1354

Biophysical Journal

Volume 97

September 20091354–1361

Contact-Mode High-Resolution High-Speed Atomic Force Microscopy
Movies of the Purple Membrane
Ignacio Casuso,† Noriyuki Kodera,‡§ Christian Le Grimellec,{ Toshio Ando,‡ and Simon Scheuring†*†

Institut Curie, Équipe Institut National de la Sante et de la Recherche Médicale Avenir, Unite Mixte de Recherche 168-Centre National de laRecherche Scientiﬁque, Paris, France; ‡Department of Physics, Kanazawa University, Kanazawa, Japan; §Core Research for Evolutional
Science and Technology, Japan Science and Technology Agency, Tokyo, Japan; and {Nanostructures et Complexes Membranaires, Centre
de Biochimie Structurale Institut National de la Sante et de la Recherche Médicale U554, Montpellier, France

ABSTRACT High-speed atomic force microscopy (HS-AFM) is becoming a reference tool for the study of dynamic biologicalprocesses. The spatial and time resolutions of HS-AFM are on the order of nanometers and milliseconds, respectively, and allow
structural and functional characterization of biological processes at the single-molecule level. In this work we present contactmode HS-AFM movies of purple membranes containing two-dimensional arrays of bacteriorhodopsin (bR). In high-resolution
movies acquired at a 100 ms frame acquisition time, the substructure on individual bR trimers was visualized. In regions in
between different bR arrays, dynamic topographies were observed and interpreted as motion of the bR trimers. Similarly, motionof bR monomers in the vicinity of lattice defects in the purple membrane was observed. Our ﬁndings indicate that the bR arrays
are in a mobile association-dissociation equilibrium. HS-AFM on membranes provides novel perspectives for analyzing themembrane diffusion processes of nonlabeled molecules.

INTRODUCTION
The atomic force microscope is a powerful tool for the characterization of biological samples. It can provide highresolution images of biological molecules, such as DNA
complexes (1) or membrane proteins (2,3), and enables
imaging in physiological buffers at room temperature andunder atmospheric pressure (4) with an outstanding signal/
noise ratio (5). Because of these features, atomic force microscopy (AFM) is believed to have great potential for observing
the dynamics of individual biological molecules. Unfortunately, however, the slowness of image acquisition (one toseveral minutes per image) by conventional AFM has
hampered that application. To observe the dynamics of biological molecules, image acquisition times of %1 s must be
attained. Therefore, a new generation of high-speed (HS)atomic force microscopes needed to be developed. The
main limiting factor of the imaging rate of AFM is the
response speed of the moving components of the atomic force
microscope, i.e., the probe and the scanner. The response
speed of these components is a function
pﬃﬃﬃofﬃ the inverse of
the square root of their mass (v  1= m). Therefore, toincrease the imaging rate of AFM, the use of lighter probes
and scanners was required, and consequently the control electronics and optical cantilever detection path of the atomic
force microscope had to be adapted. Since about 2000,
HS-AFM has been developed by three groups, known as
the Hansma (6,7), Miles (8,9), and Ando (10,11) groups.
Because of its fast feedback loop capacities, the Ando typeof HS-AFM proved to be particularly adequate for observing
single biomolecules at work (12,13).

Submitted March 6, 2009, and accepted for publication June 15, 2009.
*Correspondence: simon.scheuring@curie.fr
Editor: Peter Hinterdorfer.
Ó 2009 by the Biophysical Society
0006-3495/09/09/1354/8 $2.00

