Zeiss 2-photon (2P) LSM780 laser scanning confocal microscope

描述（由申请人提供）：我们正在申请资金购买卡尔蔡司780非线性光学共聚焦显微镜，我们将耦合到相干变色龙超飞秒钛：蓝宝石激光器和国际空间站快速荧光寿命成像探测器。该系统将是唯一的倒置双光子显微镜一般可在安舒茨医学校园，将是唯一的荧光寿命成像系统提供给研究界在大丹佛地区。蔡司LSM 780 NLO将对NIH资助的研究产生巨大影响，因为它能够进行荧光寿命成像显微镜（FLIM），双光子激发（TPE）和二次谐波发生（SHG）显微镜，以及高光谱成像。荧光寿命成像显微镜（FLIM）：FLIM正在成为一种非常强大的技术，用于线粒体代谢和氧化应激动态变化的体内时空成像。在癌症生物学中，最近重新出现的瓦尔堡效应的原始概念也与糖酵解的改变有关 与线粒体氧化磷酸化的对比。代谢的动态成像部分通过监测NAD（P）H和FAD寿命的变化来实现。FLIM也是一种非常敏感的技术来执行福斯特（或荧光）共振能量转移（FRET），一种强大的技术，用于监测发生在10 nm或更短距离内的生物学感兴趣的蛋白质-蛋白质相互作用。与广泛使用的强度技术不同，该技术受到光谱渗透和光谱重叠的限制，寿命的变化可以检测蛋白质-蛋白质相互作用中的真实的变化，而不会受到与基于强度的技术相关的限制。双光子激发（TPE）和二次谐波发生（SHG）显微镜：SHG是一种非常强大的技术，可以对胶原蛋白进行无标记成像。导致组织纤维化的纤维状胶原蛋白发生在与组织纤维化相关的许多病理生理学状况中，包括肾病、肝病、肺病和心脏病。在癌组织中，所谓的上皮向间充质转化也与细胞外基质蛋白的积累有关。相反，在晚期动脉粥样硬化中，组织基质金属蛋白酶的激活可导致局部基质蛋白的减少。光谱分解：Zeiss LSM 780提供的这种新的和改进的模式是一种强大的技术，可以同时对与几种蛋白质，DNA分子或脂质相对应的几种荧光团进行成像，而不会出现我们过去遇到的出血并发症。

DESCRIPTION (provided by applicant): We are requesting funds to purchase a Carl Zeiss 780 NLO Confocal microscope, which we will couple to a Coherent Chameleon Ultra femto-second Ti: sapphire laser and ISS fast-fluorescence lifetime imaging detector. This system will be the only inverted two-photon microscope generally available at the Anschutz Medical Campus and will be the only fluorescence lifetime imaging system available to the research community in the Greater Denver area. The Zeiss LSM 780 NLO will have tremendous impact on NIH funded research as it is capable of performing Fluorescence Lifetime Imaging Microscopy (FLIM), Two-Photon Excitation (TPE) and Second Harmonic Generation (SHG) Microscopy, and also Hyperspectral Imaging. Fluorescence Lifetime Imaging Microscopy (FLIM): FLIM is becoming a very powerful technique for in vivo spatiotemporal imaging of dynamic changes in mitochondrial metabolism and oxidative stress. In cancer biology the recently reemerging original concept of Warburg effect is also related to alterations in glycolysis versus mitochondrial oxidative phosphorylation. Dynamic imaging of metabolism is achieved in part through monitoring changes in NAD(P)H and FAD lifetimes. FLIM is also a very sensitive technique to perform Foster (or Fluorescence) Resonance Energy Transfer (FRET), a powerful technique to monitor protein-protein interactions of biological interest that occur within a distance of 10 nm or less. Unlike the widely used intensity techniques that are limited by spectral bleed through and spectral overlaps, changes in lifetime can detect real changes in protein-protein interactions without suffering from the limitations associated with the intensity based techniques. Two Photon Excitation (TPE) and Second Harmonic Generation (SHG) Microscopy: SHG is a very powerful technique that allows for label-free imaging of fibrillary collagens. Fibrillary collagens resulting in tissue fibrosis occurs in many pathophysiological conditions associated with tissue fibrosis including kidney disease, liver disease, lung disease, and heart disease. In cancer tissues the so called epithelial to mesenchymal transformation is also associated with accumulation of fibrillary extracellular matrix proteins. In contrast in advanced atherosclerosis activation of tissue matrix metalloproteinases can result in localized decreased in matrix proteins. Spectral Unmixing: This new and improved modality available with the Zeiss LSM780 is a powerful technique that will allow simultaneous imaging of several fluorophores corresponding to several proteins, DNA molecules, or lipids of interest without the complication of bleed through that we have encountered in the past.