Heterogeneous photobleaching in confocal microscopy caused by differences in refractive index and excitation mode

The photobleaching of fluorescence emission during confocal laser scanning was studied on well-defined, stained objects [microspheres of polystyrene or fluorescent gels of fluorescein isothiocyanate (FITC)-labeled dextran] and on biological samples. X,Y laser scanning with confocal microscopy induces fundamental differences in exposure rate and time in different z-planes orthogonal to the optical axis. A heterogeneous bleaching rate was observed at different focal levels in the polystyrene spheres and in the gels. This phenomenon can be caused by refractive index differences or is correlated with a photobleaching rate, which is dependent not only on the excitation light intensity but also on the photon flux (total intensity per unit of time). Heterogeneous excitation induced by refractive index differences results in photobleaching differences but will not necessarily cause heterogeneous emission intensity. Altered emission originating from altered excitation will be annihilated if the emitted light returns to the image plane along the same inverse path, compensating for the proportional increase or decrease in excitation intensity with an increased or decreased emission intensity. High numerical aperture or increased scanning speed increases the photobleaching rate. This leads to the conclusion that photobleaching in confocal scanning laser microscopy is dependent on photon energy flux density (joule/m2s).