Second-harmonic imaging microscopy of living cells

Second harmonic generation (SHG) has been developed in our laboratories as a high-resolution nonlinear optical imaging microscopy for cellular membran es and intact tissues. SHG shares many of the advantageous features for mic roscopy of another more established nonlinear optical technique: two-photon excited fluorescence (TPEF). Both are capable of optical sectioning to pro duce three-dimensional images of thick specimens and both result in less ph otodamage to living tissue than confocal microscopy. SHG is complementary t o TPEF in that it uses a different contrast mechanism and is most easily de tected in the transmitted light optical path. It can be used to image membr ane probes with high membrane specificity and displays extraordinary sensit ivity in reporting membrane potential; it also has the ability to image hig hly ordered structural proteins without any exogenous labels. (C) 2001 Soci ety of Photo-Optical Instrumentation Engineers.