Macro-SICM: A Scanning Ion Conductance Microscope for Large

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Macro-SICM: A Scanning Ion Conductance Microscope for Large-Range Imaging Nicolas Schierbaum, Martin Hack, Oliver Betz, and Tilman E Schäffer Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b04764 • Publication Date (Web): 23 Mar 2018 Downloaded from http://pubs.acs.org on March 23, 2018

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Analytical Chemistry

Macro-SICM: A Scanning Ion Conductance Microscope for LargeRange Imaging

Nicolas Schierbaum,1 Martin Hack,1 Oliver Betz,2 and Tilman E. Schäffer1* 1

Institute of Applied Physics and 2Institute of Evolution and Ecology, University of Tübingen, 72076 Tübingen, Germany.

*

E-mail: [email protected] Telephone: +49 7071 29 76030 Fax: +49 7071 29 5093

ACS Paragon Plus Environment

Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT The scanning ion conductance microscope (SICM) is a versatile, high-resolution imaging technique that uses an electrolyte-filled nanopipet as a probe. Its non-contact imaging principle makes the SICM uniquely suited for the investigation of soft and delicate surface structures in a liquid environment. The SICM has found an ever-increasing number of applications in chemistry, physics, and biology. However, a drawback of conventional SICMs is their relatively small scan range (typically 100 µm × 100 µm in the lateral and 10 µm in the vertical direction). We have developed a Macro-SICM with an exceedingly large scan range of 25 mm × 25 mm in the lateral and 0.25 mm in the vertical direction. We demonstrate the high versatility of the Macro-SICM by imaging at different length scales: from centimeters (fingerprint, coin) to millimeters (bovine tongue tissue, insect wing) to micrometers (small cell extensions). We applied the Macro-SICM to the study of collective cell migration in epithelial wound healing.

Keywords: scanning probe microscopy, scan range, macroscopic, millimeter scale, cell migration


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INTRODUCTION The scanning ion conductance microscope (SICM)1,2 has been established in chemistry, physics, and biology as a non-invasive, contact-free, and high-resolution imaging technique particularly well suited to study soft and delicate samples in a liquid environment.3-6 The SICM is based on measuring an ion current through an electrolyte-filled nanopipet while laterally scanning the nanopipet across the sample. The ion current strongly depends on the pipet-sample distance and is used as a feedback signal for tracking sample topography.1 Next to topography imaging, the SICM has successfully been applied to the measurement of electrochemical,7-9 electrostatic,10,11 electrophysiological,12,13 and mechanical6,14-17 sample properties. Conventional SICMs, however, have a relatively small scan range of around 100 µm in the lateral (xy) and 10 µm in the vertical (z) direction.18-21 This range is sufficient to image samples such as single22-24 or a small number of cells,25 artificial membranes,26 nanostructures,7 and even single proteins.27,28 However, imaging structures on the millimeter and centimeter scale has not been possible so far. One approach toward this goal when using conventional SICMs is recording many successive images on adjacent regions on the sample and stitching the images together to obtain a larger image.29 However, this approach amounts to a difficult image acquisition and alignment procedure, only increases the effective horizontal but not the vertical scan range, and fails or produces imaging artifacts in the case of surface structures that change in time. Here, we present a Macro-SICM with an exceedingly large scan range of 25 mm × 25 mm in the lateral and 0.25 mm in the vertical direction. The Macro-SICM is capable of investigating a diversity of different surface structures on a broad range of length scales.



EXPERIMENTAL SECTION

Design of the Macro-SICM To attain the large lateral scan range of our home-built Macro-SICM (Figure 1a), we use two linear sensor-based micropositioning stages (M-683.1U4, Physik Instrumente, Karlsruhe, Germany) with a range of 25 mm each. The stages are mounted on top of each other and move the sample along the lateral (xy) scan directions. Movement of the pipet in the vertical direction is provided by a z-piezo scanner with a large range of 0.25 mm (PIHera P-622.1CL, Physik Instrumente), equipped with a capacitive sensor. The piezo is mounted on a motorized stage with a range of 25 mm (M-126.PD, Physik Instrumente), allowing automatic coarse approach of the pipet towards the sample. To provide optical access to the sample and the pipet, the Macro-SICM is combined with an inverted optical microscope (Ti-U, Nikon, Tokio, Japan) with condenser-free phase contrast illumination.30 A home-built sample stage heater allows homogenous sample heating. We used borosilicate glass pipets with an inner opening radius varying between 125-2000 nm, fabricated with a CO2-laser-based micropipet puller (P-2000, Sutter Instruments, Novato, CA). The ion current was measured by a commercial patch clamp amplifier (EPC-800, HEKA Elektronik, Lambrecht, Germany). SICM topography images were recorded in the backstep/hopping mode,18,31,32 emerging as the most common imaging mode for biological samples. In this mode, the pipet was vertically approached to the sample surface while monitoring the ion current. When the ion current reached a pre-set trigger level (“ion current trigger”) of typically 0.5-1% below the initial value of the current, the pipet was lifted off the sample by the retract distance. The sample was then laterally moved to the next point above the sample and the procedure was repeated. The sensor positions of the z-piezo at the ion current trigger points gave the topography image


