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。使用荧光蛋白技术在高分辨率下研究大样本的整体将提供前所未有的洞察整个样本中发生的相互作用和相互依赖关系。不幸的是，目前的高分辨率成像工具的视场受到严重限制，使得研究大样本变得困难。扩展体成像技术允许对多个重叠区域进行成像，以重建高分辨率，复合，三维体积，比单个微观视野大得多。然而，目前可用的最佳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嵌入程序，可以使荧光蛋白变性，从而破坏其荧光并阻止直接可视化。为了填补这一未满足的需求，一个自动化的，集成的水组织切片显微镜将被开发和部署。通过消除所有有机溶剂处理步骤，保持与荧光蛋白的相容性。此外，通过将设备小型化，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水组织切片显微镜的特点、设计和实现将在出版物和加州理工学院生物成像中心的网页上公开发布。此外，将免费分发用于安装，切片和成像各种模式生物样品的优化方案，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，而在科学和工程领域，代表性不足的学生将通过少数民族本科生研究奖学金计划增加。

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.