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Comparison of atomic force microscopy and scanning ion conductance microscopy for live cell imaging Jan Seifert, Johannes Rheinlaender, Pavel Novak, Yuri Korchev, and Tilman E Schäffer Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.5b01124 • Publication Date (Web): 26 May 2015 Downloaded from http://pubs.acs.org on May 30, 2015
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Comparison of atomic force microscopy and scanning ion conductance microscopy for live cell imaging Jan Seifert,†
Johannes Rheinlaender,†
Pavel Novak,‡,§
Yuri E. Korchev,‡
and
Tilman E. Schäffer*,† †
Institute of Applied Physics, University of Tübingen, Germany
‡
Division of Medicine, Imperial College London, UK
§
School of Engineering and Materials Science, Queen Mary University of London, UK
*Correspondence to: Tilman E. Schäffer Institute of Applied Physics Eberhard Karls University Tübingen Auf der Morgenstelle 10, 72076 Tübingen, Germany Tel.: +49 7071 29 76030 E-mail:
[email protected] ACS Paragon Plus Environment
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Abstract Atomic force microscopy (AFM) and scanning ion conductance microscopy (SICM) are excellent and commonly used techniques for imaging the topography of living cells with high resolution. We present a direct comparison of AFM and SICM for imaging microvilli, which are small features on the surface of living cells, and for imaging the shape of whole cells. The imaging quality on microvilli increased significantly after cell fixation for AFM, whereas for SICM it remained constant. The apparent shape of whole cells in the case of AFM depended on the imaging force, which deformed the cell. In the case of SICM, cell deformations were avoided, owing to the contact-free imaging mechanism. We estimated that the lateral resolution on living cells is limited by the cell’s elastic modulus for AFM, while it is not for SICM. By long-term, time-lapse imaging of microvilli dynamics we showed that the imaging quality decreased with time for AFM, while it remained constant for SICM.
1. Introduction Atomic force microscopy1,2 (AFM) and scanning ion conductance microscopy3-5 (SICM) are commonly used techniques for imaging the topography of biological samples. Living cells, which are demanding samples owing to the need of a warm, liquid environment, have successfully been studied with both AFM6-10 and SICM.4,11-14 Both techniques have also been combined.9,15,16 The imaging mechanism in AFM is based on mechanical forces between the sample and the tip of a cantilever, which is used to track the surface of the sample (Fig. 1a). All standard AFM imaging modes require a direct mechanical contact between tip and 1 ACS Paragon Plus Environment
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sample, which can cause sample damage on living cells.17,18 The most commonly used AFM imaging modes for cells are the contact and the tapping mode.19,20 Living cells can be imaged with high resolution.21 Nanometer lateral resolution can be reached.22 Surface-tracking in SICM is based on measuring the ion current through an electrolytefilled nanopipette, which strongly depends on the pipette-sample distance (Fig. 1c).3 The imaging process with SICM is contact-free,11 allowing to image fragile samples such as freely suspended phospholipid membranes.23 A common imaging mode in SICM is the backstep/hopping mode.11,24,25 A direct comparison of both techniques has already been performed for fixed cells26 and other biological samples.27 Here, we compare the live cell imaging performance of AFM (in tapping mode) and SICM (in backstep/hopping mode). We chose microvilli on the surface of living epithelial A6 cells as an example for subcellular features, as their height and width was found to be very reproducible.28 This provided a consistent condition for the comparison of the two techniques. We further compare the ability to properly image the undeformed topography of whole cells and we estimate the dependency of the lateral resolution on the cell’s elastic modulus. Finally, we compare the long-term imaging capability of microvilli dynamics, which has been studied previously with SICM.28
