A novel light source for SICM-SNOM of living cells.

We have developed a novel light source for use in a scanning near-field optical microscope (SNOM or NSOM) based on a nanopipette whose distance from the sample surface is controlled using scanning ion conductance microscopy. The light source is based on the general principle of the chemical reaction between a fluorophore in the pipette and ligand in the bath, to produce a highly fluorescent complex that is continually renewed at the pipette tip. In these experiments we used fluo-3 and calcium, respectively. This complex is then excited with an Ar+ laser, focused on the pipette tip, to produce the light source. This method overcomes the transmission problem of more traditional SNOM probes and has been used to acquire simultaneous high-resolution topographic and optical images of biological samples in physiological buffer. A resolution of approximately 220 nm topographic and approximately 190 nm optical was determined through imaging fixed sea-urchin sperm flagella. Live A6 cells were also imaged, demonstrating the potential of this system for SNOM imaging of living cells.