Construction and performance of a photobleaching recovery apparatus with microsecond time resolution.
A fluorescence recovery after photobleaching (FRAP) apparatus was constructed to measure sub-millisecond fluorescence recovery processes in living cells. The photobleaching pulse and probe beams were generated by modulating the intensity of a continuous wave Argon laser (4 W) by two acousto-optic modulators in series. The maximum intensity modulation was > 10(6):1 with a rise time of < 1 microsecond and a minimum pulse width of 6 microseconds. Fluorescence was detected by a photomultiplier, amplified by a transimpedance amplifier, and digitized at 1 MHz. During the photobleaching pulse, the photomultiplier gain was reduced by ca. 1500-fold by switching the second dynode voltage ca. 100 V negative with respect to the cathode voltage by computer control of two bidirectional Mosfet optoisolators. The switching circuit produced a optoisolators. The switching circuit produced a transient anode current (t approximately 15 microseconds) which was subtracted for measurement of recoveries of < 50-100 microseconds. The apparatus was coupled to an inverted microscope for measurement of fluorescence by epi-illumination or total internal reflection. Instrument performance was evaluated by measurement of the rapid fluorescence recoveries of fluorescein and fluorescein-dextrans in solutions and living cells.
Kao
H P
HP
Department of Medicine, University of California, San Francisco 94143-0521, USA.
Verkman
A S
AS
D000818:Animals; D002478:Cells, Cultured; D005347:Fibroblasts; D005452:Fluoresceins / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D006651:Histocytochemistry / Q000295:instrumentation*; D051379:Mice; D008856:Microscopy, Fluorescence; D012984:Software; D012996:Solutions; D013050:Spectrometry, Fluorescence / Q000295:instrumentation*
D005452:Fluoresceins; D005456:Fluorescent Dyes; D012996:Solutions