Multi-Excitation TIRF Microscope for Shared Resource

 描述(由申请人提供)：我们申请资金购买共享的多激发TIRF显微镜，该显微镜基于倒置的尼康日食Ti-E，配置用于解析活细胞表面附近动态大分子相互作用的细节。该仪器将用于可视化和测量囊泡运输过程、蛋白质-蛋白质相互作用和质膜跨细胞作用。这些数据对心脏胚胎发生、癌细胞侵袭、胰腺β细胞信号和分泌、内皮细胞空泡运输缺陷、神经元形态发生和重塑以及分子神经生理学等多方面的研究至关重要。活体细胞成像实验的必备组件包括一个60X 1.49NA的TIRF镜头，稳定的TIRF照明器，带有4通道多激光模块，可同时对准多个临界角，用于同时进行光激活和成像的双端口，高速精密自动对焦系统，非常快的高分辨率相机和显微镜培养系统。由来自范德比尔特大学医学中心(VUMC)四个部门的研究人员组成的八个主要用户小组使用成像技术研究糖尿病、癌症、心脏病和神经元发育的广泛问题。他们都有相当多的基于成像的方法来量化水泡和非水泡过程的专业知识，并对多激发TIRF成像有关键的实验需求。 该仪器根据主要用户群体的需求进行了严格的配置，在保持活细胞生理条件的同时，重点考虑了最佳的TIRF物镜、关键对准、快速调制的激发源、焦点补偿和在衍射极限下的高速图像采集。这些规范是成功实施主要用户项目中描述的多激励TIRF成像的基础。该仪器将设在光厅(LH742)，并由细胞成像共享资源(CISR)维护。这间屋子是一间为所有范德比尔特调查人员而设的显微镜设施，由该机构独家维护。这个空间是安全的，所有主要用户都可以轻松访问。 对这一文书的专门知识和机构支持是特殊的。Piston博士和Wells博士分别是科学顾问和科学研究中心主任，他们是世界著名的显微镜专家，拥有共焦、多光子激发荧光、FRAP、FRET、TIRF等先进成像方法。该研究所已有20年的历史，为300多个研究实验室提供尖端成像专业知识和合作研究支持。VUMC已承诺提供50,000美元，以支持该仪器的长期运行(见负责研究的副校长Susan Wente的信)。

 DESCRIPTION (provided by applicant): We request funds for the purchase of a shared Multi-Excitation TIRF Microscope based on an inverted Nikon Eclipse Ti-E, configured to resolve details of dynamic macromolecular interactions near living cell surfaces. The instrument will be used to visualize and measure vesicular trafficking processes, protein- protein interactions, and plasma membrane transcytosis. These data are critical to multiple investigations related to, cardiac embryogenesis, cancer cell invasion, pancreatic β-cell signaling and secretion, caveolae trafficking defects in endothelial cells, neuronal morphogenesis and remodeling, and molecular neurophysiology. Requisite components for live cell imaging experiments include, a 60X 1.49NA TIRF lens, stable TIRF illuminator with 4-channel multi-laser module aligned for multiple critical angles with dual-ports for simultaneous photo-activation and imaging, a high-speed precision autofocus system, very fast, high-resolution camera, and microscope incubation system. Eight Major User groups made up of investigators from four departments within the Vanderbilt University Medical Center (VUMC) use imaging techniques to study a broad range of problems in diabetes, cancer, heart disease, and neuronal development. All have considerable expertise with imaging-based approaches to quantify vesicular and non-vesicular processes and have critical experimental needs for multi-excitation TIRF imaging. This instrument has been rigorously configured based on the needs of the major user group with significant consideration given to optimal TIRF objective lens, critically aligned, rapidly modulated excitation sources, focus compensation, and high-speed image acquisition at the diffraction-limit, while maintaining live cell physiological conditions. The specifications are fundamental to the successful implementation of the multi-excitation TIRF imaging described in the Major User projects. The instrument will be located in Light Hall (LH 742) and maintained by the Cell Imaging Shared Resource (CISR). This room is a CISR microscopy facility, maintained exclusively by the CISR, for the benefit of all Vanderbilt investigators. This space is secure and easily accessible to all major users. The expertise and institutional support for this instrument are exceptional. Drs. Piston and Wells, Scientific Advisor and Director of the CISR, respectively, are world-renowned microscopy experts with confocal, multi-photon-excited fluorescence, FRAP, FRET, TIRF and other advanced imaging methods. The CISR has a 20-year history of assisting over 300 investigator labs with cutting-edge imaging expertise and cooperative research support. The VUMC has committed $50,000 to support the long-term operation of the instrument (see letter from Susan Wente, Associate Vice Chancellor for Research).