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Biomedical Applications of Second Harmonic Generation Imaging Microscopy

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Abstract

SHG is a second order nonlinear optical process that can only arise from media lacking a center of symmetry, e.g. an anisotropic crystal or at an interface. Since SHG is a nonlinear optical phenomenon, this form of imaging shares many of the features of two-photon excited fluorescence (TPEF) microscopy1. Due to its intrinsic 3- dimensionality and greatly reduced out-of-plane photobleaching and phototoxicity, the latter methodology has gained considerable popularity as an ideal method for live cell and tissue imaging. In addition, the use of near infrared excitation results in the ability to penetrate deeply into turbid and thick tissue because of reduced Rayleigh scattering and as well as reduced absorption by endogenous chromophores. Here we first show how SHG can be produced from the membranes of living cells stained with voltage sensitive styryl chromophores. The use of different dyes allows the elucidation of the underlying contrast mechanism. We also show efficient SHG from endogenous structural proteins, including collagen and myosin from several tissue types. These abundant proteins comprise such tissues as tendon, bone, and muscle, and form highly-ordered, birefringent, supra-molecular arrays. Comparison of the SHG images with two-photon excited fluorescence of Green Fluorescent Protein (GFP) provides the basis for identifying the molecular origin of SHG signals. Polarization anisotropy further probes the lateral and radial symmetry of the protein assemblies. We will discuss how SHG can be potentially applied to the study of diseases related to defects in these proteins.

© 2002 Optical Society of America

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