2. Experimental 2.1.
AFM setup
AFM images were recorded with a commercial AFM setup (MFP3D-BIO, Asylum Research, Santa Barbara, CA), equipped with a sample heater and mounted on an 2 ACS Paragon Plus Environment
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inverted optical microscope (Ti-S, Nikon, Chiyoda, Japan). A cantilever with a nominal spring constant of 6 pN/nm (Biolever B, Olympus, Shinjuku, Japan) was used in tapping mode. In this mode the cantilever is oscillated near its resonance frequency and the oscillation amplitude is used for distance control between cantilever and sample. Unlike contact mode, tapping mode is insensitive to drift in cantilever deflection, which can occur especially during long-term measurements in liquid environments. Additionally, the lateral forces on the sample during imaging are reduced in tapping mode. A typical oscillation frequency of 9 kHz was used in this study. The free oscillation amplitude just before the contact point between cantilever and sample was set to 100 nm using amplitude-distance-curves. The amplitude setpoint was initially set to 100% of the free amplitude and then consecutively reduced while scanning until microvilli became visible, resulting in an imaging setpoint between 98% and 95%. The force on the sample was thereby kept as low as possible, allowing stable scanning of the cell surface without major damage to the cell. The lateral scan speed was 5 µm/s in all images. To avoid evaporation of culture medium, the measurements were done in a closed fluid cell. Zeroforce height sections of sample topography were recorded in force mapping mode.29,30 The imaging force in tapping mode was estimated from the mean cantilever deflection in amplitude-distance-curves. SICM setup SICM images were recorded with two self-built SICM setups. The first setup (used for Figs. 1–3, 5–6) consisted of a 25 µm z-piezo scanner (P-753.21C, Physik Instrumente, Karlsruhe, Germany) for vertical positioning of the pipette and a 200 µm xy-piezo scanner (P-527.3CL, Physik Instrumente) for lateral positioning of the sample. A 3 ACS Paragon Plus Environment
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patchclamp amplifier (EPC-800, HEKA Elektronik, Lambrecht/Pfalz, Germany) was used for measuring the ion current. Nanopipettes with a typical opening diameter of 40 – 80 nm were fabricated with a CO2-laser-based micropipette puller (P-2000, Sutter Instrument, Novato, CA). The Ag/AgCl electrodes were made from silver wire, incubated in a sodium hypochlorite solution for 20 minutes. The second SICM setup (used for Fig. 4) is described in detail elsewhere.26 In both setups, a self-built closed-loop controlled heater was used to heat the sample.13 For SICM imaging we used backstep/hopping mode,11,24,25 where the pipette is vertically approached to the sample surface until the ion current drops below a preset trigger value, thereby defining the height of the surface at this position. Repeating this procedure for many positions on the sample allows generating an image of sample topography. Scan area, duration per frame, and number of lines per image were chosen identical for AFM and SICM imaging. 128 and 64 pixels per line were recorded in AFM and SICM, respectively. In post-processing, all AFM and SICM topography images were interpolated to 256×256 pixels. Cell culture and sample preparation Xenopus laevis A6 cells were cultured at 28°C and 1% CO2. The culture medium consisted of 35% Leibovitz’s L-15 medium (Life Technologies, Carlsbad, CA), 35% Hams F-12 medium (Life Technologies), 18.9% sterile water, 0.3% sodium bicarbonate solution at 7.5% in water (Life Technologies), 9.1% fetal calf serum (PAA Laboratories, Pasching, Austria) and 1.7% L-glutamine solution (200mM) with penicillin/streptomycin (10000 U/ml) (PAA Laboratories). The cells were seeded in fibronectin-coated (5 µg/ml, Roche Applied Science, Indianapolis, IN) culture dishes (#351006, BD Falcon, VWR, 4 ACS Paragon Plus Environment
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Darmstadt, Germany) 3 – 4 days before measurements. AFM and SICM measurements on A6 cells were performed in CO2-independent medium (L-15) at room temperature. Mouse embryonic fibroblasts (MEFs)31 were cultured at a temperature of 37°C in a 5% CO2 humid atmosphere. Culture medium was Dulbecco’s Modified Eagle Medium (Life Technologies) supplemented with 10% fetal calf serum and 1% L-glutamine solution (200mM) with penicillin/streptomycin (10000 U/ml). MEFs were seeded in fibronectincoated culture dishes 24 – 48 h before measurements, which were performed in CO2independent L-15 medium at 37°C. MEFs do not tend to form a monolayer, allowing to measure the vertical position of the underlying substrate (needed for cell height measurements). For cell shape analysis, the cell height was measured as the 95th height percentile and the cell width was measured at 500 nm above the substrate. For some measurements, cells were fixed by adding formaldehyde (Sigma Aldrich, St. Louis, MO) to the culture medium to a final concentration of 2% and incubating for 20 minutes. Statistics Data are provided as arithmetic means ± SEM (standard error of the mean). Data were tested for significance using Student’s unpaired t-test, as indicated in the figure legends. Results with p-values < 0.05 were considered significantly different.
