Fluorescence photobleaching recovery in the confocal scanning light microscope

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Abstract
SUMMARY: Measurement of mobilities of species in liquid systems is of great importance for understanding a number of dynamic phenomena. A well known method for measuring mobilities driven by diffusion is fluorescence photobleaching recovery (FPR), also known as fluorescence recovery after photobleaching (FRAP). New FPR recovery equations for three‐dimensional (3‐D) apertured scanning using a Gaussian approximation for the axial beam profile have been successfully developed and found to provide a solid basis for extraction of the lateral diffusion coefficient from confocal scanning light microscopy (CSLM)‐FPR experimental data. The 2‐D diffusion coefficients of fluorescent species can be successfully measured by FPR in the CSLM, which has the great advantage that bleaching can be targeted at a well‐defined volume element in bulk samples. Two‐dimensional diffusion coefficients of 45‐nm latex spheres, of FITC molecules and of a 2·45‐nm protein‐FITC complex in water‐glycerol mixtures, measured by FPR in the CSLM, are in close agreement with those calculated from the size of the diffusing species and viscosity of the medium. Diffusion coefficients as high as 2 times 10−6 cm2/s can be measured.