HETEROGENEOUS PHOTOBLEACHING IN CONFOCAL MICROSCOPY CAUSED BY DIFFERENCES IN REFRACTIVE-INDEX AND EXCITATION MODE

The photobleaching of fluorescence emission during confocal laser scan ning was studied on well-defined, stained objects [microspheres of pol ystyrene or fluorescent gels of fluorescein isothiocyanate (FITC)-labe led dextran] and on biological samples. X,Y laser scanning with confoc al microscopy induces fundamental differences in exposure rate and tim e in different z-planes orthogonal to the optical axis. A heterogeneou s bleaching rate was observed at different focal levels in the polysty rene spheres and in the gels. This phenomenon can be caused by refract ive index differences or is correlated with a photobleaching rate, whi ch is dependent not only on the excitation light intensity but also on the photon flux (total intensity per unit of time). Heterogeneous exc itation induced by refractive index differences results in photobleach ing differences but mill not necessarily cause heterogeneous emission intensity. Altered emission originating from altered excitation will b e annihilated if the emitted Light returns to the image plane along th e same inverse path, compensating for the proportional increase or dec rease in excitation intensity with an increased or decreased emission intensity. High numerical aperture or increased scanning speed increas es the photobleaching rate. This leads to the conclusion that photoble aching in confocal scanning laser microscopy is dependent on photon en ergy flux density (joule/m(2)s). (C) 1998 Wiley-Liss, Inc.