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Results and Discussion Imaging sub-cellular features on live and fixed epithelial cells In typical AFM (Fig. 1b) and SICM (Fig. 1d) topography images of live A6 epithelial cells in a confluent monolayer, microvilli on the cell surface and junctional folds (arrows) between the cells were resolved. When imaged with SICM, microvilli consistently appeared with a higher contrast, compared to AFM. To further assess the imaging quality, we recorded high resolution topography images before and after fixation (Fig. 2). In the AFM image of live cells, single microvilli were resolved (Fig. 2a). However, AFM trace and retrace height sections (Fig. 2a, height profiles) revealed a lateral displacement of the microvilli by the AFM tip in scan direction (arrows). After fixation, the microvilli became more prominent and the image contrast increased (Fig. 2b). The lateral displacement of the microvilli was distinctly reduced (Fig. 2b, height profiles). A subsequent SICM image of the same area on the same cell (Fig. 2c) showed a similar image contrast. No lateral displacement was visible in the SICM trace and retrace height sections (Fig. 2c, height profiles). We assessed the image contrast by measuring the height and width (full width at half maximum height, FWHM) of the microvilli in the recorded AFM and SICM images. The average height in the AFM images significantly increased after cell fixation and was even larger in the SICM image (Fig. 2d). The average width in the AFM images was not notably affected by fixation, but was significantly larger than in the succeeding SICM image (Fig. 2e). An analogue image sequence was recorded with the order of AFM and SICM imaging switched (Fig. 3). The contrast in the SICM images before (Fig. 3a) and after (Fig. 3b) 6 ACS Paragon Plus Environment
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fixation was similar. Sporadically occurring displacements of microvilli on the live cell (Fig. 3a, height profiles, arrows) probably owed to microvilli moving between trace and retrace.28 The subsequent AFM image of the same area on the same cell (Fig. 3c) showed a similar contrast as the preceding SICM images. A slight lateral displacement of the microvilli was visible when comparing the AFM trace and retrace height sections (arrows). An analysis of microvilli height and width showed that neither height nor width in SICM images was significantly affected by fixation (Fig. 3d and e). This further suggests that fixation did not induce shrinkage or distortion of the microvilli structures. The height in the AFM image was significantly smaller than that in both SICM images (Fig. 3d), while the width was similar to that in both SICM images (Fig. 3e). In summary, the imaging contrast of AFM improved significantly after cell fixation, whereas it remained unchanged for SICM. In AFM images, microvilli generally appeared lower than in SICM images, probably owing to the non-zero AFM imaging force. The lateral displacement of microvilli observed in the AFM images was presumably caused by the lateral scanning mechanism of AFM tapping mode, which is different from the “vertical approach” scanning mechanism of SICM hopping mode.11 Lateral scanning artifacts are avoided in hopping mode, because the probe is vertically approached to the sample for each point individually instead of being laterally scanned across the sample surface. Imaging the shape of whole cells We investigated the influence of the imaging setpoint on the apparent cell shape of nonconfluent live MEFs. To keep changes in cell morphology during imaging as small as
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possible, only single lines through the center of a cell (height cross sections) were recorded. In AFM, the measured height and width of the cell were strongly affected by the amplitude setpoint. For a decreasing amplitude setpoint (increasing imaging force), the cell became increasingly indented and its height and width decreased (Fig. 4a and b). For comparison, a zero-force height section from force mapping shows the shape of the undeformed cell. For amplitude setpoints down to 75% (imaging force < 0.9 nN) the cell was tracked and slightly deformed, but the substrate beside the cell (x < 10 µm, x > 45 µm) was not tracked. Proper tracking of the substrate was only observed at smaller amplitude setpoints ( 0.9 nN), but then the cell was strongly