A Confocal Imaging Facility for Upright and Inverted Microscopy

将为六名发育生物学家建立一个共聚焦显微镜成像设备，研究非洲爪蟾早期胚胎的细胞结构、事件和基因表达模式，发育中的雌雄爪蟾神经系统，以及哺乳动物内皮细胞培养准备。共聚焦系统将配置3个灵敏光电倍增管和2个产生4条可见光线的激光器，以及一个配备长工作距离水浸物镜的复合立式显微镜，以方便涉及显微注射或显微操作的体内实验。多年来，PI （Danilchik）一直使用共聚焦系统和倒置显微镜来研究早期非洲爪蟾胚胎的发育，特别是在受精卵的植物（下侧）表面，在那里，局部分子通过与复杂和动态的细胞骨架的相互作用而重新定位。最近的研究转向了细胞分裂过程中卵子动物（上）表面的细胞骨架-膜相互作用。目前的研究计划是跟踪动物活体胚胎表面的粒子运动、膜融合事件和细胞骨架重组。由于两栖动物胚胎在上下颠倒时不能正常发育，因此直立共聚焦系统对这些研究至关重要。另外三个使用者（Copenhaver, Kent, Morton）研究了烟草角虫Manduca sexta的神经发育和神经元信号传导方面：Copenhaver研究了Manduca神经元迁移的指导，并开发了一种活的制备方法，其中神经元沿着肠道背表面明显迁移。Kent使用Manduca作为模型系统，研究了大型全载制剂中运动末梢和树突的发育变化，并在体外检测了已鉴定的运动神经元的突触特化和生长锥形态。Morton正在研究Manduca和秀丽隐杆线虫中神经肽对环GMP （cGMP）产生的调节，并将需要使用多激光线共聚焦显微镜来证明两种或多种抗原在全量制剂中的共定位。Christian将利用共聚焦系统研究非洲爪蟾早期胚胎发育过程中tgf β信号转导通路组分的亚细胞定位。Farrell研究细胞外基质成分在血管生成过程中的作用，并对开发共聚焦方法筛选转染整合素受体表达调节因子的细胞系感兴趣。获得具有多条激光线和增强光子探测的最先进的共聚焦系统将极大地加强和扩展我们的个人研究工作，并将使我们有能力开发关键的活细胞成像技术。该仪器将提供给研究生全年和本科生在夏季项目。

A confocal microscope imaging facility will be established for six developmental biologists investigating cellular structures, events, and gene expression patterns in early Xenopus laevis embryos, in developing Manduca sexta nervous systems, and in mammalian endothelial cell-culture preparations. The confocal system will be configured with 3 sensitive photomultipliers and two lasers producing four visible-light lines, and with a compound upright microscope equipped with long-working distance water-immersion objectives to facilitate in vivo experiments involving microinjection or micromanipulation. The PI (Danilchik) has used a confocal system with an inverted microscope for several years to study the development of early Xenopus embryos, particularly on the vegetal (underside) surface of the fertilized egg, where localized molecules are relocalized via interactions with a complex and dynamic cytoskeleton. Recent work has turned to cytoskeleton-membrane interactions on the animal (upper) surface of the egg during cytokinesis. Current research plans are to follow particle motions, membrane fusion events, and cytoskeletal reorganization in the animal surface of living embryos. Because amphibian embryos cannot develop normally when turned upside down, an upright confocal system will be vital for these studies. Three other users (Copenhaver, Kent, Morton) study aspects of neural development and neuronal signalling in the tobacco hornworm Manduca sexta: Copenhaver studies the guidance of neuronal migration in Manduca, and has developed a live preparation in which neurons migrate in plain view along the dorsal surface of the gut. Kent uses Manduca as a model system to study developmental changes in motor terminals and dendrites in large whole-mount preparations and to examine synaptic specializations and growth cone morphology of identified motor neurons in vitro. Morton is examining the regulation of cyclic GMP (cGMP) production by neuropeptides in Manduca and C. elegans, and will require confocal microscopy with multiple laser lines to demonstrate colocalization of two or more antigens in wholemount preparations. Christian will use the confocal system to study the subcellular localization of components of the TGFBeta signal transduction pathway in developing Xenopus early embryos. Farrell studies the role of extracellular matrix components in the process of angiogenesis, and is interested in developing confocal methods to screen cell lines transfected with regulators of integrin receptor expression. Acquisition of a state-of-the-art confocal system with multiple laser lines and enhanced photon detection will greatly enhance and expand our individual research efforts, and will give us the ability to develop crucial live-cell imaging techniques. The instrumentation will be available for graduate students throughout the year and undergraduates in summer programs.