Diffusion measurement of fluorescence-labeled amphiphilic molecules with a standard fluorescence microscope

The lateral diffusion of fluorescence-labeled amphiphilic tracer molecules dissolved within a two-dimensional matrix of lipids was measured by continuous illumination of an elongated rectangular region, The resulting spatial concentration profile of unbleached tracer molecules was observed with a standard epifluorescence microscope and analyzed with digital image-processing techniques. These concentration profiles are governed by the mobility of the tracers, their rate of photolysis, and the geometry of the illuminated area, For the case of a long and narrow illuminated stripe, a mathematical analysis of the process is given, After prolonged exposure, the concentration profile can be approximated by a simple analytical function, This fact was used to measure the quotient of the rate of photolysis and the diffusion constant of the fluorescent probe. With an additional measurement of the rate of photolysis, the mobility of the tracer was determined. As prototype experiments we studied the temperature dependence of the lateral diffusion of N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-dipalmitoylphosphatidyl ethanolamine in glass-supported bilayers of L-alpha-dimyristoylphosphatidylcholine. Because of its simple experimental setup, this technique represents a very useful method of determining the lateral diffusion of fluorescence-labeled membrane molecules.