Multispectral Imaging in Light Microscopy

Abstract

Many recent applications in light microscopy involve the use of multiple fluorophores or the delineation of signals arising from spectrally distinct sources. In microspectroscopy, it is always desirable to illuminate fluorescently-labeled microscopic specimens with monochromatic light as the narrowest possible excitation wavelength range usually results in the highest emission signal-to-noise ratio. Generation of polychromatic light from an arc lamp and selection of the excitation wavelength by interference filters or monochrometers are the most common techniques for excitation microspectrofluorometry. Emission spectroscopy is usually done with filter wheels, monochrometers, or interferometers inserted between the microscope detection port and the detector. This presentation will be directed toward other, less frequently-used, approaches for spectral scanning of the specimen in the light microscope. Three topics will be considered: 1) the use of acousto-optical tunable filters and lasers for rapid, narrow-band, excitation wavelength selection; 2) the use of holographic notch filters for rejection of unwanted excitation laser light; 3) using liquid-crystal tunable filters for emission scanning.