IDBR: A Microscope Stage-Mounted Miniaturized Tissue Sectioning Device Enabling Automated, Extended 3-D Volume Imaging of Fluorescent Protein Labeled Biological Samples

遗传编码的荧光蛋白标签是直接可视化特定细胞群的优秀工具，非常适合研究多细胞事件，如组织分化，器官生长和分散在大区域的神经连接的建立。使用荧光蛋白技术以高分辨率整体研究大样本将为整个样本中发生的相互作用和相互依赖性提供前所未有的洞察力。 不幸的是，目前的高分辨率成像工具的视野受到严重限制，难以研究大样本。扩展体积成像技术允许对多个重叠区域进行成像，以便重建比单个显微镜视场大得多的高分辨率复合三维体积。然而，目前最好的技术与荧光蛋白信号不兼容，因为它们采用基于有机溶剂的包埋程序，可以使荧光蛋白变性，破坏其荧光并阻止直接可视化。为了满足这一未满足的需求，将开发和部署自动化，集成的水性组织切片显微镜载物台。通过消除所有有机溶剂处理步骤来保持与荧光蛋白的相容性。此外，通过对该设备的重新设计，它将与生物学家、神经科学家和其他从业者目前使用的标准直立激光扫描显微镜兼容。用于三维、扩展体积成像的含水组织切片显微镜载物台的功能、设计和实现将在加州理工学院生物成像中心的出版物和网页上公开传播。此外，将免费分发用于从各种模式生物中安装、切片和成像样本的优化方案，以促进新设备的广泛采用。这种模块化显微镜配件的最终广泛可用性将允许来自世界各地的研究人员修改现有的激光扫描显微镜，以执行荧光蛋白标记的生物样品的三维扩展体积成像。本科生将通过夏季本科生研究奖学金计划参与多学科仪器开发项目，并通过少数民族本科生研究奖学金计划增加科学和工程专业代表性不足的学生的代表性。

Genetically encoded fluorescent protein tags are excellent tools for directly visualizing specific cell populations, ideally suited for studying multi-cellular events such as tissue differentiation, organ growth and the establishment of neural connectivity dispersed over large regions. Using fluorescent protein technologies to study large samples in their entirety at high resolution will provide unprecedented insight into the interactions and interdependencies that occur throughout the sample. Unfortunately, the field of view of current high-resolution imaging tools is severely restricted, making it difficult to study large samples. Extended volume imaging techniques allow multiple, overlapping regions to be imaged in order to reconstruct a high-resolution, composite, three dimensional volume much larger than a single microscopic field of view. However, the best currently available techniques are not compatible with fluorescent protein signal, because they employ an organic solvent-based embedding procedure that can denature fluorescent proteins that ruins their fluorescence and prevents direct visualization.To fill this unmet need, an automated, integrated aqueous tissue sectioning microscope stage will be developed and deployed. Compatibility with fluorescent proteins is maintained by eliminating all organic-solvent processing steps. Furthermore, by miniaturizing the device, it will be made compatible with the standard upright laser scanning microscopes that biologists, neuroscientists and other practitioners are currently using.The features, design and implementation of the aqueous tissue sectioning microscope stage for three dimensional, extended volume imaging will be publicly disseminated in both publications and on web pages of the Caltech Biological Imaging Center. In addition, optimized protocols for mounting, sectioning and imaging samples from a variety of model organisms will be freely distributed to facilitate widespread adoption of the new device. The eventual widespread availability of this modular microscope accessory will allow researchers from all over the world to modify an existing laser scanning microscope to perform three dimensional extended volume imaging of fluorescent protein labeled biological samples. Undergraduate students will be involved in the multidisciplinary instrument development project through the Summer Undergraduate Research Fellowship program, and representation of underrepresented students in science and engineering will be increased through the Minority Undergraduate Research Fellowship program.