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.
Rothery
A M
AM
Department of Chemistry, University of Cambridge, Cambridge, UK.
Gorelik
J
J
Bruckbauer
A
A
Yu
W
W
Korchev
Y E
YE
Klenerman
D
D
D000814:Aniline Compounds; D000818:Animals; D002460:Cell Line; D021621:Imaging, Three-Dimensional / Q000379:methods; D008027:Light; D008297:Male; D020527:Microscopy, Scanning Probe / Q000295:instrumentation*; D012617:Sea Urchins; D013094:Spermatozoa / Q000648:ultrastructure; D014966:Xanthenes
D000814:Aniline Compounds; D014966:Xanthenes; C059715:Fluo-3