SCANNING MICROPHOTOLYSIS - A NEW PHOTOBLEACHING TECHNIQUE BASED ON FAST INTENSITY MODULATION OF A SCANNED LASER-BEAM AND CONFOCAL IMAGING

The fluorescence photobleaching method has been widely used to study m olecular transport in single living cells and other microsystems while confocal microscopy has opened new avenues to high-resolution, three- dimensional imaging. A new technique, scanning microphotolysis (Scamp) , combines the potential of photobleaching, beam scanning and confocal imaging. A confocal scanning laser microscope was equipped with a suf ficiently powerful laser and a novel device, the 'Scamper'. This consi sted essentially of a filter changer, an acousto-optical modulator (AO M) and a computer. The computer was programmed to activate the AOM dur ing scanning according to a freely defined image mask. As a result alm ost any desired pattern could be bleached ('written') into fluorescent samples at high definition and then imaged ('read') at non-bleaching conditions, employing full confocal resolution. Furthermore, molecular transport could be followed by imaging the dissipation of bleach patt erns. Experiments with living cells concerning dynamic processes in cy toskeletal filaments and the lateral mobility of membrane Lipids sugge st a wide range of potential biological applications. Thus, Scamp offe rs new possibilities for the optical manipulation and analysis of both technical and biological microsystems.