In recent years there has been considerable interest in two and three-photon excited fluorescence in laser scanning optical microscopy. Because absorption is confined tot he focal plane of the objective, these techniques provide intrinsic optical sectioning without the use of a confocal aperture. In addition, photobleaching and phototoxicity are greatly reduced above and below the focal plane. We have adapted a two-photon microscope to utilize surface second harmonic generation (SHG) as a new contrast mechanism for nonlinear optical biological imaging.

Surface SHG was first described by Shen [1] and arises from the second order nonlinear susceptibility, χ(2). Signal will only arise from a non-centrosymmetric environment such as an interfacial region. Thus this technique has the potential to probe cellular membranes at high specificity. Further, since SHG results from an induced polarization and not absorption, photobleaching considerations are greatly reduced over fluorescence based methods.