In conventional AFM and HS-AFM systems, precise
control of the force applied by the probe on the sample iscrucial for successfully imaging biological samples. It has
been observed (in conventional AFM) that applied forces
higher than ~300 pN applied during the imaging of soft biological samples typically distort or damage the samples (14).
In a good case, when the force can be precisely controlled tominimal values (50–100 pN), a resolution high enough toobserve details on individual biological molecules withdimensions of ~1 nm can be achieved (5). In conventional
AFM, two strategies have been used to image biological molecules at high resolution with controlled minimal force: 1), in
contact mode, soft probes of low spring constant (0.01–
0.1 N/m) are used that provide a high deflection/force ratio(16–19); and 2), in oscillating mode, operation probes of~0.1 N/m are used and oscillated, reducing the friction between
the probe and the sample (20–22). In conventional AFM, thecontact mode is more commonly used for high-resolution
imaging, probably because the surface contact time is ~9 times
larger (at the same scan rate) compared to the oscillating mode,
where only the lower part of the sinusoidal tip movement
makes contact with the sample. So far, HS-AFM imaging of
biological molecules at high resolution has only been achievedin oscillating mode, probably because the mass-reduced
probes in HS-AFM have spring constants of ~0.1 N/m, andbecause intuitively the lateral forces due to friction and thefeedback loop delay may increase during fast scanning.
In this work we show that by carefully controlling theapplied force, it is possible to obtain high-resolution imagesof membrane proteins in contact-mode HS-AFM. We show
movies of individual bacteriorhodopsin (bR) molecules at a
100 ms frame acquisition time. These movies reveal thestructural and positional stability of bR inside the bR lattices,doi: 10.1016/j.bpj.2009.06.019

Molecular-Resolution Movies of PM

and molecule diffusion events at the bR lattice edges. We
demonstrate the power of HS-AFM for directly visualizing
the membrane diffusion processes of nonlabeled molecules.
MATERIALS AND METHODS
Sample preparation
Purple membranes (PMs) from Halobacterium salinarum containing wildtype bR were isolated according to Oesterhelt and Stoeckenius (23). PM
patches were deposited on freshly cleaved mica, prepared as previously
described (24). To check the cleavage quality, the mica was first imaged
in 50 mL of an adsorption buffer containing 10 mM Tris-HCl (pH 7.6),
150 mM KCl, 25 mM MgCl2. Subsequently, 3 mL of PM solution were injected into the adsorption buffer drop on the mica surface. After 15 min thesample was rinsed with 10 volumes of a recording buffer containing 10 mM
Tris-HCl (pH 7.6), 150 mM KCl.

HS-AFM
The bR molecules were observed in contact mode using Ando-type
HS-AFM (10). A high-sensitivity photodetector was implemented and thephotodetector signal was low-pass filtered (pass % 5 MHz). Probes designedfor HS-AFM (Olympus, Tokyo, Japan) were used with a spring constant of
0.1–0.2 N/m, a resonance frequency of 0.8–1.2 MHz, and a quality factor of~2 in solution (25). In contrast to the usual operation of Ando-type HS-AFM
in oscillating mode, the probes did not feature any additional electron beam
deposition (EBD) tip at the end of the microfabricated pyramidal tip (26).
The sensitivity of the AFM system to probe deflection was 0.1 V/nm. The
scan areas were 660  660 and 1136  1136 (Å)2, and the number of pixels
per frame was 200  200. The imaging rate was 10 frames/s.

Data analysis
Individual frames were flattened and the frame shift was corrected by crosscorrelation for movie assembly using the WSxM software (Nanotec, Madrid,
Spain) (27). All image analysis was performed using self-written routines for
the ImageJ image processing package (5,28,29). Averaging of aligned molecule images was achieved by addition and normalization of pixels in theimage matrix according to Ave ¼ 1=NSðxij Þ, where N is the number ofmolecules averaged, and xij stands for the gray value of pixel i in particle
image j. Concomitantly, a standard deviation (SD) map was calculated
rﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ.
according to the following equation: SD ¼ 1 N  Sðxij  xj Þ2 .

RESULTS
High-resolution contact-mode HS-AFM was performed on
the extracellular surface of native PMs containing two-dimensional (2D) arrays of trimeric bR, a light-driven proton pumpof the seven-transmembrane-helix protein family (e.g., mammalian rhodopsin and GPCRs). Image acquisition was performed at a 100 ms frame frequency on a 660 Å square fieldscanned at a 200 pixel edge width (Fig. 1 A). Fig. 1 shows
fifty consecutive image frames (5 s of a longer movie; seeMovie S1 in the Supporting Material) that reveal similar
image quality, and in which the individual bR molecules areresolved. We were able to perform contact-mode HS-AFM
imaging at minimal loading forces (~50 pN); therefore,
imaging was nondestructive to the membrane during longterm experiments. At 100 ms imaging frequency and a scan

