[3] Second harmonic imaging microscopy

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The chapter provides information on the second harmonic imaging microscopy. Over the past three decades, the physical phenomenon of optical second harmonic generation (SHG) has been used to study interfaces between materials and has been adapted for the purposes of microscopy. SHG is a nonlinear optical process that can take place in a microscope that uses illumination from ultrafast (near-infrared) laser light. One of the first demonstrations of SHG from a biological specimen was of bacteriorhodopsin in a membrane preparation. More recently, SHG microscope images are obtained when one leaflet of the lipid bilayer of the cell membrane is stained with a dye that enhances SHG. SHG is a less efficient process than 2PF but can be significantly resonance enhanced. However, because excited fluorescence (2PF) still results in some photobleaching at the focus, the best wavelengths for SHG are on the edges of two-photon excitation bands, enhancing SHG while reducing absorption.

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