A FRAP on TIRF superresolution microscopy system for bioimaging

荧光显微镜的巨大力量最近被授予2014年诺贝尔化学奖，表彰了三位策划了超分辨率成像的科学家。尽管被誉为活细胞成像的一次巨大飞跃，但世界上几乎没有实验室能够真正在活细胞中使用超分辨率。现在有其他的荧光成像模式可以提高分辨率，还可以实时获得细胞过程动力学的定量数据。本项目提出在一个显微镜上结合全内反射荧光(TIRF)和光漂白后荧光重分布(FRAP)两种成像模式。具体地说，现有的多激光TIRF显微镜将改装必要的硬件，以便能够进行FRAP并生成关于细胞表面蛋白质动态周转的数据。这一新工具对于量化合成世界上最丰富的生物聚合物纤维素的酶复合体以及位于质膜的膜受体、转运体和其他蛋白质复合体的周转率是必不可少的。

The tremendous power of fluorescence microscopy was recently recognized with the 2014 Nobel Prize in Chemistry to three scientists who have masterminded super resolution imaging. Although hailed as a quantum leap in live cell imaging, there are few labs worldwide who can actually use super resolution in living cells. Other modes of fluorescence imaging are now available that can improve resolution and also obtain quantitative data on the dynamics of cellular processes in real time. This project proposes to combine two such imaging modes, total internal reflectance fluorescence (TIRF) and fluorescence redistribution after photobleaching (FRAP) on one microscope. Specifically, an existing multi-laser TIRF microscope will be retrofitted with the necessary hardware to enable FRAP to be conducted and to generate data on the dynamic turnover of proteins at the cell surface. This new tool will be indispensable for quantifying the turnover of enzyme complexes that synthesize the worlds most abundant biopolymer cellulose, as well as membrane receptors, transporters and other proteins complexes situated at the plasma membrane.