Laserexcited confocalfluorescence gel scanner
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abstract
A high-sensitivity, laser-excited, confocal-fluorescence scanner has been developed for the detection of fluorescently labeled nucleic acids separated on slab gels. The gel is placed on a motor-driven, two-dimensional scan stage and raster scanned past the optical detection system. The 488-nm argon ion laser beam is introduced into the confocal optical system at a long-pass dichroic beam splitter and focused within the gel to an 2 m diameter spot by a high-numerical aperture microscope objective. The resulting fluorescence is gathered by the objective, passed back through the first long-pass beam splitter, and relayed to a second dichroic beam splitter that separates the red and green emissions. The fluorescence is then focused on confocal spatial filters to reduce stray and scattered light, passed through spectral filters, and detected with photomultipliers. The resulting signals are amplified, filtered, and digitized for display on a computer. This system can detect as little as 510-12 M fluorescein, the resolution as operated is 160 m, and it can scan a 6 cm6 cm gel using a scan rate of 4 cm/s in 12 min. The detection of DNA on slab gels, two-color DNA fragment sizing, and microtiter plate scanning are presented to illustrate some of the possible applications of this apparatus.
