On the Equivalence of FCS and FRAP: Simultaneous Lipid Membrane Measurements
On the Equivalence of FCS and FRAP: Simultaneous Lipid Membrane Measurements
复制标题
DOI：
10.1016/j.bpj.2016.06.001
复制
发表时间：
2016-07-12
影响因子：
3.4
通讯作者：
Wohland, Thorsten
中科院分区：
文献类型：
--
作者：
Machan, Radek;Foo, Yong Hwee;Wohland, Thorsten
荧光相关光谱法（FCS）和光漂后荧光恢复法（FRAP）是目前广泛应用的扩散系数测定方法。然而，它们通常不会产生相同的结果。随着基于相机的成像FCS的出现，它可以测量图像中每个像素的扩散系数，以及适当的漂白校正，现在可以通过FRAP和FCS在完全相同的图像中测量扩散系数。因此，我们同时对支持的脂质双层和活细胞膜进行了FCS和FRAP测量，以测试两种方法在结果上的差异，以及方法上的差异，特别是FRAP中的高漂白剂强度、漂白剂校正和两种方法的拟合程序是否解释了观察到的差异。总的来说，我们发现FRAP漂白剂强度对样品中的扩散没有可测量的影响，但漂白剂校正和拟合在FRAP中引入了很大的不确定性。我们通过模拟验证了我们的结果。
Fluorescence correlation spectroscopy (FCS) and fluorescence recovery after photobleaching (FRAP) are widely used methods to determine diffusion coefficients. However, they often do not yield the same results. With the advent of camera-based imaging FCS, which measures the diffusion coefficient in each pixel of an image, and proper bleaching corrections, it is now possible to measure the diffusion coefficient by FRAP and FCS in the exact same images. We thus performed simultaneous FCS and FRAP measurements on supported lipid bilayers and live cell membranes to test how far the two methods differ in their results and whether the methodological differences, in particular the high bleach intensity in FRAP, the bleach corrections, and the fitting procedures in the two methods explain observed differences. Overall, we find that the FRAP bleach intensity does not measurably influence the diffusion in the samples, but that bleach correction and fitting introduce large uncertainties in FRAP. We confirm our results by simulations.