An important class of photobleaching events are photodynamic, meaning they involve the interaction of the fluorophore with a combination of light and oxygen. Reactions between fluorophores and molecular oxygen permanently destroy fluorescence and yield a free radical singlet oxygen species that can chemically modify other molecules in living cells. The amount of photobleaching due to photodynamic events is a function of the molecular oxygen concentration and the proximal distance between the fluorophore, oxygen molecules, and other cellular components. Photobleaching can be reduced by limiting the exposure time of fluorophores to illumination or by lowering the excitation energy. However, these techniques also reduce the measurable fluorescence signal. In many cases, solutions of fluorophores or cell suspensions can be deoxygenated, but this is not feasible for living cells and tissues. Perhaps the best protection against photobleaching is to limit exposure of the fluorochrome to intense illumination (using neutral density filters) coupled with the judicious use of commercially available antifade reagents that can be added to the mounting solution or cell culture medium.

The culture of bovine pulmonary artery endothelial cells appearing in the digital image above was stained with MitoTracker Red CMXRos, vividly labeling the intracellular mitochondrial network. The specimen was also labeled for filamentous actin and DNA with Alexa Fluor 488 (green emission) conjugated to phalloidin and DAPI (blue emission), respectively. Images were recorded in grayscale with a QImaging Retiga Fast-EXi camera system coupled to an Olympus BX-51 microscope equipped with bandpass emission fluorescence filter optical blocks provided by Omega Optical. During the processing stage, individual image channels were pseudocolored with RGB values corresponding to each of the fluorophore emission spectral profiles.