OPENSUBJECT AREAS:
ATOMIC FORCEMICROSCOPY
TIME-LAPSE IMAGINGAPPLICATIONS OF AFM
SINGLE-MOLECULE BIOPHYSICS

Received
30 November 2014
Accepted
2 February 2015
Published
4 March 2015

Correspondence andrequests for materials

Long-tip high-speed atomic force
microscopy for nanometer-scale imaging
in live cells
Mikihiro Shibata1,2, Takayuki Uchihashi3,4,5, Toshio Ando3,4,5 & Ryohei Yasuda1,2
1

Max Planck Florida Institute for Neuroscience, Jupiter, FL 33458, USA, 2Department of Neurobiology, Duke University Medical
School, Durham, NC 27710, USA, 3Department of Physics, Kanazawa University, Kanazawa 920-1192, Japan, 4Bio-AFM Frontier
Research Center, Kanazawa University, Kanazawa 920-1192, Japan, 5CREST/JST, Tokyo 102-0075, Japan.

Visualization of morphological dynamics of live cells with nanometer resolution under physiologicalconditions is highly desired, but challenging. It has been demonstrated that high-speed atomic force
microscopy is a powerful technique for visualizing dynamics of biomolecules under physiologicalconditions. However, application of high-speed atomic force microscopy for imaging larger objects such aslive mammalian cells has been complicated because of the collision between the cantilever and samples.
Here, we demonstrate that attaching an extremely long (,3 mm) and thin (,5 nm) tip by amorphous
carbon to the cantilever allows us to image the surface structure of live cells with the spatiotemporal
resolution of nanometers and seconds. We demonstrate that long-tip high-speed atomic force microscopy iscapable of imaging morphogenesis of filopodia, membrane ruffles, pit formation, and endocytosis in COS-7,
HeLa cells and hippocampal neurons.

should be addressed to
M.S. (mikihiro.
shibata@mpfi.org) or
R.Y. (ryohei.yasuda@
mpfi.org)A

tomic force microscopy (AFM) can image surface topography of objects with the resolution of single
atoms on solid surface1. Furthermore, since AFM imaging can be performed in an aqueous solution, it has
been applied to biological samples such as proteins, nucleic acids, membrane lipids and even live cells
under physiological conditions2–7. AFM is also used for force measurements to estimate the strength of intra- andintermolecular bonds at the single molecule level8,9 and the elasticity of biological objects10,11. However, the use of
conventional AFM for imaging dynamics of live biological samples has been difficult, since it takes many minutes
to acquire an image and a tip motion often damages the sample.
To apply AFM to dynamic biological system, the speed of AFM scanning has been extensively optimized bymodifying each component of AFM12,13. The resulting high-speed AFM (HS-AFM) achieved the scanning speedorders of magnitudes faster than that of conventional AFM, opening a way to monitor the conformational
dynamics of single proteins on substrates with a temporal resolution of subseconds. To avoid damaging biologicalsamples, HS-AFM uses a tapping mode scanning system combined with an extremely soft cantilever (100–200 pN/nm). In the past few years, this technique realized various dynamic processes of biological samples
including photo-induced conformational change of bacteriorhodopsin14–16, myosin V walking on an actin filament17 and rotary catalysis of F1-ATPase18, reaction processes of DNA targeting enzymes19,20, nucleosome
dynamics21,22 and local conformational changes of DNA strands23,24. However, applications of this techniqueto imaging nano-structure of live mammalian cells has been complicated since the length scale of mammaliancells is orders of magnitude larger than that of proteins.
In order to further optimize HS-AFM for cellular imaging, we previously developed HS-AFM with a wide-areascanner, and demonstrated that HS-AFM is capable of imaging large area of mammalian cells (up to ,5 3 5 mm2)with temporal resolution of seconds25. In addition, HS-AFM has been combined with optical microscopy tofacilitate the positioning of the scanning tip to samples26–28. However, because of the collision between a cantileverand cells, it was difficult to image cell surface with high spatial resolution and long-term imaging often caused
severe damages to the cells.
In this paper, we further improved HS-AFM by fabricating an extremely long tip on the cantilever (long-tip
HS-AFM or LT-HS-AFM), and demonstrate that this modification enables us to image nanometer-scale morphogenesis in mammalian cells and hippocampal neurons for more than tens of minutes without damages. In
particular, we show that LT-HS-AFM makes analyses of cellular morphogenesis in response to extracellular
stimulation and pharmacological intervention possible.

