Laser-excited confocal-fluorescence gel scanner

Journal Article · Fri Apr 01 04:00:00 EST 1994 · Review of Scientific Instruments; (United States)

DOI:https://doi.org/10.1063/1.1144905· OSTI ID:5054009

Mathies, R A; Scherer, J R; Quesada, M A ^[1]; Rye, H S; Glazer, A N ^[2]

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1. Department of Chemistry, University of California, Berkeley, California 94720 (United States)
2. Department of Molecular and Cell Biology, University of California, Berkeley, California 94720 (United States)

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 [similar to]2 [mu]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 5[times]10[sup [minus]12] M fluorescein, the resolution as operated is 160 [mu]m, and it can scan a 6 cm[times]6 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.

• DOE Contract Number: FG03-91ER61125
• OSTI ID: 5054009
• Journal Information: Review of Scientific Instruments; (United States), Journal Name: Review of Scientific Instruments; (United States) Vol. 65:4; ISSN 0034-6748; ISSN RSINAK
• Country of Publication: United States
• Language: English

Related Subjects

550200* -- Biochemistry
59 BASIC BIOLOGICAL SCIENCES
AROMATICS
CARBOXYLIC ACIDS
COLLOIDS
DISPERSIONS
DNA
DNA SEQUENCING
DYES
ELECTRONIC EQUIPMENT
EMISSION SPECTROSCOPY
EQUIPMENT
FLUORESCEIN
FLUORESCENCE SPECTROSCOPY
GELS
HYDROXY ACIDS
HYDROXY COMPOUNDS
NUCLEIC ACIDS
OPTICAL EQUIPMENT
OPTICAL SCANNERS
ORGANIC ACIDS
ORGANIC COMPOUNDS
PHENOLS
POLYPHENOLS
SENSITIVITY
SPECTROSCOPY
STRUCTURAL CHEMICAL ANALYSIS