DETERMINATION OF LOCALIZED DIFFUSION-COEFFICIENTS IN GELS USING CONFOCAL SCANNING LASER MICROSCOPY

The development of a photobleaching technique, CFMM (continuous fluorescence multipoint microphotolysis), to measure diffusion coefficients in gel systems using a confocal scanning laser microscope is described. Diffusion coefficients (D) were determined for fluorescently labelled dextrans of varying molecular weight in agarose gels, and the results compared with two other methods. CFMM enabled diffusion coefficients to be rapidly determined from the profile across an irradiated area within a defined microscopic location of the gel, The technique was experimentally simple and produced values of D that corresponded well with classical double-diffusion cell methods.