deformed. Below a setpoint of 60% (imaging force ≈ 1.7 nN), the cell was detached from the substrate by the cantilever. A similar influence of the imaging setpoint on the measured height of soft samples in AFM was observed before.30 In SICM, proper tracking of substrate and cell without visible deformation was achieved for ion current setpoints between approximately 97% and 99.5% of the free ion current (Fig. 4c and d). At larger setpoints, the trigger frequently occurred too early because of noise in the current measurement, resulting in height spikes (arrows). The surface was only partially tracked. At smaller ion current setpoints (approximately 96% and below), the cell became slightly deformed owing to contact between pipette and cell, resulting in a decreasing height and width. Below an ion current setpoint of about 90%, the cell was detached from the substrate by the pipette.
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Lateral resolution of AFM and SICM on soft samples The lateral resolution of AFM on soft samples generally depends on the elastic modulus (Young’s modulus) of the sample.8,32 During formaldehyde-induced fixation, cells are known to stiffen due to protein cross-linking.32 We analyzed the amplitude response on a live cell before and after fixation (Fig. 5a and b). The slope in the recorded amplitude curves increased after cell fixation (Fig. 5b, top). This indicates a larger indentation on the live cell than on the fixed cell, reflecting the increase of elastic modulus upon fixation. A typical amplitude setpoint of 97% used for imaging in this study resulted in an imaging force of approximately 100 pN (Fig. 5b, bottom, dashed lines). By applying Sneddon-model33 fits to the force curves,30 the elastic modulus of the fixed cell was determined as 7.5 times larger than the elastic modulus of the live cell (97 kPa and 13 kPa, respectively). The SICM current curves, which were recorded at the same positions on the same sample as for AFM, were identical on the live cell, the fixed cell, and the substrate (Fig. 5c). This result was obtained also for larger pipette diameters. For AFM, a lower limit of the lateral resolution is the tip-sample contact radius.32,34 To estimate the lateral resolution on a soft sample, we approximated the cantilever tip as a conical indenter with a contact radius ܽ given by
ܽଶ =
2 ܨ ∙ ሺ1 − ߥ ଶ ሻ ∙ tan ߠ ∙ π ܧ
where ߠ is the half cone angle, ܨthe force exerted on the sample, and ܧthe elastic modulus of the sample.33 Cells were assumed as incompressible with a Poisson’s ratio of ߥ = 0.5. For a constant force ( ܨi.e., a constant imaging setpoint), the contact radius decreases and therefore the lateral resolution improves with an increasing elastic 9 ACS Paragon Plus Environment
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modulus of the sample34 (Fig. 5d, red dashed lines; typical imaging force used in this study were 100 pN). For SICM, the ion current response does not depend on the elastic modulus of the sample (s. Fig. 5c) and the lateral resolution can be calculated as three times the inner opening radius of the nanopipette35 (Fig. 5d, blue lines; typical radius used in this study: 30 nm). The elastic modulus of live cells is typically in the range of 0.1 – 100 kPa.36 Therefore, for very soft samples such as live cells, SICM might achieve a better lateral resolution than AFM (Fig. 5d, left side). Long-term imaging of microvilli dynamics on live A6 cells We next compared the long-term imaging capability of AFM and SICM for visualizing the dynamics of microvilli on live cells over several hours. Image sequences were recorded with AFM (Fig. 6a) and SICM (Fig. 6b) using identical scan sizes and frame rates. The setpoints were adjusted for optimum image quality (98% of the free amplitude for AFM, 99.5% of the free ion current for SICM). Every 10th image is shown. Assembling and disassembling microvilli were resolved with both AFM and SICM. The AFM image quality remained constant for about 60 min, making it possible to observe microvilli dynamics. Thereafter, the image quality continuously deteriorated and the microvilli appeared smeared out. Adjusting the setpoint did not considerably improve the image quality. Possibly, membrane debris accumulated at the cantilever tip. In the SICM image sequence, the image quality did not change (Fig. 6b). The full sequences are provided in the online supplementary information (Movies S1 and S2).
