Leica TCS SP5 MP confocal and two-photon microscope

描述（由申请人提供）：巴克年龄研究所研究的总体目标是了解衰老及其相关疾病，以延长人类的健康寿命。巴克研究所的研究已经达到了这样一个阶段，即在分离细胞上完善的技术现在可以应用于更复杂的急性切片和体内模型。这需要一个合适的显微镜设备，这个共享仪器建议寻求资金购买徕卡TCS SP5 MP直立荧光显微镜系统，用于双光子和共聚焦成像。该仪器将被用于研究与衰老、神经退行性疾病、中风、癌症和干细胞治疗有关的人类健康特定关键问题的项目。现有的活体标本共聚焦仪器包括蔡司LSM510与一个相干变色龙XR模型锁定钛：蓝宝石（双光子）激光器。由于该仪器安装在倒置显微镜系统Axiovert 200上，该系统带有透镜和内部去扫描检测器，该检测器具有基于滤光轮的光谱选择，为细胞培养和组织学工作优化，因此不适合厚切片和全动物研究。要求的徕卡TCS SP5 MP将包括一个直立显微镜，深层组织优化的浸没透镜和用于双光子显微镜的反射和透射光非扫描检测器。此外，一个442纳米和其他标准可见激光器，结合三个内部探测器光谱成像将促进通用的单光子，共聚焦显微镜的使用。该系统将与现有的相干变色龙XR双光子激光器集成，而蔡司LSM510将重新配备更适合薄样品紫外激发的405nm紫色激光器。后者的经费将从独立于本申请的预算中获得。新的徕卡仪器将立即加强巴克研究所多个研究小组的工作，他们在深层组织荧光成像或光谱共聚焦显微镜方面有重大需求。这种新仪器最关键的特点是能够(1)使用荧光探针和标记在600？M体内或离体活样品，(2)允许在1.5 mm深度处对固定样品进行三维重建，(3)便于在单个样品中对多达三个单独的荧光团进行光谱扫描和同时光谱分离，这是我们目前所缺乏的功能。具体项目将研究神经退行性疾病中的氧化应激、活性氧、细胞器运输和能量学、蛋白质聚集和树突脊柱结构；移植神经元前体细胞的迁移；肿瘤前乳腺癌的基因组场缺陷，肾脏疾病的体内果蝇模型，以及减缓人类衰老的蠕虫模型中化合物的药代动力学成像。

DESCRIPTION (provided by applicant): The overall objective of research at the Buck Institute for Age Research is to understand aging and its associated diseases in order to extend the healthy years of human life. Research at the Buck Institute is reaching the stage at which techniques perfected for isolated cells can now be applied to more complex acute slice and in vivo models. This requires an appropriate microscope facility and this shared instrumentation proposal seeks funding for the purchase of a Leica TCS SP5 MP upright fluorescence microscopy system for two-photon and confocal imaging. The instrument would be used by projects that are examining specific key issues in human health related to aging, neurodegenerative disease, stroke, cancer and stem cell therapies. The existing live specimen confocal instrumentation comprises a Zeiss LSM510 with a Coherent Chameleon XR modelocked Ti:Sapphire (two-photon) laser. Since this is mounted on an Axiovert 200 which is an inverted microscopic system, with lenses and internal, descanned detectors with filter-wheel based spectral selection optimized for cell culture and histological work, it is inappropriate for thick slice and whole animal studies. The requested Leica TCS SP5 MP will include an upright microscope, deep tissue optimized dipping lenses and reflected and transmitted light non-descanned detectors for two-photon microscopy. Additionally, a 442 nm and other standard visible lasers, in combination with three internal detector spectral imaging will facilitate versatle single photon, confocal microscopic use. The system will be integrated with an existing Coherent Chameleon XR two-photon laser, while the Zeiss LSM510 will be re-equipped with a 405nm violet laser better suited for the thin sample UV excitation. Funding for the latter will be obtained from a budget separate from this application. The new Leica instrument will immediately enhance the work of multiple research groups at the Buck Institute who have significant needs in deep-tissue fluorescence imaging or spectral confocal microscopy. The most critical features of this new instrumentation will be the capability to (1) conduct deep-tissu time lapse imaging of physiological parameters and morphology using fluorescent probes and markers at depths of up to 600?m in vivo or in ex vivo living samples, (2) allow three dimensional reconstruction of fixed samples at depths of up to 1.5 mm, and (3) facilitate spectral scanning and simultaneous spectral separation of up to three individual fluorophores in a single sample, features that we currently lack. Specific projects will investigate oxidative stress, reactive oxygen species, organelle transport and energetics, protein aggregation and dendritic spine structure in neurodegenerative diseases; migration of transplanted neuronal precursor cells; genomic field defects in preneoplastic breast cancer, an in vivo fly model of kidney disease, and pharmacokinetic imaging of a compound that slows aging in a worm model of human aging. PUBLIC HEALTH RELEVANCE: Ten independent research groups at the Buck Institute will use this new technology to improve and expand their ongoing studies into the mechanisms and basic biology of human aging and into diseases whose incidence increases significantly with age, including Huntington's, Alzheimer's and Parkinson's diseases, breast cancer, and stroke. The overarching aim is to reduce the incidence and severity of these diseases and extend the healthy years of life. The relevance of this new instrumentation to public health is significant in that the benefits to society will ultimately include both increased quality of later life, and significant decreases in healthcare costs.