Applications of Second-Harmonic Generation Microscopy

Abstract

Abstract By far the most best-known form of nonlinear microscopy is based on two-photon excited fluorescence (TPEF) (Denk et al., 1990), which is described in considerable detail in Chapters 4 and 5. However, a lesser-known formof this microscopy was used several years prior to the invention of TPEF microscopy, based on the generation of second-harmonic light either from surfaces (Hellwarth & Christensen, 1964; Sheppard et al., 1977) or fromendogenous tissue structures such as rat-tail tendons (Freund et al., 1986). Because of difficulties in signal interpretation and perhaps because of its seemingly arcane utility, at least in biological imaging, second-harmonic generation (SHG) microscopy has gone by relatively unnoticed until only very recently (Campagnola et al., 1999; Guo et al., 1997; Moreaux et al., 2001). The discovery that exogenous markers (Chemla & Zyss, 1984; Prasad, 1991) can lead to exceptionally high signal levels has been a leading cause for the revival of SHG microscopy. In particular, SHG markers, when properly designed and collectively organized, can produce signal levels comparable to those encountered in standard TPEF microscopy (Moreux et al., 2000a, 2000b). Moreover, the spatial resolutions provided by SHG and TPEF microscopies are commensurate, meaning that the two contrasts can be conveniently derived from the same instrument. Despite their similarities, SHG and TPEF are based on fundamentally different phenomena (Bloembergen, 1965; Butcher & Cotter, 1990). The first relies on nonlinear scattering, whereas the second relies on nonlinear absorption followed by fluorescence emission. In other words, the first is coherent, whereas the second is not. The consequences of this basic difference will be described below. The organization of this chapter is as follows. First, the basic principles of SHG will be qualitatively described at the molecular level. Second, typical experimental configurations for combined SHG and TPEF imaging will be briefly described, along with basic protocols for sample preparation and labeling. Finally, various applications of SHG microscopy will be addressed, with an emphasis on high-sensitivity membrane potential imaging and on endogenous imaging in thick tissue.