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size of 660 Å at a sampling of 200 pixels, the AFM probes
each pixel, (3.3 Å)2 in size, during only 2.5 ms. This is fasterby ~2 and 3 orders of magnitude compared to conventional
contact-mode or oscillating-mode imaging, respectively.
The load per area and time is consequently also diminishedby such a factor, rendering the acquisition less invasive. Individual frames (Fig. 1 B, top panel) reveal imaging quality
comparable to that of conventional slow contact-mode (30)and oscillating-mode (21) images of the extracellular surfaceof bR. As can be readily seen in raw data topographs, averaging and concomitant calculation of SD maps revealed thetrimeric architecture of the extracellular surface of the bR(Fig. 1 C, top panel). Despite the fast sampling rate on eachpixel, the height and SD values are similar to those acquiredin low-speed high-resolution imaging (31). Similarly, themajority of the molecules found in the image have a crosscorrelation value (CCV (5)) between 0.6 and 0.74, witha noise-free ensemble average used as reference (Fig. 1 D,
top panel). The symmetry of the trimers is also nicely
preserved, with the majority of the molecules revealing aninternal symmetry (IS) (5) between 0.7 and 0.8 (Fig. 1 E,
top panel). To learn more about the influence of the scanand sampling rates, we merged two, five, and 10 subsequent
images in the movie, resulting in image (and pixel) samplingtimes of 200 ms (5 ms), 500 ms (12.5 ms), and 1 s (25 ms),
respectively (Fig. 1 B, bottom three panels). The corresponding CCV (Fig. 1 C, bottom three panels) and IS (Fig. 1 D,bottom three panels) analysis of the molecules revealed thatboth characteristics increased with longer acquisition times
toward a CCV and IS of 0.8. From this, we learn that ourimage frames at the 100 ms rate contain some minor noise,
which we may learn to eliminate in the future. Nevertheless,the best molecules in the 100 ms acquisition time images revealed a CCV of 0.75 and IS of 0.8, indicating the feasibilityof high-speed, low-noise image acquisition. Whatever the
sampling rate and the quantity of noise present in the images,
the averages all show the same features, and we estimate that
all averages have the same lateral resolution of ~20 Å, using
our stringent resolution criterion (5). Indeed, the averages
show the same features as averages presented by subnanometer resolution contact-mode and oscillating-mode imaging(21,30). Another aspect that merits close examination is theimage stability. Distortions may be introduced on the bR
lattice, for three major reasons: 1), piezo drift, creep, andimperfections can distort the entire image; 2), lattice disruption due to the scan mechanism can alter the distance between
the molecules; and 3), real protein movement may also resultin distance changes between molecules. To analyze this, we
performed a pair correlation function (PCF) analysis of fiveimages (Fig. 1 F). PCF analysis graphs the probability offinding another molecule as a function of the distance within
an image; it is therefore sensitive to short- and long-range
variations between molecules. In Fig. 1 F we plot the average(black line) and SD (gray lines) of the intermoleculardistances found. In all analyzed images, a sharp highBiophysical Journal 97(5) 1354–1361

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Casuso et al.

FIGURE 1 High-resolution contact-mode HS-AFM of the extracellular surface of individual bacteriorhodopsin (bR) molecules. (A) Fifty aligned images(660 Å  660 Å; 200 pixel  200 pixel) recorded at 100 ms time intervals, from 5 s of a longer HS-AFM movie. The image quality is consistent and depicts
the trimeric assembly of individual bR molecules in the purple membrane (PM). (B) From top to bottom: one individual frame (frame sampling time: 100 ms;
pixel sampling time: 2.5 ms), and merge of two, five, and 10 subsequent frames with 200 ms, 500 ms, and 1 s acquisition sampling time. (C) Averages and SD
maps (n ¼ 46) corresponding to the images shown in B; the averages have a full color range of 0.5 Å < height < 6.7 Å, and the SD maps have a full color scaleof 0.5 Å < SD < 2.3 Å, 0.5 Å < SD < 2.1 Å, 0.4 Å < SD < 1.8 Å, and 0.4 Å < SD < 1.6 Å, respectively. (D) Histogram showing the number of individual
molecules found in the images shown in B as function of their CCV with a noise-free ensemble average. (E) Histogram showing the number of individual
Biophysical Journal 97(5) 1354–1361

