Shared TIRF and Patterned Illumination Fluorescence Microscope

描述(由申请人提供)：本申请申请资金购买一台多功能荧光显微镜，由斯坦福大学医学院生物化学系的三名NIH R01支持的首席研究人员和一名NIH K99支持的研究人员共享。Jim Spudich、Suzanne Pfeffer、Aaron Straight和Rajat Rohatgi的实验室在基于显微镜的细胞系统动态分析以及通过生化分析对这些系统进行机械剖析方面有着共同的兴趣。我们实验室研究的中心是使用荧光显微镜研究细胞蛋白质和细胞器动力学。为了分析复杂的细胞生物学过程，我们的实验室将细胞的光学、化学和遗传扰动与高分辨率免疫荧光成像和活细胞时间推移成像相结合。为了了解单个细胞成分如何参与细胞功能，我们将基于显微镜的分析应用于重组的生化系统和基于细胞提取的反应，这些反应概括了细胞的过程。具体地说，我们使用了一系列方法，从光学捕获和TIRF显微镜对细胞骨架聚合物上马达蛋白运动的单分子分析，到细胞提取物中染色体分离和囊泡运输的荧光成像。我们正在申请资金，以购买能够活细胞时移荧光和亮场显微镜、TIRF显微镜、图案化荧光光漂白和光激活以及迭代去卷积的共享显微镜。这些组合模式将通过提供共享资源来满足我们的显微镜需求，从而显著增强NIH支持的我们实验室的研究。

DESCRIPTION (provided by applicant): This application requests funds to purchase a multi-use fluorescence microscope to be shared by three NIH R01 supported Principal Investigators and one NIH K99 supported investigator in the Department of Biochemistry at Stanford University School of Medicine. The laboratories of Jim Spudich, Suzanne Pfeffer, Aaron Straight and Rajat Rohatgi share a common interest in microscopy based dynamic analysis of cellular systems and mechanistic dissection of those systems through biochemical analysis. Central to the research in our laboratories is the use of fluorescence microscopy to study cellular protein and organelle dynamics. In order to analyze complex cell biological processes, our laboratories combine optical, chemical, and genetic perturbation of cells with high-resolution immunofluorescence imaging and live cell timelapse imaging. To understand how individual cellular components participate in cellular functions we apply microscopy-based analysis to reconstituted biochemical systems and cell extract based reactions that recapitulate cellular processes. Specifically, we use methods ranging from single molecule analysis of motor protein movement on cytoskeletal polymers by optical trapping and TIRF microscopy to epifluorescence imaging of chromosome segregation and vesicle trafficking in cell extracts. We are requesting funds to purchase a shared microscope capable of live cell time-lapse fluorescence and brightfield microscopy, TIRF microscopy, patterned fluorescence photobleaching and photoactivation and iterative deconvolution. These combined modalities will significantly enhance the NIH supported research in our laboratories by providing a shared resource to address our microscopy needs.