Second Harmonic Generation Signal from Biological Materials Using Multi-Functional Two-Photon Laser Scanning Microscopy

The nonlinear optical effect, such as two-photon (Denk et al., 1990) and three-photon (Maiti et al., 1997; Schrader et al., 1997; Wokosin et al., 1996) fluorescence significantly improved the depth resolution and reduced the background noise. The nonlinear optical techniques have been used to develop a new generation of optical microscopes with novel capabilities. These new capabilities include the ability of using near-infrared light to induce absorption, and hence fluorescence from fluorophores that are absorbed in the ultraviolet region. Other capabilities of nonlinear microscopes include: improved spatial and temporal resolution without the use of pinholes or slits for spatial filtering, improved signal strength, deeper penetration into thick, highly scattering tissue and confinement of photo-bleaching to the focal volume (Denk et al. 1990). The invention of nonlinear laser microscopy opened new opportunity to noninvasively examine the structure and function of living cells or tissues (Denk et al., 1990). Among different multiphoton implementations (Zipfel et al., 2003; Zumbusch et al., 1999), second harmonic generation imaging (Roth and Freund 1980; Freund et al., 1986; Campagnola et al., 2001; Yeh et al., 2002; Campagnola and Lowe, 2003; Cox et al., 2003) is particularly suitable for investigating non-centrosymmetric structures. Second harmonic generation (SHG) is a nonlinear optical process that occurs only at the focal point of a laser beam (Shen, 1989). SHG is not much younger than laser, but the application of such a weak process to the imaging of cellular structures and functions is quite new and notable (Campagnola and Loew., 2003). Advancement in mode-locked laser (instead of a continuous wave, mode-locked laser emits short pulses in the range of ns to fs) makes SHG imaging of cells possible since one do not need such a high intensity which in fact cooks the cells right a way. Using chiral chromophores, chiral SHG imaging can be applied to otherwise impossible symmetric structures (Yan et al., 2006). Second harmonic imaging microscopy (SHIM) is based on nonlinear optical effect known as second harmonic generation. A laser scanning microscope using second harmonic generation as a probe is shown to produce high-resolution images of transparent biological specimens. SHIM has