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of the sample. For imaging we have used the following pipet inner opening radii, retract distances, approach/retract velocities, ion current triggers, and pixel resolutions, respectively: 2 µm, 100 µm, 400 µm/s, 0.8%, 200×200 pixels (Figure 1b); 2 µm, 120 µm, 720 µm/s, 1%, 240×240 pixels (Figure 1c); 2 µm, 50 µm, 400 µm/s, 1%, 200×200 pixels (Figure 2a); 2 µm, 50 µm, 300 µm/s, 0.7%, 200×140 pixels (Figure 3b); 250 nm, 25 µm, 250 µm/s, 0.7%, 100×100 pixels (Figure 3c); 125 nm, 10 µm, 100 µm/s, 0.8% (Figure 4b,d); 200×200 pixels (Figure 4b); 50×50 pixels (Figure 4d); 350 nm, 15 µm, 300 µm/s, 0.8%, 125×50 pixels (Figure 5a); 125 nm, 10 µm, 100 µm/s, 1% (Figure S1a and Figure S3b); 50×50 pixels (Figure S1a and Figure S3b, top and bottom right); 100×100 pixels (Figure S3b, top and bottom left). All shown SICM topography images were linearly interpolated by a factor of 3.

Sample Preparation The top surface of a bovine tongue and a hindwing of the pentatomid bug Graphosoma italicum (O.F. Müller, 1766) (Insecta, Heteroptera: Pentatomidae) were fixed in a culture dish each using vacuum grease. SICM imaging was performed in PBS at room temperature. Mouse embryonic fibroblasts (MEFs) were cultured at 37°C and 5% CO2 in stable Lglutamine Dulbecco’s modified Eagle’s medium (DMEM) (Biochrom, Berlin, Germany), supplemented with 10% fetal bovine serum (FBS) (Biochrom), and 1% penicillin/streptavidin (Pen/Strep) (Biochrom). Cells were seeded on culture dishes at sparse density and fixed after 24 h hours in 3.7% formaldehyde solution for 20 min. After three washing steps with PBS, cells were imaged in PBS at room temperature. Epithelial frog (Xenopus laevis) kidney A6 cells were cultured at 28°C and 1% CO2 in a mixture of 35% Leibovitz L-15 medium (Biochrom), 35% Ham’s F-12 medium (Biochrom), 18.9% deionized sterile water, 0.3% sodium bicarbonate solution (Biochrom), 9.1% fetal bovine serum (FBS) (Biochrom), 1.7% L-glutamine (Biochrom) (200 mM), and Pen/Strep (Biochrom). For the wound healing assay we placed a 2-well silicone culture-insert (ibidi, ACS Paragon Plus Environment


Martinsried, Germany) in a culture dish and filled each well with 75 µL of an A6 cell suspension. After cells grew to confluency in both wells, the culture-insert was removed. Prior to SICM measurements, the cell culture medium was replaced by CO2-independent Leibovitz L-15 medium, containing the same supplements as the cell culture medium. SICM measurements were conducted at room temperature.

Data Analysis To assess the cell height and the cell volume from SICM topography images, first, the substrate slope was line-by-line removed in the images (“flattening”). Then, the images were vertically offsetted such that the substrate height was zero. All pixels above 150 nm were considered as part of the cell (denoted as cell pixel). The cell area was then obtained by multiplying the number of cell pixels with the calibrated pixel area. The cell height was calculated as the average of the height values at each cell pixel. The cell volume was calculated as the sum of the height values at each pixel multiplied with the calibrated pixel area in accordance with previous studies.33,34

RESULTS AND DISCUSSION

Large-Range Imaging To demonstrate the large-range imaging capability of the Macro-SICM, we imaged the topography of a human finger print on a glass surface with its characteristic friction ridges (Figure 1b) and the surface of a metal coin (Figure 1c) within a huge lateral scan range of 20 × 20 mm² and 12 × 12 mm², respectively.

