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Scanning ion conductance microscopy (SICM) using a nanopipette as a probe and ionic current as a feedback signal was introduced as a novel technique to study live cells in a physiological environment. To avoid contact between the pipette tip and cells during the conventional lateral scanning mode, we adopted a standing approach (STA) mode in which the probe was moved vertically to first approach and then retracted from the cell surface at each measurement point on an XY plane. The STA mode ensured non-contact imaging of the topography of live cells and for a wide range of uneven substrates (500 x 300 microm to 5 x 5 microm). We also used a field-programmable gate array (FPGA) board to enhance feedback distance regulation. FPGA dramatically increased the feedback speed and decreased the imaging time (450 s per image) with enhanced accuracy and quality of live cell images. To evaluate the potential of the STA mode for SICM, we carried out imaging of a convoluted surface of live cell in various scan ranges and estimated the spatial resolutions of these images.

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Scanning electrochemical microscopy (SECM) was employed to quantitatively characterize the oxygen permeation behaviors of poly(dimethylsiloxane) (PDMS) and surfacemodified PDMS. The mass-transfer process of oxygen from the PDMS substrate to the tip electrode is diffusion limited, whereas the oxygen permeability of PDMS subjected to oxygen plasma treatment or albumin adsorption is critically restricted. Our results suggest that the oxygen permeability of PDMS is possibly affected by O 2 plasma irradiation and albumin adsorption at the PDMS surfaces.

A membrane protein on the surface of a single living mammalian cell was imaged by scanning electrochemical microscopy (SECM). The epidermal growth factor receptor (EGFR) is one of the key membrane proteins associated with cancer. It elicits a wide range of cell-type-specific responses, leading to cell proliferation, differentiation, apoptosis, and migration. To estimate EGFR expression levels by SECM, EGFR was labeled with alkaline phosphatase (ALP) via an antibody. The oxidation current of PAP (p-aminophenol) produced by the ALP-catalyzed reaction was monitored to estimate the density of cell surface EGFR. EGFR measurement by SECM has three advantages. First, a single adhesion cell can be measured without peeling it from the culture dish; second, it is possible to optimize labeling antibody concentrations by using living cells because detection of faradaic current is suitable for quantitative estimation in situ; and third, SECM measurements afford information on the expression state at the cell membrane at the single-cell level. In this study, we optimized the concentration of labeling antibody for EGFR at the cell surface and confirmed distinct differences in EGFR expression levels among three types of cells. SECM measurements were compatible with the results of flow cytometry.