LASER-EXCITED CONFOCAL-FLUORESCENCE GEL SCANNER

A high-sensitivity, laser-excited, confocal-fluorescence scanner has b een developed for the detection of fluorescently labeled nucleic acids separated on slab gels. The gel is placed on a motor-driven, two-dime nsional scan stage and raster scanned past the optical detection syste m. The 488-nm argon ion laser beam is introduced into the confocal opt ical system at a long-pass dichroic beam splitter and focused within t he gel to an approximately 2 mum diameter spot by a high-numerical ape rture 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 spat ial filters to reduce stray and scattered light, passed through spectr al 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 5 X 10(-12) M fluorescein, the resolut ion as operated is 160 mum, and it can scan a 6 cm x 6 cm gel using a scan rate of 4 cm/s in 12 min. The detection of DNA on slab gels, two- color two-color DNA fragment sizing, and microtiter plate scanning are presented to illustrate some of the possible applications of this app aratus.