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

描述(申请人提供)：我们正在申请资金购买卡尔蔡司780NLO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ISS快速荧光寿命成像探测器。该系统将是安舒茨医学院唯一普遍可用的倒置双光子显微镜，也将是大丹佛地区研究社区唯一可用的荧光寿命成像系统。蔡司LSM 780 NLO将对NIH资助的研究产生巨大影响，因为它能够执行荧光寿命成像显微镜(FLIM)、双光子激发(TPE)和二次谐波(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，可以在体内对线粒体代谢和氧化应激的动态变化进行时空成像。在癌症生物学中，最近重新出现的华宝效应的原始概念也与糖酵解的变化有关。 而不是线粒体氧化磷酸化。代谢的动态成像部分是通过监测NAD(P)H和FAD寿命的变化来实现的。FliM也是执行Foster(或荧光)共振能量转移(FRET)的一种非常敏感的技术，FRET是一种强大的技术，可以监测发生在10 nm或更短距离内的生物感兴趣的蛋白质之间的相互作用。与受到光谱溢出和光谱重叠限制的广泛使用的强度技术不同，寿命的变化可以检测到蛋白质-蛋白质相互作用的真正变化，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(SHG)显微镜：SHG是一种非常强大的技术，允许对纤维胶原进行无标记成像。导致组织纤维化的纤维性胶原在许多与组织纤维化相关的病理生理条件下发生，包括肾脏疾病、肝脏疾病、肺病和心脏病。在癌症组织中，所谓的上皮向间充质转化也与纤维状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基质蛋白的局限性减少。光谱分解：蔡司LSM780提供的这一新的改进模式是一项强大的技术，它将允许同时对与几种感兴趣的蛋白质、DNA分子或脂类相对应的几个荧光团进行成像，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.