The use of scanning ion conductance microscopy to image A6 cells

Abstract

Background: Continuous high spatial resolution observations of living A6 cells would greatly aid the elucidation of the relationship between structure and function and facilitate the study of major physiological processes such as the mechanism of action of aldosterone. Unfortunately, observing the micro-structural and functional changes in the membrane of living cells is still a formidable challenge for a microscopist. Method: Scanning ion conductance microscopy (SICM), which uses a glass nanopipette as a sensitive probe, has been shown to be suitable for imaging non-conducting surfaces bathed in electrolytes. A specialized version of this microscopy has been developed by our group and has been applied to image live cells at high-resolution for the first time. This method can also be used in conjunction with patch clamping to study both anatomy and function and identify ion channels in single cells. Results: This new microscopy provides high-resolution images of living renal cells which are comparable with those obtained by scanning electron microscopy (SEM) and atomic force microscopy (AFM). Continuous 24 h observations under normal physiological conditions showed how A6 kidney epithelial cells changed their height, volume, and reshaped their borders. The changes in cell area correlated with the density of microvilli on the surface. Surface microvilli density ranged from 0.5 μm⁻² for extended cells to 2.5 μm² for shrunk cells. Patch clamping of individual cells enabled anatomy and function to be correlated. Conclusions: Scanning ion conductance microscopy provides unique information about living cells that helps to understand cellular function. It has the potential to become a powerful tool for research on living renal cells.

Introduction

The apical membrane of renal cells is a highly dynamic structure, which contains important signaling transducers such as ion channels and receptors (Nelson et al., 1992, Rehn et al., 1998). It also often contains microvilli, dynamic membranous protrusions filled with actin cytoskeletal components. Various molecules and molecular complexes are believed either to move about freely in the plane of the membrane, or to be restricted to some specific microdomains, which in turn seem to be dynamic (Nelson, 1992b). Continuous high spatial resolution observations of renal cells would help to elucidate many of these structure/function relationships and also facilitate investigation of major physiological processes such as hormone mediated ion transport or volume regulation. For example, the time necessary to observe the full effect of aldosterone on the regulation of Na⁺ transport is known to be up to 24 h (Booth et al., 2002).

Investigation of the micro-structural and functional changes in the membrane of living cells is a formidable challenge (White et al., 2001). Furthermore, there are specific problems associated with studying renal cells. Cultured renal epithelial cells are used for studying renal functions and display the key features of polarized epithelial cells with distinct apical and basolateral membranous compartments separated by tight junctions between cells (Duchatelle et al., 1992, Nelson, 1992a). They also tend to display more differentiated phenotypes when cultured on specially treated surfaces, like filter supports (Candia et al., 1993). However, most of these surfaces are not transparent, which makes it difficult to study such cultures by conventional optical microscopy. Alternatively, the cell membranes can be visualized by fluorescent microscopy. However, addition of fluorophors causes other problems such as photobleaching and photoinduced damage, especially if a long period of observation is required.

In principle scanning, a fine probe over the surface to determine the topography, as generally applied in scanning probe microscopy (SPM), offers a way to visualize the cell membrane. One such technique, atomic force microscopy (AFM), has been used to image living cells in real time on a nanometer scale (Fritz et al., 1993, McElfresh et al., 2002). These studies have provided valuable data, but also perhaps serve to highlight the difficulties of using AFM on intact living cells. AFM has also been used for imaging microvilli on the surface of epithelial cells (Braet et al., 1998, Lesniewska et al., 1998). However, all authors noted that it was difficult to resolve microvilli due to technical problems and reported reliable high-resolution imaging only on fixed cell preparations.

Hansma et al. (1989) demonstrated that a new kind of SPM, scanning ion conductance microscopy (SICM), which uses a glass micropipette as the sensitive probe, was suitable for imaging non-conducting surfaces bathed with electrolytes. A specialized version of this kind of SICM has been developed by our group and has been applied to the high-resolution imaging of living cells (Korchev et al., 1997a, Korchev et al., 1997b).

In the present work, we used SICM for long-term imaging of the nano-structures of living renal cell membranes under physiological conditions. A well-studied renal tubular epithelial A6 cell line, derived from Xenopus laevis distal nephron, was used as a model to investigate the potential of this modified SICM for long-term studies of renal cells.