Zeiss LSM 710 Confocal Microscopy System for Imaging of Mineralized Tissues

描述（由申请人提供）：我们从宏观到微观到纳米甚至到原子尺度理解健康和疾病中的细胞功能的能力取决于先进仪器的能力。本申请中描述的NIH资助项目需要提高成像分辨率和多光谱能力，以使其明场和荧光显微镜应用提高到下一个水平。需要一台蔡司lsm71034通道激光扫描共聚焦显微镜。该仪器将作为共享资源运行，以支持NIH资助的UMKC牙科学院、UMKC护理学院和堪萨斯大学医学中心的主要和次要用户核心小组的研究项目。用户组的研究重点是矿化组织和肌肉骨骼研究。为了适应项目应用的范围，该仪器将配置为用于固定细胞和组织标本的基本共聚焦显微镜，以及活细胞成像，多光谱成像，光漂白后荧光恢复（FRAP）和光学切片/3D成像。其中一个将采用该技术的项目是研究骨细胞外基质（ECM）的组装机制，使用新型荧光探针对各种骨细胞外基质成分进行延时成像。这些研究为骨基质组装的动态性质和细胞运动在组装过程中的作用提供了新的见解。骨细胞在调节骨骼对机械负荷的反应中的作用是三个参与项目的重点。直到最近，人们对这种难以捉摸的细胞类型的功能知之甚少，因为它难以接近，嵌入在骨骼中。随着最近骨细胞分化标记物的鉴定和具有骨细胞荧光谱系报告的转基因小鼠的发展，将这些细胞在矿化环境中的成像达到前所未有的水平成为可能。一个项目使用谱系报告结合时间推移成像来检查成骨细胞和骨细胞的动态特性，以深入了解成骨细胞分化成骨细胞的过程。另一个项目研究骨细胞蛋白E11/gp38在树突形成和骨细胞对机械负荷的反应中的作用。第三个项目研究骨细胞感知机械负荷信号并将其转化为骨形成反应的分子机制。其他项目包括肌肉钙成像，确定调节磷酸盐稳态的分子机制，以及确定小天使病患者骨炎性病变的分子发病机制。蔡司LSM 710系统将通过提供矿化组织中骨细胞网络三维成像所需的无与伦比的分辨率来推进这些项目的进展。LSM 710的多光谱成像能力及其增强的实时成像灵敏度对于将该研究推进到下一个水平至关重要，并且将允许同时对多个分子探针进行成像，最大限度地从每个实验中收集信息。这项技术的获得将推动有关肌肉骨骼健康的发现，并将对骨质疏松症、骨软化症和遗传性结缔组织疾病等疾病产生影响。

DESCRIPTION (provided by applicant): Our capacity to understand cellular function in health and disease from the macro to micro to nano and even to the atomic scale depend on the capabilities of advanced instrumentation. The NIH funded projects described in this application require increased imaging resolution and multi-spectral capabilities for their brightfield and fluorescence microscopy applications to advance to the next level. A Zeiss LSM 710 34 channel laser scanning confocal microscope is requested. The instrument will be operated as a shared resource to support the research programs of a core group of NIH funded major and secondary users in the UMKC School of Dentistry, UMKC School of Nursing and University of Kansas Medical Center. The user group has a research emphasis on mineralized tissues and musculoskeletal research. To accommodate the range of project applications, the instrument will be configured for basic confocal microscopy on fixed cell and tissue specimens, and for live cell imaging, multispectral imaging, fluorescence recovery after photobleaching (FRAP) and optical sectioning/3D imaging. One of the projects that will employ this technology is investigating the mechanisms of assembly of bone extracellular matrix (ECM), using time lapse imaging with novel fluorescent probes for various bone ECM components. These studies are providing novel insights into the dynamic nature of bone matrix assembly and the role of cell motility in the assembly process. The role of the osteocyte in regulating skeletal responses to mechanical loading is the focus of three of the participating projects. Until recently, little was known about the function of this elusive cell type, due to its inaccessibility, embedded within bone. With the recent identification of osteocyte differentiation markers and development of transgenic mice with fluorescent osteocyte lineage reporters, it is possible to image these cells within their mineralized environment to an unprecedented level. One project examines the dynamic properties of osteoblasts and osteocytes using lineage reporters combined with time lapse imaging to gain insight into the process by which osteoblasts differentiate into osteocytes. Another project examines the role of the osteocyte protein, E11/gp38, in dendrite formation and in osteocyte responses to mechanical loading. A third project examines the molecular mechanisms by which osteocytes perceive mechanical loading signals and translate them into bone formative responses. Other projects involve calcium imaging in muscle, determining the molecular mechanisms regulating phosphate homeostasis and determining the molecular pathogenesis of bone inflammatory lesions in Cherubism patients. The Zeiss LSM 710 system will advance the progress of these projects by providing an unsurpassed level of resolution that is needed for 3D imaging of osteocyte networks in mineralized tissues. The multi-spectral imaging capabilities of the LSM 710 and its enhanced sensitivity for live imaging, are essential to advance this research to the next level, and will allow for simultaneous imaging of multiple molecular probes, maximizing the information gathered from each experiment. Acquisition of this technology will advance discoveries regarding musculoskeletal health and will have implications for diseases, such as osteoporosis, osteomalacia and inherited connective tissue disorders. PUBLIC HEALTH RELEVANCE: This application is for a Zeiss LSM 710 34 channel laser scanning confocal microscope, to be operated as a shared resource that will support the research programs of a core group of NIH funded major users and secondary users, located in the UMKC School of Dentistry, UMKC School of Nursing and University of Kansas Medical Center. The research emphasis of the core users is on mineralized and musculoskeletal tissues and this state of the art instrumentation will enhance their ability to generate high resolution images of muscle cells and bone cells in culture and in situ within the mineralized tissue to advance the research goals of the participating projects. Acquisition of this technology will result in new discoveries regarding bone and muscle health and will have major implications for diseases of mineralized tissues, such as osteoporosis, osteomalacia and inherited connective tissue disorders.