A novel light source for SICM-SNOM of living cells

J Microsc. 2003 Feb;209(Pt 2):94-101. doi: 10.1046/j.1365-2818.2003.01122.x.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aniline Compounds
  • Animals
  • Cell Line
  • Imaging, Three-Dimensional / methods
  • Light*
  • Male
  • Microscopy, Scanning Probe / instrumentation*
  • Sea Urchins
  • Spermatozoa / ultrastructure
  • Xanthenes

Substances

  • Aniline Compounds
  • Xanthenes
  • Fluo-3