Fluorescence loss in photobleaching

Two related fluorescence microscopy techniques serve the purpose to examine active movement or diffusion of intracellular or membrane bound molecules: Fluorescence-Loss-In-Photobleaching (FLIP) and Fluorescence-Recovery-After-Photobleaching (FRAP). Typically a specific area of a cell membrane previously labelled with a floating fluorescent dye is bleached with a confocal laser scanning microscope and the loss or recovery of fluorescence over time is recorded to determine the lateral membrane fluidity. In addition FLIP is useful in verifying the continuity of membranous organelles. By continuous bleaching of a small circumscribed organelle area the fluorescence from the non-illuminated part slowly vanishes, if unbleached fluorophores can laterally diffuse into the illuminated spot until all fluorescent molecules are depleted.