CORRELATED IMAGING OF SUPRAMOLECULAR COMPLEXES ANDCELLULAR COMPARTMENTS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 在NCMIR，我们试图了解蛋白质如何映射到复杂的细胞微域，如突触，以及这些微域如何反过来与更高级别的结构相关。尽管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，但这种方法通常不适用于细胞生物学中许多最有趣的问题，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推动二氨基联苯胺(DAB)被氧化成一种不溶于水的聚合物，该聚合物可以通过四氧化臭氧处理而变得电子致密。我们使用荧光素的溴化衍生物曙红使荧光光氧化与免疫标记和原位杂交兼容。这种方法的优点是将光学显微镜的低分辨率分析与电子显微镜可获得的更精细的超微结构细节相关联。与基于酶的方法相比，光氧化标记和定位的特异性很突出，因为活性氧物种相对较短，不会扩散到离生产现场很远的地方，组织的广泛交联会最大限度地减少反应产物的扩散。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，以突出和剖析细胞和组织中的大分子特化区域，并通过电子断层扫描绘制三维排列图。我们正在开发高压冷冻脆弱的神经组织和光转换样品的方法。利用我们的能量过滤显微镜的能力和改善断层重建的对准和分割的计算方法，我们不断提高我们成像技术的分辨率，以弥合细胞动力学的光学显微成像和直接显示大分子结构之间的差距。

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 enzymebased 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.