Imaging of Biological Tissue The capability of the SICM to study macroscopic structures such as biological tissue has so far been limited not only by the lateral, but also by the vertical scan range. Using the MacroACS Paragon Plus Environment

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SICM with its large available vertical scan range (0.25 mm), we managed to image the rough surface of a bovine tongue tissue sample. The topography image (Figure 2a) shows a dense distribution of micrometer-sized projections that presumably are filiform and fungiform papillae,35 giving the tongue a rough texture. The height profile along the dashed line shows large height variations of up to 140 µm (Figure 2b), which is much larger than the available vertical scan range of conventional SICMs (typically 10 µm). Investigating such relatively rough tissues with conventional SICMs would therefore require specific sample preparation to ensure that the sample surface is sufficiently flat with respect to the available vertical scan range. For example, tissue samples have been cryosectioned36-39 for imaging with conventional atomic force microscopes (AFMs), which have a similar scan range as conventional SICMs. In other cases, several small-range images have been stitched together to obtain a large-range image of the sample.36,38 We demonstrated that the Macro-SICM can image biological tissue with a large-range without the need for any specific sample preparation such as cryo-sectioning and without the need for image stitching.

Imaging of Biological Composite Structures The hindwing of the pentatomid bug Graphosoma lineatum italicum is a composite structure consisting of chitin nanofibers embedded in a protein matrix.40 The surface of the hindwing is formed by an expanded membrane that is longitudinally traversed by tubular buttressing veins (Figure 3a). The large-range scanner of the Macro-SICM allowed us to successively zoom in on these structures, resolving fold zones laterally bounded by false veins (Figure 3b) and local conical protrusions on the wing membrane with a resolution on the micrometer scale (Figure 3c). The exoskeleton of insects known as insect cuticle is a living structure and thus affected by desiccation after separation from the insect body; therefore, it is best imaged under hydrated conditions, which can be ensured by immersing the cuticle in a buffer (e.g. PBS) or water. Imaging the hindwing topography under hydrated conditions can help to better



understand the structure of insect cuticle with respect to the dimensions and geometries of its surface protuberances. Such structures can have quite different functions in insect cuticle that are still not well understood such as self-cleaning (Lotus effect),41 structural coloration,42 or adhesion and friction enhancement or reduction.43 In particular, mapping the stiffness16 on resilin-containing regions within a large range will reveal important functional data that help to understand the wing’s function in (foldable) deployable constructions.40

Imaging of Many Single Cells For phenotyping in single cell experiments, it is often desirable to have a statistically sufficient number of cells to account for the cell-to-cell variability within a cell population. Owing to its non-contact imaging principle, the SICM was previously used to accurately measure morphological parameters such as cell height44 or cell volume.33,45 As the lateral size of adherent cells approaches the scan range of conventional SICMs (100 µm), a separate image needed to be acquired for each cell in these previous studies - a cumbersome process for the large number of cells needed for obtaining good statistical values. Here, we selected a suitable region containing many fibroblast cells (MEFs) using the optical microscope (Figure 4a) and recorded one single SICM image of this region with a lateral scan range of 1×1 mm2 and an imaging duration of 2 h (Figure 4b). This allowed us to obtain cell height, cell volume, and their averages for n=85 cells (Figure 4c). To investigate the effect of drift during imaging, we repeatedly imaged a fixed fibroblast cell over the duration of 1 h (Figure S1). The drift was below 0.5 µm/h in both lateral (x, y) and vertical (z) directions and therefore did not significantly affect the height and volume measurements. The precision with which height and volume of a single cell can be measured was determined as 0.07 µm (height) and 0.02 pL (volume) (Figure S2). These values for the precision are much smaller than the measured respective standard deviations of height and


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volume of the cell population in Figure 4c, indicating that these standard deviations are caused by biological cell-to-cell variability. The large scan range of the Macro-SICM allowed us to accurately move to different regions for high-resolution imaging of single fibroblast cells (Figure 4d). The cells showed the typical polarized morphology of fibroblasts with small fingerlike cellular extensions originating at the lamellipodium. The precision with which the pipet can be positioned on the millimeter scale was determined as about 1 µm (Figure S3).