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Results
We optimized HS-AFM for live-cell imaging by making following
modifications. First, we fabricated an extremely long (,3 mm), andthin (5–8 nm in diameter) tip by amorphous carbon grown vertically on top of a soft cantilever (100–200 pN/nm) of AFM by using aphenol deposition technique under a scanning electron microscope29 (Fig. 1a). This new tip design enables imaging of cells withhigh spatial resolution with minimum collision to the cell when
scanned under a tapping-mode (,100 nm free amplitude, 800 kHz).
Also, we adapted a wide-area, high speed piezo scanner, which has
the maximum scan rage of 40 3 40 mm2 and is capable of scanning,5 3 5 mm2 area within ,5 s (Ref. 25). In addition, to facilitate
locating the long-tip to the target area, we combined LT-HS-AFM
with fluorescence microscopy (Fig. 1b,c and Supplementary Fig. 1).
Using LT-HS-AFM, we first imaged a COS-7 cell transfected withmEGFP (Fig. 2). We placed the cantilever tip at the leading edge ofthe cell using fluorescence microscopy (Fig. 1c) and scanned over
,10 3 10 mm2 area to observe the dynamics of the surface topologyof the cell at 10 s per frame. LT-HS-AFM imaging of the cell could be
performed for more than 1 hour without any obvious cellular
damage. At the leading edge, the LT-HS-AFM movie showed membrane ruffling and extension and retraction of filopodia over a few
seconds (Fig. 2a, b and Supplementary Movie 1). After the application of an inhibitor of actin polymerization, cytochalasin D30, thesestructural dynamics were abolished (Fig. 2c, d and Supplementary
Movie 1). Subsequently, after the cell was washed out for ,30 min

with the imaging solution, the dynamic movement of the leadingedge was recovered (Fig. 2e, f and Supplementary Movie 2). These
results indicate that the observed membrane dynamics requires actin
polymerization.
It is known that membrane dynamics are regulated by growth
factors and hormones31. Thus, we imaged the leading edge of COS7 cells before and after application of insulin (Fig. 3a). We found that,in response to insulin application, the ruffling at the leading edge wasenhanced (Fig. 3c–f and Supplementary Movie 3). In addition, we
imaged the leading edge of HeLa cells (Fig. 3b), since it is known that
HeLa cells display a retrograde actin flow at the leading edge32.
Consistent with the previous reports32, we observed relatively homogeneous flow of the membrane with speeds of 14.7 6 0.90 nm/s
(before), 3.29 6 0.064 nm/s (after cytochalasin D) and 14.9 6
1.1 nm/s (washout) with LT-HS-AFM (Fig. 3g, h, k and Supplementary Movie 4). Compared to COS-7 cells, the membrane movement was much more directional and uniform. When EGF wasapplied to the cell, the averaged membrane flow was accelerated from
13.5 6 0.83 nm/s (before) to 22.8 6 6.8 nm/s (after EGF) (Fig. 3i, j, l
and Supplementary Movie 4). Thus, these results demonstrate that
LT-HS-AFM is capable of imaging ligand-induced membrane
morphogenesis.
Next, we observed the membrane dynamics near the nucleus of
COS-7 cells (Fig. 4a). In this area, we observed pits cycling between
the open and closed forms on the same area of the plasma membrane(0.474 6 0.044 pits/min/mm2; Supplementary Fig. 2 and Supple-