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Conclusion We used microvilli on the surface of living A6 cells as an example for subcellular sample features for our comparison of AFM and SICM for live cell imaging. Living microvilli were resolved with both AFM and SICM. The quality in AFM images significantly improved after cell fixation (Fig. 2), while it was unaffected in SICM images (Fig. 3). The measured height and width of whole cells were strongly dependent on the imaging setpoint (imaging force) in AFM, while they were constant within a large setpoint range in SICM (Fig. 4). Imaging the accurate, undeformed topography of living cells in AFM is so far only possible using the force mapping mode, which (besides mechanical sample properties) provides a “zero-force” or “contact height” image of the cell. However, the imaging speed of force mapping in liquid is usually limited to a few pixels per second, requiring tens of minutes for images with a pixel resolution as used in this study. Force mapping with a comparable imaging speed is possible, but requires a specialized setup.37 We estimated that the lateral resolution depends on the elastic modulus of the sample for AFM, while it is independent of the elastic modulus for SICM (Fig. 5). Long-term microvilli dynamics of live A6 cells could be visualized with both AFM and SICM (Fig. 6). However, while the image quality deteriorated with time for AFM, it remained unchanged for SICM. In summary, on very soft samples such as living cells, SICM can be advantageous to AFM in terms of imaging quality, accuracy in topography measurements, lateral resolution, and long-term imaging stability.
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Acknowledgment Financial support of the DFG (KFO274, TP06) is gratefully acknowledged. We thank Asylum Research for technical support.
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into Three-dimensional Collagen Matrices. J. Biol. Chem. 2010, 285 (17), 1312113130. Braet, F.; Rotsch, C.; Wisse, E.; Radmacher, M. Comparison of fixed and living liver endothelial cells by atomic force microscopy. Appl. Phys. A 1998, 66 (1), 575-578. Sneddon, I. N. The relation between load and penetration in the axisymmetric boussinesq problem for a punch of arbitrary profile. Int. J. Eng. Sci. 1965, 3 (1), 47-57. Radmacher, M.; Fritz, M.; Hansma, P. K. Imaging soft samples with the atomic force microscope: gelatin in water and propanol. Biophys. J. 1995, 69 (1), 264270. Rheinlaender, J.; Schäffer, T. E. Image formation, resolution, and height measurement in scanning ion conductance microscopy. J. Appl. Phys. 2009, 105 (9), 094905. Alonso, J. L.; Goldmann, W. H. Feeling the forces: atomic force microscopy in cell biology. Life Sci. 2003, 72 (23), 2553-2560. Braunsmann, C.; Seifert, J.; Rheinlaender, J.; Schäffer, T. E. High-speed force mapping on living cells with a small cantilever atomic force microscope. Rev. Sci. Instrum. 2014, 85.
