By adapting a laser scanning microscope with a titanium sapphire femtosecon
d pulsed laser and transmission optics, we are able to produce live cell im
ages based on the nonlinear optical phenomenon of second harmonic generatio
n (SHG). Second harmonic imaging (SHIM) is an ideal method for probing memb
ranes of living cells because it offers the high resolution of nonlinear op
tical microscopy with the potential for near-total avoidance of photobleach
ing and phototoxicity. The technique has been implemented on three cell lin
es labeled with membrane-staining dyes that have large nonlinear optical co
efficients. 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 si
gnal intensity.