Kitamura Akira, Kinjo Masataka
Laboratory of Molecular Cell Dynamics, Faculty of Advanced Life Science, Hokkaido University, Sapporo, Hokkaido 001-0021, Japan.
Biophys Physicobiol. 2018 Jan 19;15:1-7. doi: 10.2142/biophysico.15.0_1. eCollection 2018.
Fluorescence recovery after photobleaching (FRAP) enables characterization of quantitative dynamic properties such as diffusion coefficients of fluorescent molecules in living cells by analyzing the recovery of fluorescence intensity after photobleaching in a specific cellular compartment or area. To quantitatively determine high intracellular diffusion coefficients, a suitable optical system as well as an appropriate model for fast diffusion analysis is necessary. Here, we propose a procedure to quantify the diffusion coefficient of rapidly-diffusing fluorescent molecules that makes use of an epi-fluorescence microscope with a photobleaching laser in combination with established models for diffusion analysis. Analysis for the diffusion coefficients of tandemly oligomerized green flurescent proteins (GFPs) in living cells when changing the photobleaching times showed that photobleaching with shorter times than the diffusion speed indicated not the only way to obtain appropriate diffusion coefficients of fast-moving molecules. Our results also showed that the apparent spreading of the effective radius of the photobleached area works as a correction factor for determining appropriate diffusion coefficients of fast-moving molecules like monomeric GFPs. Our procedure provides a useful approach for quantitative measurement of diffusion coefficients in living cells. This procedure is relevant for characterizing dynamic molecular interactions, especially of fast-moving molecules, and is relevant for studies in many biological fields.
光漂白后荧光恢复（FRAP）通过分析特定细胞区室或区域光漂白后荧光强度的恢复情况，能够对诸如活细胞中荧光分子扩散系数等定量动态特性进行表征。为了定量测定高细胞内扩散系数，需要一个合适的光学系统以及一个用于快速扩散分析的适当模型。在此，我们提出一种程序来量化快速扩散荧光分子的扩散系数，该程序利用配备光漂白激光的落射荧光显微镜并结合已建立的扩散分析模型。当改变光漂白时间时，对活细胞中串联寡聚化绿色荧光蛋白（GFP）的扩散系数进行分析表明，光漂白时间短于扩散速度并非获得快速移动分子合适扩散系数的唯一方法。我们的结果还表明，光漂白区域有效半径的明显扩展可作为确定像单体GFP这样快速移动分子合适扩散系数的校正因子。我们的程序为活细胞中扩散系数的定量测量提供了一种有用的方法。该程序对于表征动态分子相互作用，特别是快速移动分子的相互作用具有重要意义，并且与许多生物学领域的研究相关。
