Zeiss Laser TIRF 3 microscope and live cell imaging system

描述(申请人提供)：这是一个蔡司激光TIRF 3显微镜和活细胞成像系统的申请，将允许奥尔巴尼医学院的5名研究人员和纽约州立大学公共卫生学院的1名研究人员选择性地成像发生在细胞质膜内或非常接近细胞膜的分子事件，因为细胞位于玻璃盖玻片表面附近。在TIRF显微镜中，一个非常薄的、深约100 nm的亚分辨率光学体积被逝去波照射，只有位于该空间内的荧光分子被激发，而位于该空间之外的荧光团对背景或荧光信号没有贡献，从而产生高对比度、高分辨率的荧光图像。该用户组的成员将在几个不同的研究领域应用这一能力：了解整合素如何在间期和有丝分裂期间调节中心体功能和微管组装；在平滑肌细胞迁移和肿瘤细胞侵袭期间，通过跟踪活细胞中荧光蛋白成分的动态相互作用，调节细胞和细胞外基质中分子之间焦点接触的组装和拆解；使用基于FRET的生物传感器Raichu，评估RAC、CDC42和RhoA在迁移细胞中的分布；将吞噬杯组装为囊泡，将新膜带到组装位置、接近并融合到吞噬体内；用基于FRET的生物传感器检测巨噬细胞吞噬过程中信号分子和肌动蛋白细胞骨架的相互作用；适配蛋白与人FSHR的相互作用，导致刺激几个信号级联反应，并在其配体FSH结合后通过内吞作用使受体脱敏；使用基于FRET的生物传感器Raichu，评估RAC、CDC42和RhoA在迁移细胞中的分布；TIRF-FRET方法的优化。购买该仪器将极大地加强正在进行的用户组成员的研究工作。目前，他们无法接触到TIRF显微镜。TIRF显微镜的出现将成为机构研究界的中心资源，并导致新项目的启动，因为使用该技术的用户群体的成功变得广为人知。

DESCRIPTION (provided by applicant): This is a request for a Zeiss Laser TIRF 3 microscope and live cell imaging system that will permit a group of 5 researchers at the Albany Medical College and 1 at the SUNY School of Public Health to selectively image molecular events that occur within or very close to the plasma membrane of cells as they lie close to a glass coverslip surface. In TIRF microscopy, a very thin, sub-resolution optical volume about 100 nm in depth is illuminated by the evanescent wave and only fluorescent molecules located within this space are excited, while fluorophores located outside this space do not contribute to the background or the fluorescence signal, resulting in the production of high-contrast, high-resolution fluorescence images. The members of the user group will apply this capability in several diverse areas of study: understanding how integrins regulate centrosome function and microtubule assembly during interphase and mitosis; the regulation of the assembly and disassembly of focal contact between cells and molecules in the extracellular substrate by following the dynamic interaction of fluorescent-protein components in live cells during smooth muscle cell migration and tumor cell invasion; use of the FRET based biosensor, Raichu, to assess the distribution of Rac, CDC42 and RhoA in migrating cells; the assembly of the phagocytic cup as vesicles bringing new membrane to the assembly site, approach and fuse into phagosomes; the interaction of signaling molecules and the actin cytoskeleton during phagocytosis by macrophages as detected with FRET-based biosensors; the interaction of adapter proteins with the human FSHR that leads to stimulation of several signaling cascades and receptor desensitization via endocytosis following binding of its ligand FSH use of the FRET based biosensor, Raichu, to assess the distribution of Rac, CDC42 and RhoA in migrating cells; the optimization of TIRF-FRET methodology. Acquisition of this instrument will dramatically enhance the research endeavors of members of the user group that are in progress. Currently they have no access to a TIRF microscope. The presence of the TIRF microscope will become a central resource in the institutional research community and lead to initiation of new projects as success of the user group using this technology becomes widely known.