Figure 1 | LT-HS-AFM system combined with fluorescence microscopy. (a) Scanning electron microscopy (SEM) images of the end of thecantilever without an electron-beam-deposit (EBD) tip (left) and with ,3 mm EBD tip (right). (b) A schematic illustration of LT-HS-AFM system. An arc
lamp for fluorescence excitation and a CCD camera to capture bright-field and fluorescence images were installed under the LT-HS-AFM system.
(c) Fluorescence images of a COS-7 cell transfected with mEGFP. The white broken lines show the shadow of the cantilever. The area indicated with thewhite square was subjected to LT-HS-AFM imaging. LT-HS-AFM images corresponding to c is shown in Figure 2.
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Figure 2 | LT-HS-AFM images of a living COS-7 cell. (a) A LT-HS-AFM topographical image acquired from the area indicated in Figure 1c
before the addition of cytochalasin D. The corresponding movie (10 s per frame) is in Supplementary Movie 1. (b) LT-HS-AFM images taken at theindicated times (green) overlaid with the image at 0 s (magenta). Filled and open white arrows indicate newly appeared and disappeared structures at theleading edge of the cell, respectively. Yellow and red arrows indicate the movement of protrusions and vesicles, respectively. (c-f) LT-HS-AFM image after
the application of cytochalasin D (20 ng/mL) (c, d) and following washout (e, f). See also Supplementary Movies 1 and 2.

Figure 3 | LT-HS-AFM images of living COS-7 and HeLa cells in response to extracellular stimuli. (a, b) Fluorescence image of a COS-7 and HeLa celltransfected with mEGFP. The white dotted lines indicate the shadow of the cantilever. The white square corresponding to the LT-HS-AFM scanning area.
LT-HS-AFM images corresponding to a and b are shown in c-f and g-j, respectively. (c, e) A LT-HS-AFM topographical image of the same cell as in a
before (c) and after (e) insulin application (20 mg/mL). Cells were starved with serum-free medium for ,1 hour before the experiments.
(d, f) LT-HS-AFM images taken at the indicated times (green) overlaid with the image at 0 s (magenta) before (d) and after (f) insulin application. Filled
and open white arrows indicate newly appeared and disappeared structures at the leading edge of the cell, respectively. The corresponding movie (10 s perframe) is in Supplementary Movie 3. (g, i) A LT-HS-AFM topographical image of the same cell as in b before (g) and after (i) EGF application (20 ng/mL).
(h, j) LT-HS-AFM images taken at the indicated times (green) overlaid with the image at 0 s (magenta) before (h) and after (j) EGF application. The
corresponding movie (10 s per frame) is in Supplementary Movie 4. (k) Flow speed of retrograde actin flow at the leading edge of HeLa cells observed by
LT-HS-AFM before and after application of cytochalasin D. (mean 6 s.d.) (l) Flow speed of retrograde actin flow at the leading edge of HeLa cells
observed by LT-HS-AFM before and after EGF application.
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Figure 4 | Pits formation on the plasma membrane of a living COS-7 cell. (a) Fluorescence images of a COS-7 cells transfected with mEGFP. The whitebroken lines show the shadow of the cantilever. The area indicated with the white square was subjected to LT-HS-AFM imaging. (b) The number ofobserved pits per min per mm2 area before, after and washout of dynasore application. (c) A sequence of magnified LT-HS-AFM images of a living COS-7
cell, taken at 6 s per frame, during the pit formation and the closure of the pit with a cap. Blue arrows indicate the formation of the pit. Dotted red circles
indicate the formation of the closure cap. (d) A sequence of magnified LT-HS-AFM images during pit formation and closure of a pit without a cap. Bluearrows indicate the formation of the pit. (e) Time courses of the depth of pits with and without closure caps. More data are shown in Supplementary
Figure 4. Red bars indicate the formation of closure caps. Blue arrows indicate pit formation.

