CONSTRUCTION AND PERFORMANCE OF A PHOTOBLEACHING RECOVERY APPARATUS WITH MICROSECOND TIME RESOLUTION

A fluorescence recovery after photobleaching (FRAP) apparatus was cons tructed 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 modulati on was > 10(6):1 with a rise time of < 1 mu s and a minimum pulse widt h of 6 mu s. Fluorescence was detected by a photomultiplier, amplified by a transimpedance amplifier, and digitized at 1 MHz. During the pho tobleaching pulse, the photomultiplier gain was reduced by ca. 1500-fo ld by switching the second dynode voltage ca. 100 V negative with resp ect to the cathode voltage by computer control of two bidirectional Mo sfet optoisolators. The switching circuit produced a transient anode c urrent (t approximate to 15 mu s) which was subtracted for measurement of recoveries of < 50-100 mu s. The apparatus was coupled to an inver ted microscope for measurement of fluorescence by epi-illumination or total internal reflection. Instrument performance was evaluated by mea surement of the rapid fluorescence recoveries of fluorescein and fluor escein-dextrans in solutions and living cells.