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probability peak appears at 63 Å, corresponding to the nearest
neighbors in the bR lattice (a ¼ b ¼ 62.5 Å, g ¼ 120 (32));
also, the long-distance peaks (multiples of the lattice) are wellpreserved. This analysis illustrates the stability of the image
acquisition, corroborates the preservation of the bR latticeunder the scan conditions employed here, and affirms thehigh lateral stability of the bR molecules within their 2D array.
Finally, we also analyzed the IS of the average (n ¼ 46) offrame 038 (shown at the top of Fig. 1 B), which peaks at
0.86 for threefold symmetry, indicating a minor tip asymmetry (Fig. 1 G).
One aspect that merits particular attention in the high-resolution analysis movie is the detection of a lattice vacancy
consisting of one missing bR monomer (Fig. 2, cell D4).
HS imaging of the area of the vacancy showed an induced
instability of the surrounding bR monomers, where sporadic
motion of the adjacent monomers into the vacant area isobserved (Fig. 2 C, cell D4). Nonetheless, the vacancy
remains unoccupied most of the time, which may suggest
a structural malformation of the area of the vacancy, occupation of the vacant area with lipid molecules, or restrictedrotational freedom of the molecules and consequent weakassociation of invading molecules within the vacancy. The
sporadic motion of the adjacent monomers to the vacancy
was recorded over a time span of 700 ms (Fig. 2, A–D; cells
D4–D5); this measurement illustrates the power of the technique for membrane diffusion analysis.
At medium resolution, we imaged two differently orientedbR lattices and a nonordered membrane region in between.
Image acquisition was performed at a 100 ms frame
frequency on a 1136 Å square field scanned at a 200 pixeledge width (Fig. 3 A). The fifty consecutive image frames(pixel sampling (5.7 Å)2) represent 5 s of a longer movie(Movie S2). The high imaging rate did not disturb thefeatures, and all images in Fig. 2 A and throughout the movie
reveal a similar topography. Most of the individual image
frames depict the different lattice orientations of the bR
lattice in the top left and the one on the bottom of the frame(Fig. 3 B). Lateral alignment of all image frames with respect
to each other allowed the calculation of an average image(Fig. 3 C). In this figure, although the features are somewhat
blurred by the averaging process, the two independent bR
lattices in the membrane, the lattice edges, and the area inbetween are well visible. At a 100 ms frame acquisitiontime, movements of proteins can be observed by HS-AFM(13,33). Knowing that except for some defects, the bR molecules inside the 2D lattice are essentially immobile (34), we
focused on the membrane region in between the two bR
arrays (Fig. 3, B and C; yellow dashed outline). Indeed, in
this region fine topographical changes as a function of timeare visible in the images acquired at the 100 ms frame rate

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(Fig. 3 D), as documented in the corresponding movie(Movie S3). For example, in the frames shown in Fig. 3 D,
a topographical feature appears in frames 5–8. Although
our resolution is currently insufficient to assign this topography unambiguously to bR, its dimension of ~60 Å corresponds well to the size of a bR trimer. Furthermore, thepresence of the topographical feature clearly localized during
four subsequent frames (400 ms) is a strong argument that
we are not looking at imaging noise, but at a real topographic
feature in the PM. We hypothesize that individual bR trimers
diffuse between the edges of the two bR lattices, and thiscan be observed by high-resolution contact-mode HS-AFM.
This finding indicates that the native bR 2D arrays in themembranes are in association-dissociation equilibrium withneighboring bR arrays, as was recently shown in a study using
oscillating-mode HS-AFM (35).
DISCUSSION
Because of the speed of the analog feedback control and thehigh sensitivity of the photodiode (10,26), it is possible toprecisely control (precision: ~10 pN) the force applied tothe tip during contact-mode HS-AFM imaging with cantilevers with a spring constant of ~0.1 N/m. Using high-resolution contact-mode HS-AFM, not only are we able to observe
individual bR molecules in the PM, we can also detect the
motion of the bR molecules. Investigators have attempted
to use conventional AFM to analyze the movement of
membrane proteins (36,37), but at a frame acquisition timeof ~1 min only snapshots of stable states can be acquired.
Now, with the use of HS-AFM, protein motion can be visualized, and the myosin motor movement (11), the chaperonin
cycle of GroEL (38), and the association-dissociation
process of streptavidin in 2D arrays (33) have been observed.
Here we present evidence for the observation of membraneprotein diffusion using contact-mode HS-AFM. The
contact-mode HS-AFM methodology introduced here was
successfully tested on PMs. This methodology is appropriate
for samples with low corrugation; in these samples, blunt tips
provide the best results because they distribute the applied
force over a broader area due to electrostatic balancing(14). This situation contrasts with oscillating-mode HS-AFM
studies of biological samples, in which probes featuringhigh-aspect-ratio EBD tips provided the best results (26).
However, these probes are inconvenient in that growing
the EBD tips is a time-consuming process that demands
sophisticated electron microscope technology and expertise.
Finally, in our study, the aspect ratio of the EBD tips proved
to be unsuitable for contact-mode HS-AFM. These probes,
when used in contact mode, often lead to destruction of themembranes during imaging.