mentary Movie 5). After the application of dynasore, an inhibitor ofdynamin33, these pit dynamics were abolished (0.0454 6 0.014 pits/
min/mm2). Subsequently, the dynamics of pits were partially recovered by washing out the drug for ,30 min (0.238 6 0.044 pits/min/
mm2; Fig. 4b, Supplementary Fig. 2 and Supplementary Movies 5, 6).
These results suggest that the closure of the pits reflects dynamindependent endocytosis. Further, we often observed ‘‘cap-type’’ endocytosis, in which pits are closed by protrusions formed near thepits27,34 (Fig. 4c–e). When we overexpressed the constitutive active
mutant of Rab5, which positively regulates endocytosis35,36, the lifetime of pits became shorter (31.5 6 0.5 s for cells expressing mEGFP
and 14.4 6 0.3 s for cells expressing mEGFP-Rab5(Q79L)), further
suggesting that the pits formation and closure are the processes of
endocytosis (Supplementary Fig. 3 and Supplementary Movie 7).
Next we applied our LT-HS-AFM to visualize morphologicalchanges of live neurons. We plated hippocampal neurons from P0
rats at low density on top of a layer of glia cells placed on a glass stage
and cultured for 9–15 days37,38. We identified a neuron transfectedwith mEGFP using fluorescence imaging and acquired high-resolution topographical images using LT-HS-AFM (Fig. 5a, 6a, 7a). On
thin dendrites, we often observed rapid filopodia extension andretraction (Fig. 5b and Supplementary Movie 8). Also, around dendritic shafts, we observed a thin and sheet-like ruffling structure on topof glia cells. This structure would be difficult to image under an
optical microscope due to its small width (,150–500 nm wide)
and thickness (,70 nm high), which are beyond the resolution of
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optical microscopes (Fig. 6b and Supplementary Movie 9). Pits
undergoing cycles between open and closed states were also observedon the dendritic surface (Fig. 6c, d). These events presumably indicate spontaneous endocytosis events. Some of the closure events wereassociated with cap formations similarly to endocytosis observed in
COS-7 cells. Notably, in relatively mature neurons at 15 days in vitro(DIV), we succeeded in imaging protrusions from dendrites thatappear to be dendritic spines. These images revealed highly dynamic
morphogenesis of the spine-like structure over the time scale ofseconds (Fig. 7b and Supplementary Movie 10).

Discussion
As demonstrated above, the development of LT-HS-AFM clearlyshows the high-resolution AFM movies of retrograde actin flow on
the plasma membrane, morphogenesis of filopodia, membrane ruffles, pit formation, and endocytosis in COS-7, HeLa cells and hippocampal neurons without any significant cellular damages for several
tens of minutes. This is because the long AFM tip robustly steps overcells on the substrate without collisions between a cantilever and cells
during HS-AFM scanning. Furthermore, the sharpness of the tip(,5 nm) provided lateral resolution of nanometers. This long-term
stability of imaging with LT-HS-AFM permitted us to visualize morphological dynamics of cells in response to external stimuli such as
drugs and ligands.
LT-HS-AFM also has limitations. Although LT-HS-AFM canimage nanometer scale structural information, it does not provide
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Figure 5 | LT-HS-AFM images of a living cultured hippocampal neuron at 9 days in vitro. (a) Fluorescence image of a hippocampal neuron transfectedwith mEGFP. The white dotted lines show the shadow of the cantilever. The white square indicates the LT-HS-AFM imaging area. (b) A sequence of LTHS-AFM topographical images of a dendrite captured from the area indicated in a. The white arrows indicate the growth of filopodia. The size of thedendrite is ,400 nm with ,200 nm height. The corresponding movie is in Supplementary Movie 8.

Figure 6 | LT-HS-AFM images of a living cultured hippocampal neuron at 13 days in vitro. (a) Fluorescence image of a hippocampal neuron transfectedwith mEGFP. Corresponding LT-HS-AFM images for a is shown in b. (b) A sequence of LT-HS-AFM topographical images at 5 s per frame. White arrows
indicate the thin, sheet-like ruffling structure. White dotted box at 360 s indicates the magnified region shown in c. The corresponding LT-HS-AFM
movie is shown in Supplementary Movie 9. (c) A sequence of magnified LT-HS-AFM images during the formation and the closure of pits. Blue arrows
indicate the pit formation and dotted red circles indicate the formation of the closure cap. (d) Time course of the depth and height of the pit with andwithout the closure cap. Red bars indicate the formation of the closure cap. Blue arrows indicate pit formations.
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Figure 7 | LT-HS-AFM images of a living cultured hippocampal neuron at 15 days in vitro. (a) Fluorescence image of a hippocampal neuron transfectedwith mEGFP. Corresponding LT-HS-AFM images for a is shown in b. (b) A sequence of LT-HS-AFM topographical images at 5 s per frame. White arrows
indicate the dynamics of spine-like structure. The corresponding LT-HS-AFM movie is shown in Supplementary Movie 10.

