
recognition forces. Colloids Surf., B, 54, 10–19.
39. Xu, W., Mulhern, P. J., Blackford, B. L., Jericho, M. H.,
Firtel, M. & Beveridge, T. J. (1996). Modeling andmeasuring the elastic properties of an archaeal surface,
the sheath of Methanospirillum hungatei, and theimplication of methane production. J. Bacteriol. 178,
3106–3112.
40. Radmacher, M. (2002). Measuring the elastic properties of living cells by the atomic force microscope.
Methods Cell. Biol. 68, 67–90.
41. Hoh, J. H. & Schoenenberger, C. A. (1994). Surface
morphology and mechanical properties of MDCK
monolayers by atomic force microscopy. J. Cell Sci.
107, 1105–1114.
42. Radmacher, M., Fritz, M., Kacher, C. M., Cleveland,
J. P. & Hansma, P. K. (1996). Measuring the viscoelastic properties of human platelets with the atomic
force microscope. Biophys. J. 70, 556–567.
43. Blakemore, R. P., Maratea, D. & Wolfe, R. S. (1979).
Isolation and pure culture of a freshwater magnetic
spirillum in chemically defined medium. J. Bacteriol.
140, 720–729.
44. Yamamoto, D., Uchihashi, T., Kodera, N., Yamashita,
H., Nishikori, S., Ogura, T. et al. (2010). High-speed
atomic force microscopy techniques for observing
dynamic biomolecular processes. Methods Enzymol.
475, 541–564.
45. Asano, T. & Nishiuchi, T. (2011). Comparative
analysis of phosphoprotein expression using 2DDIGE. Methods Mol. Biol. 744, 225–233.
46. Mizushima, S. & Yamada, H. (1975). Isolation andcharacterization of two outer membrane preparations
from Escherichia coli. Biochim. Biophys. Acta, 375, 44–53.
47. Laemmli, U. K. (1970). Cleavage of structural proteinsduring the assembly of the head of bacteriophage T4.
Nature, 227, 680–685.
48. Taoka, A., Asada, R., Sasaki, H., Anzawa, K., Wu, L. F.
& Fukumori, Y. (2006). Spatial localizations of Mam22
and Mam12 in the magnetosomes of Magnetospirillum
magnetotacticum. J. Bacteriol. 188, 3805–3812.