Multicellular Dynamics Next, we demonstrate the potential of the Macro-SICM for monitoring the dynamics of multicellular systems by imaging wound healing of an epithelial monolayer. A wound healing assay is a frequently used method to study the dynamics of collective cell migration under defined conditions, giving insights into the complex processes of wound repair,46 tissue homeostasis,47 and cancer invasion.48 SICM topography images (Figure 5a) recorded with a frame rate of approximately 40 min show collective cell migration from the left and the right wound edges into the cell free region. To evaluate the wound closure we determined the wound area (Figure 5b) and the wound volume (Figure 5c) as a function of time. When the wound area was completely covered again by the monolayer at around 19 h (Figure 5b), the wound volume was not yet fully regrown (Figure 5c).

Advantages and Limitations of the Macro-SICM The major advantage of the presented Macro-SICM over conventional SICMs is the capability for large-range imaging and large-range pipet positioning. Owing to its large scan range, the Macro-SICM has some limitations concerning imaging speed and resolution. In backstep/hopping mode, imaging speed is determined by the velocity with which the z-piezo vertically approaches and retracts the pipet toward and away from the sample surface and by



the velocity with which the xy-scanner laterally moves to the next point on the sample. The zpiezo’s intrinsic response delay time causes the piezo to overshoot when the ion current trigger occurs (Figure S4a). The overshoot distance linearly increases with the z-piezo velocity (Figure S4b). To avoid the pipet touching the sample surface, the piezo velocity has to be chosen such that the overshoot distance does not exceed the pipet-sample distance at the ion current trigger (e.g., about one pipet inner opening radius at an ion current trigger of 1%)49,50. Generally, a piezo with a larger total travel range (used in the Macro-SICM) has a longer response delay time and therefore overshoots more at a given velocity than a piezo with a shorter range (used in conventional SICMs). The imaging speed is therefore smaller for the Macro-SICM than for conventional SICMs when using the same pipet. We used approach velocities and ion current triggers such that the overshoot distance was always smaller than the pipet-sample distance at the ion current trigger. To further reduce the probability of pipet-sample contact, the piezo can be decelerated near the sample surface during the approach phase (as was done in the present study).51 In conventional SICMs, pipets with inner opening radii in the range of 50-100 nm are typically used.24,26,33 Here, we also used larger pipets with an inner opening radius of about 2 µm. Larger pipets have the advantage that the ion current is larger, which reduces the influence of current noise. Larger pipets are also less easily clogged with debris. Furthermore, they have a larger pipet-sample distance at a given ion current trigger and can therefore tolerate a larger overshoot distance. For larger pipets, however, the imaging speed is not necessarily higher, because the ion current has a weaker dependency on the pipet-sample distance and therefore requires a larger retract distance (Figure S5). We estimated the maximum step rate of the used xy-micropositioning stages as 22-40 Hz (Figure S6), which is much slower than the performance of piezo-based stages used in conventional SICMs. ACS Paragon Plus Environment

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The lateral resolution of the Macro-SICM is technically limited by the minimal step size of the micropositioning stages (0.3 µm) and fundamentally limited by three times the pipet’s inner opening radius.52 In our study, the pipet inner opening size was much smaller than the pixel size, possibly leading to a certain extent of error in morphological parameters such as average height or volume of objects (depending on the sample roughness). However, this is a common situation e.g. in AFMs, where nanometer-sized tips are routinely used for large-range (e.g. 100 µm) imaging and subsequent object shape quantification. Using smaller pipets has the advantage of higher vertical and lateral resolution, which allows zooming-in on regions of interest in a large-range image (as done in Figure 4). There are several other microscope techniques that can be used to image sample topography, for example, the AFM,53 the scanning electron microscope (SEM),54 or various advanced optical techniques such as the digital holographic microscope (DHM).55 The AFM, which typically has a similar scan range as conventional SICMs, can also operate in a liquid environment, making it useful for studying living cells.56,57 However, the AFM tip gets into mechanical contact with the sample, possibly leading to damage of delicate surface structures.58 It has been shown for soft samples that the SICM can be advantageous to the AFM in terms of imaging quality and long-term imaging stability.44 The SEM is a widely used technique to image samples on the centimeter down to the nanometer scale. However, for biological samples it requires a costly preparation protocol including fixation, dehydration, and metal coating, which can cause surface artifacts.59 In addition, the SEM does not directly provide quantitative information about the vertical sample height. The DHM is based on recording an image of the optical phase shift, from which morphological parameters such as height and volume can be derived.60 Like the SICM, the DHM has the advantage that it provides contact-free and non-invasive imaging. It has been applied, for example, to study



cell dynamics during cell division61 or during wound healing.62 Compared to optical techniques, however, the SICM has the outstanding advantage that the pipet serves as a multifunctional device, for example, to deliver or uptake molecules,63 to sense electrochemical surface properties,7-9 or for mechanical measurements.14,16