the identity of the structure observed. Thus, the combination LT-HSAFM with advanced optical microscopy could open the new window
to cell biology. For example, by comparing the timing of endocytosis
events in fluorescence image with LT-HS-AFM structural information, it is possible to identify the protein functions and cellular event.
Further, combining LT-HS-AFM with fluorescence resonance energy
transfer (FRET) imaging39 or optical nanoscopy techniques40,41 could
add further information about molecular interactions and intracellular signal transduction.
In summary, we demonstrated the potential of LT-HS-AFM provides to directly visualize nanometer-scale morphological changes inlive cells. In particular, the successful observations of nano-structuraldynamics in live neurons will open the possibility of visualizing themorphology of plasticity of synapses at nanometer resolution in realtime in the near future.

Methods
Fabrication of long AFM tips on original AFM tips of cantilevers. We usedcantilevers measuring ,9 mm long, ,2 mm wide and ,0.13 mm thick with a spring
constant of 100–200 pN/nm and ,0.8 mm tip at the end of the cantilever (BLAC10DS, Olympus). To fabricate an additional tip on the original AFM tip by aphenol deposition technique29, we fixed the cantilever on a cylindrical chamber withsmall through-holes on its lid (,0.1 mm diameter) using both-side tapes (PELCO
Tabs). In the holder ,10 mg of phenol crystal (Sigma) was included. Under ascanning electron microscope (Quanta 250, FEI), the focus of an electron beam wasparked onto the cantilever tips at 30 kV, 5 mm working distance with 30 mm aperturesize in a high-vacuum mode. A stylus-shaped deposition of amorphous carbon was
produced at a growth rate of ,600 nm/min. The focused electron beam wasirradiated for 1 min and repeated 5–7 times. The focus position was reset after eachcycle to compensate for the mechanical drift of the instrument. After fabrication of
EBD tips, cantilevers were further sharpened in the RF-plasma etcher chamber (PE2000, South Bay Technology) filled with ,90% argon gas for ,10 s. Typical power ofthe RF plasma is about 10 W and the chamber pressure is ,50 mTorr. It reduced the

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apex radius of EBD tip to 5–8 nm. The AFM tip was used in 2–3 experiments until the
tip accumulates contamination and the spatial resolution visibly degrades.
Combined LT-HS-AFM and fluorescence microscopy setup. We built HS-AFM
apparatus equipped with the wide-range HS-AFM scanner as reported25. We put
the fabricated long-tip cantilever to HS-AFM for cellular imaging. The cantileverdeflection was detected by detecting the position of a red laser beam (670 nm)
reflected by the cantilever with a position-sensing quadrant photodiode13. The
laser beam was focused onto a cantilever using a 360 objective lens (CFI S Plan
Fluor ELWD, Nikon). To combine the fluorescence microscopy, the excitation
lamp (M470LM, Thorlab), dichroic filters (MDF-GFP, Thorlabs) and a CCD
camera (DS-Fi2, Nikon) are installed under the HS-AFM set up (Fig. 1b). A celltransfected with mEGFP was located to the LT-HS-AFM scanning area (,40 3
40 mm2) using fluorescence imaging and subjected for nanometer imaging with
LT-HS-AFM (Supplementary Fig. 1). HS-AFM images were acquired in tapping
mode (100 nm free amplitude, ,800 kHz). LT-HS-AFM resolutions were set to200 3 200 pixels2. The scan rate was set to 5 s per frame (40 lines/s) or 10 s perframe (20 lines/s). There was no obvious change in the spatial resolution during
an experiment unless the tip was contaminated. The tip was brought to the sample
at the speed of 1.5 mm/s using a motor (RK543AW, Orientalmotor) while theoscillation amplitude was monitored. When the oscillation amplitude became,10% of the free amplitude, the motor was stopped and an imaging session wasstarted29.
Cell culture, transfection and LT-HS-AFM observation of COS-7 and HeLa cells.
COS-7 cells (ATCC #: CRL-1651) and HeLa cells (ATCC #: CCL-2) were cultured inDubelco’s modified eagle medium (DMEM) (GIBCO) supplemented with 10% fetal
bovine serum (FBS) (GIBCO) at 37uC in 5% CO2, and transfected with plasmids using
Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions.
Approximately 3–4 hours after transfection, cells were spread to a poly-L-ornithine(Sigma) coated AFM stage for at least 20 hours. LT-HS-AFM imaging were
performed ,24 hours after transfection. The AFM stage is made of glass and shaped
to be a hexagonal cylinder with 0.8 mm side length and 2.0 mm height. LT-HS-AFM
imaging was performed in imaging solution containing 15 mM HEPES (pH 7.4),
142 mM NaCl, 5 mM KCl, 2 mM CaCl2, 2 mM MgCl2 and 25 mM glucose. The
temperature of imaging solution was kept at 32–34uC by heating the LT-HS-AFM
chamber. Imaging solution was perfused for every 10 min. In some experiments, cells