molecules found in the images shown in B as function of their IS. (F) Average (black line) and SD (gray lines) of the PCF between bR trimers, peaking at 63 Å
(FWHH: 5 Å) of five individual image frames, showing the high precision and stability of the scanner. (G) Symmetry analysis of the average calculated from
one image frame (B and C, top) peaking for threefold symmetry at 0.85.
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FIGURE 2 Sporadic motion of a bR monomer adjacent to a lattice vacancy. Images acquired at different times are shown: (A) t ¼ 0, (B) t ¼ 200 ms, (C) t ¼
500 ms, and (D) t ¼ 700 ms. The four columns represent i), fully acquired images (660 Å  660 Å; 200 pixel  200 pixel); ii), digital zoom images in theregion of the vacancy (409 Å  409 Å; 124 pixel  124 pixel), where a mesh is included for identifying individual bR lattice cells; iii), selection of relevant
cells for the observation of the motion of bR monomers; and iv), schematic of the positions of bR monomers. From this sequence, it can be concluded that backand-forth motion of a single bR monomer takes place between the adjacent bR trimer (cell D5) and the vacancy (cell D4).

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Molecular-Resolution Movies of PM

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FIGURE 3 Medium-resolution contact-mode HS-AFM of the extracellular surface of bR lattices and their interface. (A) Fifty aligned images (1136 Å 
1136 Å; 200 pixel  200 pixel) recorded at 100 ms time intervals, from 5 s of a longer HS-AFM movie. The image quality is consistent and reveals two differently
oriented bR lattices interspaces by protrusions and depressions. (B) One individual frame (frame sampling time: 100 ms; pixel sampling time: 2.5 ms) of the movie.
The different orientations of the two lattices (top left and bottom of the image) are visible. (C) Average of the aligned images in the movie depicts with stronger
contrast the two bR lattices (white dashed outlines) with different orientation and the interspaces comprising a protruding and a lipid region. (D) Ten aligned
images of the region (yellow dashed outline in B and C) in between the two bR lattices. Protein movement is visible in frames 5–8 (white dashed circles).

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The imaging conditions implemented (100  100 nm2,
100 ms per frame) open the door to diffusion tracking ofmultiple membrane proteins. Considering hr2i ¼ 4Dt (39),where hr2i is the mean-square displacement, D is the diffusion coefficient, and t is the time, we find that the fastest
diffusion coefficient observable at an image rate of 100 ms
would be 2.5  104 nm2/s (0.025 mm2/s). Proteins with diffusion coefficients below this limit include the cystic fibrosis
transmembrane conductance regulator (D ¼ 0.005 mm2/s(40)), a glycosylphosphatidylinositol-anchored protein (D ¼
0.0035–0.0800 mm2/s (41)), the transferrin receptor (D ¼
0.005mm2/s (42)), and the glycoproteins in macrophages(D ¼ 0.003mm2/s (43)). Therefore, we are confident that
HS-AFM will soon provide the first structural movies ofnonlabeled membrane protein diffusion.
SUPPORTING MATERIAL
Three movies are available at http://www.biophysj.org/biophysj/supplemental/
S0006-3495(09)01152-7.
This study was supported by Agence National de la Recherche grants ANR08-NANO-010 and ANR-PCV08_343399, and by the Ville de Paris (Appelà projets de la Ville de Paris en Recherche Médicale et en Santé).

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Biophysical Journal 97(5) 1354–1361