"Development of a Fluorescence Recovery after Photobleaching Method to Measure Diffusion in Biological Samples"

Abstract:

Fluorescence recovery after photobleaching (FRAP) is a primary method of measuring diffusion coefficients within living cells. A FRAP method was developed and tested using an Olympus FV1000 confocal laser scanning microscope. AlexaFluor 488 fluorescent probes conjugated with 10 kDa dextran molecules were employed to measure diffusion coefficients in model media. An aqueous solution (KH2PO4), four concentrations of glycerol (10, 25, 50 and 100%), and agarose gels of three densities (1, 5 and 10%) were used. During each experiment, circular regions of interest (ROI) of 30 µm, 45 µm, 60 µm and 120 µm diameters were photobleached, and fluorescence recovery was then analyzed in the ROI to determine the diffusion coefficient. Equations in Yguerabide et al. (1982) were utilized to analyze the data. The diffusion coefficients in the aqueous and glycerol solutions were within the range observed in previous studies, and there was a strong inverse correlation between the viscosity and the diffusion coefficient in these solutions. These findings indicate that the method developed was effective and is suitable for use in living cells. In contrast, in the agarose gels, the diffusion coefficients were higher in the higher percent gels, which is contrary to the expected patterns. However, this discrepancy probably results from the manner in which the sample was prepared, and not due to bias inherent in the FRAP protocol.