CONCLUSION We described a Macro-SICM with the largest so far reported scan range of 25 mm × 25 mm in xy- and 0.25 mm in z-direction. Its large scan range makes the Macro-SICM a versatile tool for investigating structures at different length scales, as we demonstrated by imaging structures on the centimeter (fingerprint, coin), millimeter (bovine tongue, insect wing), and micrometer (small cellular extensions of single fibroblast cells) scale. Further, we have shown that the Macro-SICM is a promising tool to significantly improve throughput, which we demonstrated by imaging 85 single cells in one single image (many more cells are possible since here we used only 4 % of the full scan range). The SICM has a large variety of possible advanced applications beyond topography imaging, because the pipet is a multifunctional device such as for molecule delivery, electrochemistry, or mechanical measurements. We anticipate that the Macro-SICM will improve the applicability and throughput of such applications, because the pipet can easily and reproducibly be positioned within a large scan range.


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ASSOCIATED CONTENT

Supporting Information Supporting Information available as noted in the text. The Supporting Information is available free of charge on the ACS Publications website at DOI: … Imaging stability (Figure S1), precision of measuring cell height and cell volume (Figure S2), precision and repeatability of large-range pipet positioning (Figure S3), characterization of zpiezo overshoot (Figure S4), ion current vs. distance behavior (Figure S5), and step rate of the xy-micropositioning stages (Figure S6).

ACKNOWLEDGEMENTS We acknowledge support by the Ministry for Science, Research, and Art of BadenWürttemberg (MWK) within the Industry on Campus (IoC) program. The authors thank Timo Ziegler for building an early version of the Macro-SICM and Lars Hanke for help with construction. We thank Johannes Rheinlaender, Jan Seifert, and Stefan Simeonov for helpful discussions.

AUTHOR INFORMATION Email: [email protected]

NOTES The authors declare no competing financial interest.



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FIGURES

Figure 1. Macro-SICM for large-range imaging. (a) The Macro-SICM is equipped with two linear micropositioning stages (x-, y-scanner) for lateral scanning the sample with a scan range of 25×25 mm2. The pipet is vertically positioned over the sample using a z-piezo with a scan range of 0.25 mm. The z-piezo is attached to a motorized stage with 25 mm range. The Macro-SICM is equipped with phase contrast illumination and a heated sample stage. (b) SICM topography of a human fingerprint on a glass surface, recorded with a lateral scan range of 20×20 mm2. (c) SICM topography of a Greek 5 Eurocent coin, recorded with a lateral scan range of 12×12 mm2.


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Figure 2. Bovine tongue tissue imaged with the Macro-SICM. (a) 3d-rendered SICM topography of the surface of a bovine tongue. (b) Height profile along the dashed line in panel (a).



Figure 3. Hindwing of the pentatomid bug Graphosoma lineatum italicum imaged at different length scales with the Macro-SICM. (a) Optical image of the whole wing. The wing membrane (me) and transition zones between rigid and flexible cuticle as connected to fold zones (fz) and associated false veins (fv) are visible. (b) SICM topography of the region marked with the blue box in panel (a) and height profile along the black dotted line showing height variations on the order of 100 µm. (c) SICM topography of a region on the wing membrane and height profile of local conical protrusions along the black dotted line.


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Figure 4. Single cell imaging with the Macro-SICM. (a) Optical phase contrast of fixed fibroblast cells seeded at sparse density and (b) the corresponding SICM topography of the same region. (c) Cell height and cell volume for n = 85 cells approximately followed normal distributions (solid traces, plotted by using the mean µ and the standard deviation σ from the distributions). (d) SICM topography of single fibroblast cells within the red outlined regions marked in panels (a) and (b).



Figure 5. Time-lapse SICM imaging of epithelial wound healing over 19 h. (a) Series of SICM topography images of A6 cells and corresponding average height profiles, averaged over all horizontal scan lines. During wound healing, cells collectively migrate from the left and the right wound edge into the cell free region, thereby continuously decreasing the (b) wound area and (c) wound volume. The wound boundaries at time 00:00 h are marked with red dotted lines in panel (a).


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FOR TOC ONLY


Macro-SICM: A Scanning Ion Conductance Microscope for Large

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