High-resolution nonlinear optical imaging of live cells by second harmonicgeneration

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.