CORRELATED IMAGING OF SUPRAMOLECULAR COMPLEXES ANDCELLULAR COMPARTMENTS

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。在NCMIR，我们试图了解蛋白质如何映射到复杂的细胞微结构域，如突触，以及这些结构域如何与更高层次的结构相关。虽然未染色材料的冷冻断层扫描提供了原位研究天然分子结构的机会，但这种方法对于细胞生物学中许多最有趣的问题通常不可行，特别是在神经系统中。NCMIR率先使用荧光光氧化作为相关光和电子显微镜成像的染色技术。当荧光化合物被强烈激发时产生的活性氧驱动二氨基联苯胺（DAB）氧化成不溶性聚合物，该聚合物可以通过用四氧化锇处理而呈现电子致密。我们使用荧光团曙红（一种溴化的荧光素衍生物）使荧光光氧化与免疫标记和原位杂交兼容。这种方法的优点是相关的低分辨率分析，在光学显微镜的电子显微镜获得更精细的超微结构的细节。与基于酶的方法相比，光氧化产生的标记和定位的特异性是突出的，因为活性氧物质的寿命相对较短，并且不会扩散到远离产生部位，并且组织的广泛交联使反应产物的扩散最小化。我们正在应用选择性对比方法，包括四半胱氨酸（TC）技术，免疫细胞化学和组织学染色技术，突出和解剖细胞和组织中大分子专业化的领域，并绘制三维排列与电子断层扫描。我们正在开发脆弱神经组织的高压冷冻和光转换样品的方法。利用我们的能量过滤显微镜和计算方法的能力来改善断层重建的对齐和分割，我们不断推动我们的成像技术的分辨率，以弥合细胞动力学的光学显微镜成像和大分子结构的直接可视化之间的差距。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. At NCMIR, we seek to understand how proteins map onto complex cellular microdomains like synapses, and how these domains in turn relate to higher orders of structure. Although cryo-tomography of unstained material offers the opportunity to study native molecular structure in situ, this approach is not often feasible for many of the most interesting questions in cell biology, particularly in the nervous system. NCMIR has pioneered the use of fluorescence photooxidation as a staining technique for correlated light and electron microscopic imaging. Reactive oxygen, generated when fluorescent compounds are strongly excited, drives the oxidation of diaminobenzidine (DAB) into an insoluble polymer that can be rendered electron-dense by treatment with osmium tetroxide. We made fluorescence photooxidation compatible with both immunolabeling and in situ hybridization using the fluorophore eosin, a brominated derivative of fluorescein. This method has the advantage of correlating low resolution analysis at the light microscope to the finer ultrastructural details obtainable at the electron microscope. The specificity of the photooxidation-generated labeling and localization was outstanding compared to enzyme¿based methods because the reactive oxygen species are relatively short-lived and do not diffuse far from the site of production and the extensive cross-linking of tissue minimizes the spread of the reaction product. We are applying selective contrasting methods, including tetracysteine (TC) technology, immunocytochemical and histological staining techniques, to highlight and dissect areas of macromolecular specialization in cells and tissues and map the three dimensional arrangement with electron tomography. We are developing methods for high pressure freezing of fragile nervous tissue and for photoconverted samples. Using the capabilities of our energy filtering microscope and computational methods for improving the alignment and segmentation of tomographic reconstructions, we continually push the resolution of our imaging techniques to bridge the gap between light microscopic imaging of cellular dynamics and direct visualization of macromolecular structure.