Simplified Equation to Extract Diffusion Coefficients from Confocal FRAP Data |
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Authors: | Minchul Kang Charles A. Day Anne K. Kenworthy Emmanuele DiBenedetto |
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Affiliation: | 1. Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, , Nashville, TN, 37232 USA;2. Current address: School of Science, Technology, and Engineering Management, St. Thomas University, , 16401 NW 37th Avenue,, Miami Gardens, FL, 33054 USA;3. Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, , Nashville, TN, 37232 USA;4. Chemical and Physical Biology Program, Vanderbilt University School of Medicine, , Nashville, TN, 37232 USA;5. Department of Mathematics, Vanderbilt University, , Nashville, TN, 37240 USA |
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Abstract: | Quantitative measurements of diffusion can provide important information about how proteins and lipids interact with their environment within the cell and the effective size of the diffusing species. Confocal fluorescence recovery after photobleaching (FRAP) is one of the most widely accessible approaches to measure protein and lipid diffusion in living cells. However, straightforward approaches to quantify confocal FRAP measurements in terms of absolute diffusion coefficients are currently lacking. Here, we report a simplified equation that can be used to extract diffusion coefficients from confocal FRAP data using the half time of recovery and effective bleach radius for a circular bleach region, and validate this equation for a series of fluorescently labeled soluble and membrane‐bound proteins and lipids. We show that using this approach, diffusion coefficients ranging over three orders of magnitude can be obtained from confocal FRAP measurements performed under standard imaging conditions, highlighting its broad applicability. |
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Keywords: | confocal laser scanning microscope diffusion coefficient fluorescence microscopy FRAP half time of recovery |
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