When in DIC mode, the WU Filter Cube radio button displays a fluorescence image of rat retina optic ganglion cells stained with the chromophore Fast Blue. The Choose A Sample pull-down menu allows the visitor to select between rat retina tissue and cat brain tissue infected with cryptococcus. When the cat brain tissue is selected, and the WIB Filter Cube radio button is checked, a photomicrograph of the tissue imaged with fluorescence illumination through an Olympus WIB filter cube is displayed. Note the pseudo three-dimensional appearance of the photomicrograph. The rate of photobleaching is dependent upon several factors, including the chemical reactivity of the chromophore, the intracellular chemical environment, and the intensity and wavelength of the excitation light.
Some fluorescent dyes are readily susceptible to photobleaching while other are relatively stable.
In many instances, the specimen may recover from the effects of photobleaching, particularly if it is kept cool and in a dark environment. Sometimes quenching results from the transfer of energy to other acceptor molecules residing physically close to the excited fluorochromes, a phenomenon known as resonance energy transfer. This particular phenomenon has become the basis for a newer technique of measuring distances far below the lateral resolution of the light microscope. Impurities in the chromophore may also reduce fluorescence intensity either by photobleaching or quenching. Neutral density filters can be placed in the light path before the light reaches the excitation filter, thus diminishing the excitation light intensity.