Andrew I. Shevchuk

Nanoscale live cell imaging using hopping probe ion conductance microscopy

We describe hopping mode scanning ion conductance microscopy that allows noncontact imaging of the complex three-dimensional surfaces of live cells with resolution better than 20 nm. We tested the effectiveness of this technique by imaging networks of cultured rat hippocampal neurons and mechanosensory stereocilia of mouse cochlear hair cells. The technique allowed examination of nanoscale phenomena on the surface of live cells under physiological conditions.

Simultaneous Noncontact Topography and Electrochemical Imaging by SECM/SICM Featuring Ion Current Feedback Regulation

We described a hybrid system of scanning electrochemical microscopy (SECM) and scanning ion conductance microscopy (SICM) with ion current feedback nanopositioning control for simultaneous imaging of noncontact topography and spatial distribution of electrochemical species. A nanopipette/nanoring electrode probe provided submicrometer resolution of the electrochemical measurement on surfaces with complex topology. The SECM/SICM probe had an aperture radius of 220 nm. The inner and outer radii of the SECM Au nanoring electrode were 330 and 550 nm, respectively. Characterization of the probe was performed with scanning electron microscopy (SEM), cyclic voltammetry (CV), and approach curve measurements. SECM/SICM was applied to simultaneous imaging of topography and electrochemical responses of enzymes (horse radish peroxidase (HRP) and glucose oxidase (GOD)) and single live cells (A6 cells, superior cervical ganglion (SCG) cells, and cardiac myocytes). The measurements revealed the distribution of activity of the enzyme spots on uneven surfaces with submicrometer resolution. SECM/SICM acquired high resolution topographic images of cells together with the map of electrochemical signals. This combined technique was also applied to the evaluation of the permeation property of electroactive species through cellular membranes.

Multifunctional Nanoprobes for Nanoscale Chemical Imaging and Localized Chemical Delivery at Surfaces and Interfaces

Double take: Double-barrel carbon nanoprobes with integrated distance control for simultaneous nanoscale electrochemical and ion conductance microscopy can be fabricated with a wide range of probe sizes in less than two minutes. The nanoprobes allow simultaneous noncontact topographical (left image) and electrochemical imaging (right) of living neurons, as well as localized K+ delivery and simultaneous neurotransmitter detection.

Simultaneous measurement of Ca2+ and cellular dynamics: combined scanning ion conductance and optical microscopy to study contracting cardiac myocytes.

We have developed a distance modulated protocol for scanning ion conductance microscopy to provide a robust and reliable distance control mechanism for imaging contracting cells. The technique can measure rapid changes in cell height from 10 nm to several micrometers, with millisecond time resolution. This has been demonstrated on the extreme case of a contracting cardiac myocyte. By combining this method with laser confocal microscopy, it was possible to simultaneously measure the nanometric motion of the cardiac myocyte, and the local calcium concentration just under the cell membrane. Despite large cellular movement, simultaneous tracking of the changes in cell height and measurement of the intracellular Ca2+ near the cell surface is possible while retaining the cell functionality.

Topographical and electrochemical nanoscale imaging of living cells using voltage-switching mode scanning electrochemical microscopy

We describe voltage-switching mode scanning electrochemical microscopy (VSM-SECM), in which a single SECM tip electrode was used to acquire high-quality topographical and electrochemical images of living cells simultaneously. This was achieved by switching the applied voltage so as to change the faradaic current from a hindered diffusion feedback signal (for distance control and topographical imaging) to the electrochemical flux measurement of interest. This imaging method is robust, and a single nanoscale SECM electrode, which is simple to produce, is used for both topography and activity measurements. In order to minimize the delay at voltage switching, we used pyrolytic carbon nanoelectrodes with 6.5–100 nm radii that rapidly reached a steady-state current, typically in less than 20 ms for the largest electrodes and faster for smaller electrodes. In addition, these carbon nanoelectrodes are suitable for convoluted cell topography imaging because the RG value (ratio of overall probe diameter to active electrode diameter) is typically in the range of 1.5–3.0. We first evaluated the resolution of constant-current mode topography imaging using carbon nanoelectrodes. Next, we performed VSM-SECM measurements to visualize membrane proteins on A431 cells and to detect neurotransmitters from a PC12 cells. We also combined VSM-SECM with surface confocal microscopy to allow simultaneous fluorescence and topographical imaging. VSM-SECM opens up new opportunities in nanoscale chemical mapping at interfaces, and should find wide application in the physical and biological sciences.

Dynamic assembly of surface structures in living cells

Although the dynamics of cell membranes and associated structures is vital for cell function, little is known due to lack of suitable methods. We found, using scanning ion conductance microscopy, that microvilli, membrane projections supported by internal actin bundles, undergo a life cycle: fast height-dependent growth, relatively short steady state, and slow height-independent retraction. The microvilli can aggregate into relatively stable structures where the steady state is extended. We suggest that the intrinsic dynamics of microvilli, combined with their ability to make stable structures, allows them to act as elementary “building blocks” for the assembly of specialized structures on the cell surface.

Ion channels in small cells and subcellular structures can be studied with a smart patch-clamp system.

