Tao Antai, Zhang Rongjing, Yuan Junhua
Hefei National Laboratory for Physical Sciences at the Microscale and Department of Physics, University of Science and Technology of China, Hefei 230026, Anhui, China.
J Phys Chem B. 2020 Mar 12;124(10):1892-1897. doi: 10.1021/acs.jpcb.9b11028. Epub 2020 Feb 27.
Photoactivatable fluorescent proteins (PA-FPs) are widely used in photoactivated localization microscopy (PALM), and information about their blinking properties is important for precise assembling of PALM images and for quantitative molecular counting with PALM. As the photophysical properties of fluorescent proteins depend sensitively on the local physiochemical environment, we developed a series of procedures to characterize the properties of the PA-FPs in fixed cells. As an example, we characterized the properties of Dendra2 in and found that they are appreciably different from those in vitro. This highlights the importance of characterizing the photophysical properties of PA-FPs under the same experimental conditions as subsequent PALM imaging or molecular counting experiments. Other PA-FPs could be characterized using this strategy.
光激活荧光蛋白（PA-FPs）广泛应用于光激活定位显微镜（PALM），其闪烁特性信息对于精确组装PALM图像以及用PALM进行定量分子计数非常重要。由于荧光蛋白的光物理性质敏感地依赖于局部物理化学环境，我们开发了一系列程序来表征固定细胞中PA-FPs的性质。例如，我们表征了Dendra2在[具体条件未给出]中的性质，发现它们与体外性质明显不同。这突出了在与后续PALM成像或分子计数实验相同的实验条件下表征PA-FPs光物理性质的重要性。其他PA-FPs也可以使用这种策略进行表征。