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Figures
Figure 1: Schematics of AFM and SICM and topography images on live A6 cells. (a) In AFM, the sample topography is imaged by direct mechanical contact between the sample (cell) and the tip of the cantilever. The dashed curve denotes the path of tip over the sample. (b) AFM topography image of a confluent monolayer of live A6 cells recorded in tapping mode. Microvilli can be observed on the cell surfaces. Junctional folds between the cells are marked with arrows. (c) In SICM, an ion current I through an electrolyte-filled nanopipette is used to image the sample topography without direct mechanical contact. (d) SICM topography image of a confluent monolayer of live A6 cells recorded in backstep/hopping mode.
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Figure 2: Comparison of live and fixed cell imaging (AFM first, then SICM). (a) AFM image of live A6 cells before fixation. Height sections at the position of the dashed line are shown below the image. Trace (scan direction from left to right) and retrace (scan direction from right to left) sections do not overlap, revealing a lateral displacement of the microvilli by the tip during scanning (arrows). (b) AFM image of the same area on the same cell after fixation. The image appears with an increased contrast and less lateral displacement (arrows). (c) Subsequent SICM image of the same area on the same cell. No lateral displacement is visible in the height sections. The images were processed by applying a 3rd order plane fit to allow for a better visual comparison. (d, e) Average
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height and average width (FWHM) of 30 analyzed microvilli in the AFM and SICM images a-c. * (p < 0.01) indicates statistically significant difference.
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Figure 3: Comparison of live and fixed cell imaging (SICM first, then AFM). (a) SICM image of a live A6 cell surface before fixation. Height sections at the position of the dashed line are shown below the image. A displacement of the microvilli occurred only sporadically, probably owing to microvilli dynamics (arrows). (b) SICM image of the same area on the same cell after fixation. No lateral displacement is visible in the height sections. (c) Subsequent AFM image of the same area on the same cell. A slight lateral displacement of the microvilli occurred (arrows). The images were processed by applying a 3rd order plane fit. (d, e) Average height and average width (FWHM) of 30 analyzed microvilli in the SICM and AFM images a-c. * (p < 0.01) indicates statistically significant difference using Student’s unpaired t-test. 19 ACS Paragon Plus Environment
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Figure 4: Influence of the imaging setpoint on sample tracking and cell shape. (a) AFM height profiles of a live mouse embryonic fibroblast (MEF) for different tapping mode amplitude setpoints (relative to the free amplitude) and the corresponding estimated imaging forces. The zero-force height (gray) was obtained in the force mapping mode. (b) Height and width of the cell from panel a as a function of the amplitude setpoint. For amplitude setpoints above 75% (imaging force < 0.9 nN), the cell was tracked and slightly deformed, but the substrate beside the cell (x < 10 µm and x > 45 µm) was not tracked. For amplitude setpoints below 75% (imaging force > 0.9 nN), the substrate was tracked, but the cell was strongly deformed. The dashed lines 20 ACS Paragon Plus Environment
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in the graph mark height and width, respectively, of the zero-force height trace in panel a. (c) SICM height profiles of a live MEF for different ion current setpoints (relative to the free ion current). At current setpoints of 99.75% and above, early triggers occurred, resulting in height spikes (arrows). (d) Height and width of the cell from panel c as a function of the ion current setpoint. The cell was tracked without deformation at current setpoints between 97% and 99.5%. At larger current setpoints, frequent spikes occurred and the surface was only partially tracked. At smaller ion current setpoints (96% and below), the cell became slightly deformed. The dashed lines in the graph mark height and width, respectively, at a current setpoint of 99.5%. Insets: Optical images of the cells; the dashed lines mark the locations of the cross sections.
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Figure 5: Lateral resolution on soft samples. (a) Optical image of a live MEF. At the marked positions, amplitude-distance curves for AFM and current-distance curves for SICM were recorded before and after fixation. (b) AFM amplitude (relative to the free amplitude) and force as a function of the cantilever’s z-position on the live cell (bright red curve), on the same position after fixation (dark red curve), and on the substrate (black curve). A typical amplitude setpoint of 97% used in this study gave an imaging force of approximately 100 pN (dashed line). The slope of the amplitude curves in the contact region (z
