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，以延长人类寿命的健康年数。巴克研究所的研究正处于这样一个阶段，即针对分离细胞而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。这需要一个适当的显微镜设施，这个共享仪器的建议寻求资金购买徕卡TCS SP 5 MP直立荧光显微镜系统的双光子和共焦成像。该仪器将用于研究与衰老、神经退行性疾病、中风、癌症和干细胞疗法有关的人类健康具体关键问题的项目。现有的活体样本共聚焦仪器包括Zeiss LSM 510和Coherent Chameleon XR锁模Ti：Sapphire（双光子）激光器。由于这是安装在Axiovert 200上，Axiovert 200是一种倒置显微镜系统，具有透镜和内部去扫描检测器，具有基于滤光轮的光谱选择，针对细胞培养和组织学工作进行了优化，因此不适合厚切片和整个动物研究。所要求的Leica TCS SP 5 MP将包括一个正置显微镜、深层组织优化的浸渍镜头以及用于双光子显微镜的反射和透射光非去扫描探测器。此外，442 nm和其他标准可见光激光器与三个内部检测器光谱成像相结合，将有助于多用途单光子共焦显微镜的使用。该系统将与现有的Coherent Chameleon XR双光子激光器集成，而蔡司LSM 510将重新配备更适合薄样品紫外激发的405 nm紫光激光器。后者的资金将从与本申请分开的预算中获得。新的徕卡仪器将立即加强巴克研究所的多个研究小组的工作，这些研究小组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。这种新仪器的最关键的特点将是能够（1）进行深组织的生理参数和形态的时间推移成像，使用荧光探针和标记物在深度高达600？在体内或离体活体样品中，（2）允许在高达1.5mm的深度处对固定样品进行三维重建，以及（3）促进光谱扫描和单个样品中高达三个单独荧光团的同时光谱分离，这是我们目前缺乏的特征。具体项目将研究氧化应激，活性氧，细胞器运输和能量学，神经退行性疾病中的蛋白质聚集和树突棘结构;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，肾脏疾病的体内苍蝇模型，以及在人类衰老的蠕虫模型中减缓衰老的化合物的药代动力学成像。 公共卫生相关性：巴克研究所的10个独立研究小组将利用这项新技术改进和扩大他们正在进行的研究，研究人类衰老的机制和基础生物学，以及发病率随年龄增长而显著增加的疾病，包括亨廷顿氏病、阿尔茨海默氏病和帕金森氏病、乳腺癌和中风。总体目标是降低这些疾病的发病率和严重程度，延长健康寿命。这一新仪器与公共卫生的相关性在以下方面具有重要意义： 对社会的好处最终将包括晚年生活质量的提高和医疗保健费用的显著降低。

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.