We have developed a scanning patch-clamp technique that facilitates single-channel recording from small cells and submicron cellular structures that are inaccessible by conventional methods. The scanning patch-clamp technique combines scanning ion conductance microscopy and patch-clamp recording through a single glass nanopipette probe. In this method the nanopipette is first scanned over a cell surface, using current feedback, to obtain a high-resolution topographic image. This same pipette is then used to make the patch-clamp recording. Because image information is obtained via the patch electrode it can be used to position the pipette onto a cell with nanometer precision. The utility of this technique is demonstrated by obtaining ion channel recordings from the top of epithelial microvilli and openings of cardiomyocyte T-tubules. Furthermore, for the first time we have demonstrated that it is possible to record ion channels from very small cells, such as sperm cells, under physiological conditions as well as record from cellular microstructures such as submicron neuronal processes.

Plasma membrane topography and interpretation of single-particle tracks.

To the Editor: Many contemporary models of the plasma membrane are based on single-particle tracking (SPT) by light microscopy on live cells. Whereas the analysis of single-particle tracks typically presumes that the cell surface is locally flat, both the ability of cells to rapidly swell and electron microscopy evidence suggest considerable folding1. Even the interface between the plasma membrane and a coverslip is not necessarily flat2. However, there is a lack of hard evidence about the topography of living cells. Accordingly, we examined cell topography using hopping-probe ion conductance microscopy (HPICM)3, a highresolution, noncontact method. HPICM, like all scanning-probe microscopes, has a limited speed (Supplementary Fig. 1), but by scanning many small square blocks, rather than sequential lines, HPICM ensures that the relative heights of adjacent pixels are accurate. Of the 70 cell types we examined using this approach on living cells (Supplementary Table 1), none had flat plasma membrane subregions (Fig. 1a,b and Supplementary Figs. 1–3). This observation has serious implications for SPT. It is well established that molecules on cell surfaces appear to diffuse appreciably more slowly than on planar artificial membranes. Similarly, when we analyzed simulated movement over the non-flat surface of a live cell and a fixed cell in two dimensions (2D), the standard form of SPT analysis, the apparent rate of diffusion dropped; the effect varied with the local topography (Fig. 1b,c). Diffusion over simulated geometric surfaces also had a reduction in apparent movement4 (Supplementary Note and Supplementary Figs. 4,5) and diffusion over a real anisotropically textured surface proved to be similarly anisotropic5. When analyzed in 2D, pillars emulated features of hop diffusion6, where barriers divide a surface into separate domains in which movement is locally unconstrained (Fig. 1d) but from which particles may be excluded or within which they may be confined. The vertical sides of pillars appeared to trap particles (Fig. 1d), a phenomenon we call apparent topographical trapping; for thin pillars, this resembled binding (Supplementary Fig. 6). In these simulations, exclusion, confinement and locally unconstrained movement are artifacts produced by following movement over a non-flat surface in 2D. On membrane blebs, hop diffusion vanishes and diffusion rates are similar to those on reconstituted planar membranes7, which is compatible both with the loss of diffusion barriers and with smoothing of the membrane. The impressive precision with which particles can be localized, down to ±1.5 nm but more typically tens of nanometers, depends generally on localization of a tag rather than the molecule of interest. Cellular topography could cause an underappreciated problem in this respect as well. On a smooth surface the tag and molecule probably maintain a stable alignment, but topography produces an offset of up to ± the tag’s radius (Fig. 1e), which is substantial given the typical diameter of tags (5–40 nm). Membrane folds may also trap or exclude tags. Analysis in 2D of movement on non-flat cellular membranes can cause simple diffusion to show apparently complex patterns that necessitate complex explanations. High-resolution tracking in 3D is possible8. As the topography of live cells is measurable, a topographical map might be used to provide a general 40 × 40 × 2.5 μm

Scanning surface confocal microscopy for simultaneous topographical and fluorescence imaging: application to single virus-like particle entry into a cell.

We have developed a method for simultaneous recording of high-resolution topography and cell surface fluorescence in a single scan which we call scanning surface confocal microscopy. The resolution of the system allows imaging of individual fluorescent particles in the nanometer range on fixed or live cells. We used this technique to record the interaction of single virus-like particles with the cell surface and demonstrated that single particles sink into the membrane in invaginations reminiscent of caveolae or pinocytic vesicles. This method provides a technique for elucidating the interaction of individual viruses and other nanoparticles, such as gene therapy vectors, with target cells. Furthermore, this technique should find widespread application for studying the relationship of fluorescently tagged molecules with components of the cell plasma membrane.

The scanned nanopipette: a new tool for high resolution bioimaging and controlled deposition of biomolecules

The boundary between the physical and biological sciences has been eroded in recent years with new physical methods applied to biology and biological molecules being used for new physical purposes. We have pioneered the application of a form of scanning probe microscopy based on a scanned nanopipette, originally developed by Hansma and co-workers, for reliable non-contact imaging over the surface of a live cell. We have found that the nanopipette can also be used for controlled local voltage-driven application of reagents or biomolecules and this can be used for controlled deposition and the local delivery of probes for mapping of specific species. In this article we review this progress, focussing on the physical principles and new phenomena that we have observed, and then outline the future applications that are now possible.