Scanning Ion Conductance Microscopy: Non-Contact Imaging and Electrophysiology

Editorial

Scanning Ion Conductance Microscopy: Non-Contact Imaging and Electrophysiology

Y-K Yoo, K-D Ryang, S-J Cho, Park Systems
KANC 4F, Iui-Dong 906-10
Suwon 443-270
South Korea
www.parkafm.co.kr

Introduction

A new chapter in the study of living cells has opened with the introduction of scanning ion conductance microscopy (SICM). This advance will provide a unique and unprecedented opportunity in cell biology by enabling targeted localized stimulation and non-destructive monitoring of cellular activity heretofore inaccessible by other analytical techniques.

Scanning Ion Conductance Microscopy

In SICM a glass nanopipette filled with an electrolyte senses the ion current above specimens completely immersed in a liquid buffer. The tip-sample distance is maintained by keeping the ion current constant, instead of applying a physical force to the sample, and so ICM is an ideal tool to obtain a stable image of soft and sticky biological samples [1,2].v

One electrode is placed inside the pipette while another is located in a bath solution. When an external bias is applied between these two electrodes, a current flow is detected. Completing the overall electrical circuit, one needs to account for two electrical resistances at the channel assuming that the resistance of the bath solution is negligible. The first electrical resistance emanates from the frustum shape of the pipette while the second results from the distance between the pipette and the sample surface. When the pipette is far from the surface, the latter electrical resistance diminishes, reaching a saturated current because the resistance due to the tip shape is almost constant during the measurement. As the pipette gets closer to the sample however, the volume of the conductive ion channel between the probe and the sample becomes smaller, resulting in a rapid decrease of the ion current, which is in turn used as a reference feedback signal. One can also apply an AC modulation to the technique in order to achieve a more stable operation [1] during measurement.

Although ICM was developed many years ago, it has not been widely used during the last decade due to the instrumentation complexity and the subsequent operational instability, in particular the large Z-bandwidth requirement for proper Z-servo feedback.

Live Cell Membrane Imaging with SICM

The cell membrane is probably the most important component of a cell. Most cellular activity is mediated via the membrane. However, it is extremely difficult to monitor a live cell membrane at the nanometre scale. Furthermore, the transparency of the membrane makes it virtually impossible to observe with light microscopy.

Figure 1 shows SICM images of live COS-1 cells. The cells were alive and stable during the entire duration of ICM imaging, showing no signs of physical deterioration. The yellow arrows in Figures 1a and 1b show how fibroblasts behave when two cells collide. Often, two neighbouring cells exhibit different levels of protrusive activity, as shown in Figures 1c and 1d. A higher density of surface protrusions can be observed in fibroblast A compared to B and such different densities are even more evident in the phase image shown in Figure 1d. Such structural differences are almost impossible to observe with a light microscope or a traditional AFM.

The topography image of a mouse lung cell in Figure 2a shows the detail of a live cell. Furthermore, the ICM current-error image in Figure 2b displays the cell retraction fibres on the substratum after contraction of a live mouse muscle cell (C2C12).

 Scanning ion conductance microscopy images of live COS-1 cells.

Figure 1: Scanning ion conductance microscopy images of live COS-1 cells. (a) and (c) are SICM images whose scan sizes are 30 µm and 40 µm respectively. (b) and (d) are the corresponding phase images.The colour bar shows relative height in µm.

 ICM topography image of a live mouse lung cell.

Figure 2: (a) ICM topography image of a live mouse lung cell. Courtesy of Prof. D. Anselmetti and group at the University of Bielefeld, Germany. (b) ICM current-error image of live mouse muscle cell (C2C12). Colour scale bar is in pA.

Targeted Localized Stimulation and Monitoring of Cellular Activity

Using a fluid-filled pipette for ICM instead of a silicon cantilever for AFM opens pathways for new analytical possibilities. Ideal for imaging soft biological samples in liquid, such as living cells, ICM can easily be adapted to a host of qualitative and quantitative biochemical stimulation on single cells and cell motility studies, whose applications include targeted localized stimulation and monitoring and cellular drug delivery.

In targeted localized stimulation, one induces a cell movement by applying a localized pressure via the pipette hole and monitors the subsequent responses [2]. Furthermore, the functional capability of the ICM can be extended to the study of live-cell dynamics in response to targeted chemical or drug stimulation, achieving precisely controlled electrophysiology at the nanoscale.

References
1. Pastre, D. et al. Ultramicroscopy 90(1):13-19, 2001.
2. Korchev, Y. E. et al. Nature Cell Biology 2:616-619, 2000.
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