Two-photon microscope Adapted for Automated 3D Tissue Reconstruction at High Spat

描述（由申请人提供）：组织结构（如心脏或骨骼肌）在疾病中经历实质性重塑，包括肉眼可见的变化，例如：总体几何结构;血管网络重组;纤维和片层取向;间质纤维化;以及微观变化，例如：分子表达的重新分布;以及线粒体的能量状态。迫切需要了解这种重塑的性质，因为所有这些过程都影响这些组织在健康和疾病中的功能，但现有的组织重建方法是低分辨率的，而目前的高分辨率亚细胞成像不能扩展到组织和整个器官。在这里，我们描述了一个前所未有的方法，双光子显微镜将被用来执行整个组织的三维重建在微米级的空间分辨率，弥合从微观到宏观尺度的差距。 我们要求一个最先进的蔡司7 MP专用多光子显微镜系统，用于多光子激发荧光成像和组织的自动3D重建，供多个NIH资助的研究人员使用，以完成现有设备目前无法完成的研究。电动载物台和自动切片机的集成使该仪器能够以更高的分辨率产生图像，用于更大体积的组织，而不是已经实现的，使得大组织体积图像的三维重建成为可能。 该系统最初将有7个主要用户，并将充分用于大型组织的长时间成像;随着研究的发展，可能会增加次要用户。这将是约翰霍普金斯大学唯一的同类仪器。所有其他多光子显微镜在大学用于不同的应用，不能使用或修改为我们的目的;这是唯一一个在JHU被用于这些耗时的组织重建。该大学已经确定了这一需求，并支持我们的建议，为基础设施提供财政支持，以适应这一成像系统。相关性：拟议的系统消除了目前NIH资助的广泛研究的障碍，并为未来对人类健康产生重大影响的广泛工作打开了大门。应用包括：多个物种的健康和患病心脏图谱;以及对发生的群体水平和个体水平解剖变化的计算分析。许多下游应用涉及使用生成的高分辨率数据集来创建心脏电生理学、电动力学、血流和药物输送的复杂解剖学详细和个性化的计算模型，以模拟心脏性猝死、心脏除颤、外周动脉疾病等。事实上，我们设想获得的大多数数据至少被使用三次：作为图像;作为地图集的组成部分;以及作为至少一个计算模型的基础。

DESCRIPTION (provided by applicant): The structure of tissues (such as heart or skeletal muscle) undergoes substantial remodeling in disease, including macroscopic changes, e.g.: overall geometry; vascular network reorganization; fiber and sheet orientation; and interstitial fibrosis; and microscopic changes, e.g.: redistribution of molecular expression; and the energetic state of mitochondria. There is an urgent need to understand the nature of this remodeling, because all of these processes impact on the function of these tissues in health and disease, but existing methods for tissue reconstruction are low-resolution, while current high-resolution sub- cellular imaging cannot be expanded to tissues and whole organs. Here we describe an unprecedented approach by which two-photon microscopy will be used to perform whole-tissue 3D reconstructions at micron- level spatial resolution, bridging the gap from micro to macro scale. We request a state-of-the-art Zeiss 7MP dedicated multiphoton microscope system for the multiphoton- excited fluorescence imaging and automated 3D reconstruction of tissues, to be used by multiple NIH-funded investigators to accomplish research not currently possible with existing equipment. Integration of a motorized stage and automated microtome allow this instrument to produce images at a higher resolution, for a larger volume of tissue, than has been yet achieved, making three-dimensional reconstructions of large-tissue- volume images possible. The system will initially have 7 major users and will be fully utilized for long imaging sessions of large tissues; as research evolves, minor users may be added. This will be the only instrument of its kind at the Johns Hopkins University. All other multiphoton microscopes at the university are used for different applications and cannot be used or modified for our purposes; this is the only one at JHU to be used for these time-consuming tissue reconstructions. The University has identified this need and backs our proposal with financial support for infrastructure to accommodate this imaging system. Relevance: The proposed system removes roadblocks to a wide range of currently NIH-funded research, and opens the door to a breadth of future work of significant impact on human health. Applications include: atlases of healthy and diseased hearts in multiple species; and computational analysis of both the population-level and individual-level anatomical changes that occur. Many of the downstream applications involve using the high- resolution datasets generated to create complex anatomically-detailed and individualized computational models of cardiac electrophysiology, electromechanics, blood flow and drug delivery, to simulate Sudden Cardiac Death, Cardiac Defibrillation, Peripheral Arterial Disease and more. In fact, we envision most data acquired being used at least three times: as an image; as a component of an atlas; and as the basis of at least one computational model.