Scanning near-field optical microscopy of cell surfaces after structure conserving air-drying

摘要

Scanning near-field optical microscopy (SNOM) can simultaneously map topographic and optical properties (e.g., absorption or fluorescence) of surfaces with a spatial resolution between that of far-field light microscopy and electron microscopy, i.e. in the range of 100 nm. Since commercially available SNOM instruments came on the market, this technique has become interesting for the routine biological research laboratory especially in combination with far-field light imaging. However, due to the usually applied shear-force feedback controlling the SNOM tip, this technique still poses several challenges for biological applications. In our experiments for instance imaging of soft samples (compared to solid body surfaces), large topographical changes on structurally conserved cell surfaces (typically some μm), and in particular the requirement for completely dried specimen had to be considered. To visualise surfaces of cells fixed on standard glass slides by SNOM, an easy to handle, optimised protocol using dehydration and hexamethyldisilazane exposure before air drying was developed. Using the commercially available instrument SNOM 210 (Carl Zeiss Jena GmbH, Digital Instruments) with micro-fabricated silicon nitride tips, it was shown for several cell systems (antibody labelled lymphocytes, macrophages, breast cancer cells of the cell line T-47D) that the cellular morphology and surface structures were well preserved after this procedure of drying.