Second-Harmonic Generation Imaging Microscopy of Structural Protein Arrays in Tissue

Abstract

Abstract In the past several years, second-harmonic generation (SHG) has emerged as a powerful nonlinear optical contrast mechanism for biological imaging applications. SHG is a coherent processwhere two lower-energy photonsare up-converted to exactly twice the incident frequency (or half the wavelength). This effect was first demonstrated by Kleinman (Kleinman, 1962) in crystalline quartz in 1962, where thisadvance wasmade possible with the invention of the ruby laser. Since that discovery, SHG in uni-axial birefringent crystals has been exploited to frequency double pulsed lasers to obtain shorter wavelengths, thereby producing multiple colors from a single source. SHG from interfaces was later discovered by Bloembergen in 1968 and rapidly became a versatile spectroscopic tool to study chemical and physical processes at air–solid, air– liquid, and liquid–liquid interfaces (for reviews, see Eisenthal, 1996; Shen, 1989). The first integration of SHG and optical microscopy was achieved in 1974 by Hellwarth and coworkers, who used SHG as an imaging tool to visualize the microscopic crystal structure in polycrystalline ZnSe (Hellwarth & Christensen, 1974). Sheppard then implemented the method on a scanning microscope in 1977 (Sheppard et al., 1977).