Multiparametric deep tissue microscope for in vivo and in vitro imaging

项目摘要： 每个细胞和组织生物医学研究计划都需要研究标本的显微镜可视化。 其中一些需求可以通过常规的宽视场和共焦显微镜相对薄（< 50 μ m） 组织切片或组织培养物。然而，对分子、细胞和组织水平动力学的生物学理解 最近，先进的多参数显微镜程序的爆炸式发展使其成为可能， 体内和离体成像。这些程序包括多光子显微镜结合谐波 用于在厚（100微米）组织和实体肿瘤中成像的产生的信号（SHG），表达多个 荧光蛋白，以及用于光学透明组织（例如， Clarity，iDISCO）用多种荧光标记物染色。这些技术现在是一个必要的部分， 当代生物医学细胞和组织研究。要成功应用这些方法， 多功能、先进的成像系统，结合了快速扫描、多光子和单光子激发， 多波长、多检测通道的高灵敏度和光谱选择性、快速成像 和环境控制。这些国家的最先进的显微镜方法的要求，为两个专业 设备和专门知识往往超过了单个研究实验室的现有资源。 因此，这些需求最好通过共享资源设施中的共享工具来满足。为这些 我们申请资金以取代斯坦福大学细胞科学成像设施的12- 一年前的体内多光子/共聚焦显微镜（Leica SP 5），配备最先进的新型Stellaris 8 潜水显微镜。这结合了多光子、共聚焦、体内和体外成像以及体外成像 显微镜将是一个共享资源，位于一个完善的，多用户的光和电子显微镜 设备：细胞科学成像设备（http：microscopy.stanford.edu）。该设施可供 斯坦福大学的整个研究界以及周围的生物技术公司。Stellaris 8 DIVE显微镜将支持11个用户的研究项目，其中10个由NIH资助。学业 研究各种模式生物和组织中的关键功能和结构问题，并涵盖 生物医学研究领域与人类健康和疾病的各个方面的影响。这些 项目包括：干细胞和肿瘤起始细胞的克隆命运和谱系追踪， 肺泡干细胞龛，小细胞肺癌和基底细胞皮肤癌的分子和细胞机制 骨生成中骨骼干细胞的发育、表征、骨髓的表征 微环境，遗传性疾病的基因治疗和触摸的生物物理学以及表征。 所有这些项目都迫切需要先进的仪器来成像活的和固定的组织和细胞样本， 只有Leica Stellaris 8 DIVE显微镜才能满足这一需求。

Project Summary: Every cell and tissue biomedical research program requires microscopic visualization of research specimens. Some of this need can be addressed by routine widefield and confocal microscopy of relatively thin (< 50 um) tissue slices or tissue culture. However, biological understanding of molecular, cell and tissue-level dynamics has been uniquely enabled by the recent explosion of advanced, multi-parametric microscopy procedures for in vivo and ex vivo imaging. These procedures include multiphoton microscopy combined with harmonically generated signals (SHG) for imaging in thick (100s of microns) tissue and solid tumors, expressing multiple fluorescent proteins, as well as for deep (>1 mm), high resolution imaging of optically cleared tissues (e.g. Clarity, iDISCO) stained with multiple fluorescent markers. These techniques are now a required part of contemporary biomedical cell and tissue research. To be applied successfully, these methods require a versatile, advanced imaging system that incorporates fast scanning, multi-photon and 1-photon excitation at multiple wavelengths, high sensitivity and spectral selectivity over multiple detection channels, rapid imaging, and environmental control. The demands of these state-of-the-art microscopy methods for both specialized equipment and specialized expertise often outstrip the resources available to individual research laboratories. As such, these needs are best met with a shared instrument housed in a shared resource facility. For these reasons we are requesting funding to replace Stanford University's Cell Sciences Imaging Facility's 12- year-old in vivo multi-photon/confocal microscope (Leica SP5) with the new, state-of-the-art Stellaris 8 DIVE microscope. This combined multi-photon, confocal, in vivo, and ex vivo as well as in vitro imaging microscope will be a shared resource located in a well-established, multi-user light and electron microscopy facility: The Cell Sciences Imaging Facility (http://microscopy.stanford.edu). This facility is accessible to Stanford University's entire research community as well as surrounding biotech companies. The Stellaris 8 DIVE microscope will support research projects from 11 users, 10 of which are NIH funded. Their studies investigate critical functional and structural questions in a variety of model organisms and tissues and cover areas of biomedical research with implications for diverse aspects of human health and disease. These projects include: clonal fate and linage tracing of stem cells and tumor initiating cells, interrogation of the alveolar stem cell niche, molecular and cellular mechanisms of small cell lung and basal cell skin cancers development, characterization of skeletal stem cells in osteogenesis, characterization of the bone marrow microenvironment, gene therapy for inherited diseases and the biophysics of touch as well as characterizing. All these projects critically require advanced instrumentation for imaging live and fixed tissue and cell samples, a need which can only uniquely be met by the requested Leica Stellaris 8 DIVE microscope.