High-resolution nonlinear optical imaging of live cells by second harmonic generation.
By adapting a laser scanning microscope with a titanium sapphire femtosecond pulsed laser and transmission optics, we are able to produce live cell images based on the nonlinear optical phenomenon of second harmonic generation (SHG). Second harmonic imaging (SHIM) is an ideal method for probing membranes of living cells because it offers the high resolution of nonlinear optical microscopy with the potential for near-total avoidance of photobleaching and phototoxicity. The technique has been implemented on three cell lines labeled with membrane-staining dyes that have large nonlinear optical coefficients. The images can be obtained within physiologically relevant time scales. Both achiral and chiral dyes were used to compare image formation for the case of single- and double-leaflet staining, and it was found that chirality plays a significant role in the mechanism of contrast generation. It is also shown that SHIM is highly sensitive to membrane potential, with a depolarization of 25 mV resulting in an approximately twofold loss of signal intensity.
Campagnola
P J
PJ
Department of Physiology and Center for Biomedical Imaging Technology, University of Connecticut Health Center, Farmington, Connecticut 06030 USA. campagno@panda.uchc.edu
Wei
M D
MD
Lewis
A
A
Loew
L M
LM
D016475:3T3 Cells; D000818:Animals; D055592:Biophysical Phenomena; D001703:Biophysics; D002477:Cells / Q000166:cytology*; D004396:Coloring Agents; D007834:Lasers; D008564:Membrane Potentials; D051379:Mice; D008853:Microscopy / Q000295:instrumentation*; D017711:Nonlinear Dynamics; D055095:Optics and Photonics / Q000295:instrumentation*; D014407:Tumor Cells, Cultured
D004396:Coloring Agents