Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, TN 37232, USA.
Traffic. 2012 Dec;13(12):1589-600. doi: 10.1111/tra.12008. Epub 2012 Oct 10.
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.
扩散的定量测量可以提供有关蛋白质和脂质如何与其在细胞内的环境相互作用以及扩散物质的有效大小的重要信息。共聚焦荧光漂白后恢复（FRAP）是测量活细胞中蛋白质和脂质扩散的最广泛应用的方法之一。然而，目前缺乏直接用于根据绝对扩散系数来量化共聚焦 FRAP 测量的方法。在这里，我们报告了一个简化的方程，可以使用恢复的半衰期和圆形漂白区域的有效漂白半径来从共聚焦 FRAP 数据中提取扩散系数，并针对一系列荧光标记的可溶性和膜结合蛋白和脂质验证该方程。我们表明，使用这种方法，在标准成像条件下进行的共聚焦 FRAP 测量可以获得三个数量级范围内的扩散系数，突出了其广泛的适用性。
