Confocal Live Cell Imaging Instrument

本申请的目的是获得一个共聚焦显微镜系统，该系统将专门设计用于在受控培养条件下对活细胞和胚胎进行成像。荧光蛋白变体的重要性日益增加，它可以作为蛋白质表达和亚细胞定位的体内报告，加速了对仪器的需求，这将使KUMC的研究人员能够在活细胞中更多地利用这项技术。需要 对卵和胚胎中的细胞内钙流进行成像也提高了对专用于活细胞成像的系统的需求。本建议书中要求的设备已被选定，以满足KUMC十二个主要用户的需求，并将作为生殖科学成像核心中心的一个组成部分进行操作。该请求是尼康多平台四线共聚焦显微镜，包括三个光电倍增管检测器，钙 比率成像系统，图像采集和分析软件，封闭的温度/湿度控制系统，以及用于卵和单细胞显微注射的双显微操作器。该系统的预期用途主要是对活脊椎动物细胞或胚胎进行长达24小时的延时观察，并将其放置在专用于此目的的单独房间中。活细胞成像系统将提供给所有研究人员， 堪萨斯大学系统和周围机构的成员。然而，使用该设施的大多数研究项目将来自12个NIH资助的实验室，这些实验室被确定为主要用户。William Kinsey博士将使用该仪器对注射荧光钙报告分子的斑马鱼和小鼠卵进行成像。De莱尔博士将使用GFP构建体研究分泌囊泡动力学。戈德温博士需要 在活的斑马鱼胚胎中成像GFP和RFP构建体。Hunt和Petroff博士将使用比率钙成像能力以及GFP构建体的成像。Little博士和Rongish博士将对鸟类胚胎和哺乳动物胚胎外植体中的血管内皮细胞或心脏细胞进行延时分析。Sarras博士研究细胞外基质成分在斑马鱼发育中的作用，并将利用该设施对GFP进行延时成像 在活的胚胎中构建。Soares博士将使用该设备进行荧光酶抑制剂的显微注射和滋养层细胞的钙成像。Stephens博士将在HIV-1发病模型中将GFP构建体成像为与Vpu基因中的高尔基体加工相关。Tash博士将使用该设备注射和定量精子发生的荧光抑制剂。Terranova博士将使用该设备将GFP构建体注入哺乳动物卵母细胞并成像，以研究Src酪氨酸激酶在早期发育中的作用。他还将研究在体外用肿瘤坏死因子α诱导分裂的颗粒细胞中的钙瞬变。

The purpose of this application is to acquire a confocal microscope system that will be specifically designed for imaging of live cells and embryos under controlled culture conditions. The growing importance of fluorescent protein variants which can act as in vivo reporters for protein expression and subcellular location has accelerated the demand for an instrument that will allow investigators at KUMC to make more use of this technology in live cells. The need to image intracellular calcium fluxes in eggs and embryos have also raised the demand for a system dedicated to live cell imaging. The equipment requested in this proposal has been selected to meet the needs of a group of twelve major users at KUMC and will be operated as a component of the Center for Reproductive Sciences Imaging Core. The request is for a Nikon multiplatform four line confocal microscope that includes a three PMT detector, calcium ratiometric imaging system, image acquisition and analysis software, an enclosed temperature/humidity control system, and dual micromanipulators for microinjection of eggs and single cells. The intended use of this system will be primarily for time lapse observation of live vertebrate cells or embryos for periods of up to 24 hrs, and it will be housed in a separate room dedicated to this purpose. The live cell imaging system will be available to all investigators within the University of Kansas System and to members of surrounding institutions. However, the majority of the research projects using this facility will come from twelve NIH-funded laboratories identified as major users. Dr. William Kinsey will use the instrument for imaging zebrafish and mouse eggs injected with fluorescent calcium reporter molecules. Dr. De Lisle will study secretory vesicle dynamics using GFP constructs. Dr. Godwin will require the ability to image GFP and RFP constructs in living zebrafish embryos. Drs. Hunt and Petroff will use the ratiometric calcium imaging capabilities as well as imaging of GFP constructs. Dr. Little and Dr. Rongish will be doing time-lapse analysis of vascular endothelial or cardiac cells in avian embryos and mammalian embryo explants. Dr. Sarras studies the role of extracellular matrix components in zebrafish development, and will use this facility for time lapse imaging of GFP constructs in live embryos. Dr. Soares will use the facility for microinjection of fluorescent enzyme inhibitors and for calcium imaging in trophoblast cells. Dr. Stephens will image GFP constructs as related to Golgi processing in the Vpu gene in HIV-1 pathogenesis models. Dr. Tash will use the equipment to inject and quantitate fluorescent inhibitors of spermatogenesis. Dr. Terranova will use the equipment to inject and image GFP constructs into mammalian oocytes in the study of the role of Src tyrosine kinase in early development. He will also study calcium transients in granulosa cells induced to divide in vitro with tumor necrosis factor alpha.