Construction and performance of a photobleaching recovery apparatus with microsecond time resolution

Abstract

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 μs and a minimum pulse width of 6 μs. 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 transient anode current ( t ≈ 15 μs) which was subtracted for measurement of recoveries of < 50–100 μs. 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.