University of Minnesota Multi-User Total Internal Reflection Fluorescence Microsc

描述（由申请人提供）：所有生物学功能均依赖于分子相互作用。因此，通过研究分子何时、何地以及如何相互作用，我们可以阐明生物学的基本原理。该基金的主要研究人员来自明尼苏达大学的三个不同部门，研究各种各样的主题，从磷脂受体激活的动力学到控制阿米巴细胞运动的机制。这些研究项目各自使用不同的模型系统，并需要自己的专用试剂。然而，这些项目是统一的事实，每一个都将受益于全内反射荧光显微镜（TIRFM）的使用。该提案的长期目标是通过提供最先进的共享TIRFM设备设施以及技术和科学支持，加强明尼苏达大学的基础和转化研究。 TIRFM是一种特殊类型的荧光显微镜，其中仅在玻璃盖玻片的约100 nm内的荧光分子被激发（即激活以发出荧光）。使用传统的落射荧光显微镜，荧光激发发生在样品的整个深度;这会产生焦点外的背景荧光，产生差的信噪比并模糊可视化。此外，样品暴露于大量的荧光，这可能导致光漂白（导致成像能力丧失），并且在活细胞成像的情况下，光毒性和细胞死亡。使用激光共聚焦显微镜，激发光束聚焦在相当窄的区域（直径：250 - 800 nm，深度：500-1500 nm），提高信噪比并降低光毒性;然而，使用连续成像，光漂白和光毒性仍然是一个问题。 该提案中描述的所有项目都涉及活细胞，荧光标记蛋白质的高速或延时成像，以跟踪细胞内单个蛋白质的运输，组装和/或加工。这些实验需要由TIRFM实现的荧光激发的极窄区域;在每种情况下，研究人员尝试了标准的落射荧光或共聚焦显微镜，发现这些技术不足，因为它们未能提供必要的分辨率和/或样品保存。这些实验的数据将阐明细胞分化、运动和信号传导的机制，这些过程对所有生物体都是至关重要的，如果调节不当，可能会严重影响个体的健康和寿命。这项建议的具体目标是：目标1）提供调查员个人无法获得的专门和尖端资源。获得这些资源将扩展NIH资助的研究人员的研究能力和专业知识。 目标2）促进新的合作，特别是那些扩大研究方向和NIH资助的研究人员的影响。

DESCRIPTION (provided by applicant): All biological functions are dependent on molecular interactions. Therefore, by studying when, where and how molecules interact, we can elucidate the fundamental principles of biology. The Principle Investigators on this grant come from three different departments at the University of Minnesota and study a wide variety of topics, ranging from the dynamics of phospholipid receptor activation to the mechanisms that control amoeboid cell motility. These research projects each use different model systems and require their own specialized reagents. Yet, these projects are unified by the fact that each would benefit immensely from the use of Total Internal Reflection Fluorescence Microscopy (TIRFM). The long term goal of this proposal is to enhance basic and translational research at the University of Minnesota by providing state-of-the art, shared TIRFM equipment facilities with technical and scientific support. TIRFM is a specialized type of fluorescent microscopy in which only fluorescent molecule(s) within approximately 100 nm of the glass coverslip are excited (i.e. activated to fluoresce). With conventional epifluorescence microscopy, fluorescent excitation occurs though the entire depth of the sample; this generates out of focus background fluorescence, creating a poor signal-to-noise ratio and obscuring visualization. In addition, the sample is exposed to a considerable amount of fluorescent light, which can cause photobleaching (which leads to loss of imaging capacity) and, in the case of live cell imaging, phototoxicity and cell death. With laser confocal microcopy, the excitation beam is focused on a considerably narrower region (diameter: 250 -800 nm and depth: 500-1500 nm), improving the signal to noise ratio and reducing phototoxicity; however, with continuous imaging photobleaching and phototoxicity can still be a problem. All of the projects described in this proposal involve live cell, high speed or time-lapse imaging of fluorescently tagged proteins in order to follow the transport, assembly and/or processing of individual proteins within the cell. These experiments require the extremely narrow region of fluorescent excitation made possible by TIRFM; in each case, the investigators have attempted standard epifluorescence or confocal microscopy and found these techniques insufficient because they failed to provide the necessary resolution and/or sample preservation. Data from these experiments will elucidate the mechanisms that govern cell differentiation, motility and signaling, processes that are fundamental to all living organisms and, when mis-regulated, can deleteriously affect the health and lifespan of the individual. The specific aims of this proposal are: Aim 1) to provide access to specialized and cutting-edge resources not available to individual investigators. Having access to these recourses will extend the research capabilities and expertise of NIH-funded investigators. Aim 2) to foster new collaborations, particularly those that expand the research directions and impact of NIH-funded investigators.