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were treated with 20 ng/mL cytochalasin D (Sigma), 20 mg/mL insulin (Sigma),
20 ng/mL epidermal growth factor (EGF) (Sigma) or 80 mg/mL dynasore (Sigma).
Primary culture, transfection and LT-HS-AFM observation of hippocampal
neurons. Dissociated hippocampal cultures were prepared from newborn Sprague
Dawley rats at postnatal day 0–1, as described previously38. Briefly, the hippocampi
were dissected in HBSS-HEPES (GIBCO), washed several times with HBSS-HEPES.
Then, hippocampi were digested with papain (100 units, Worthington Biochemical)
at 37uC for 15 minutes and gently pipetted in solution containing DNase I type IV(0.6 mg/mL, Sigma) until the tissue was mostly dissolved. Undissociated tissue wasremoved from the solution by passing the mixture through a cell strainer (40 mm, BD
Biosciences). Dissociated neurons were collected in 50 mL conical tube andprecipitated with centrifugation (1000 rpm for 5 min). The precipitate wasresuspended in a culture medium consisting of Minimum Essential Medium (MEM)(GIBCO) and 10% FBS to various cell concentrations. The neurons were placed onto
poly-L-lysine/laminin-coated AFM stage for 1 hour. The medium was replaced witha medium containing Neurobasal Medium (GIBCO), B-27 Supplements (GIBCO),
and GlutaMAX (GIBCO) and cultured at 37uC in 5% CO2. Cultured hippocampal
neurons were infected with lenti virus encoding synapsin promoted mEGFP at 2–3
days in vitro.
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Acknowledgments
The authors thank H. Watanabe for providing the wide-area scanner, J. Nishiyama forproviding the plasmid of mEGFP-Rab5(Q79L) , N. Hedrik for critical reading and D.
Kloetzer for lab management. This work was supported by the Human Frontier Science
Program (HFSP grant RGP0069/2011), JSPS Postdoctoral Fellowships for Research Abroad(M.S.), JST CREST (T.A.), Grants-in-Aids for Scientific Research from the MEXT Japan(No. 24227005, T.A.; No. 23115008 and 26104514, T.U).

Author contributions
M.S. and R.Y. conceived and designed the experiments. M.S. and T.U. made the software for
HS-AFM instrument. M.S. developed the LT-HS-AFM instrument. M.S. performed the all
experiments. M.S. and R.Y. analyzed the data. T.A. and R.Y. supervised the experiments.
M.S. and R.Y. wrote the paper. All authors discussed the results and commented on the
manuscript.

Additional information
Supplementary information accompanies this paper at http://www.nature.com/
scientificreports
Competing financial interests: The authors declare no competing financial interests.
How to cite this article: Shibata, M., Uchihashi, T., Ando, T. & Yasuda, R. Long-tip
high-speed atomic force microscopy for nanometer-scale imaging in live cells. Sci. Rep. 5,8724; DOI:10.1038/srep08724 (2015).
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