CORRELATED IMAGING OF SUPRAMOLECULAR COMPLEXES ANDCELLULAR COMPARTMENTS

超分子复合物和细胞区室的相关成像

• 批准号: 7358103
• 负责人: GINA E SOSINSKY
• 金额: $ 10.17万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-05-01 至 2007-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7358103
• 关键词: CORRELATED; IMAGING; SUPRAMOLECULAR; COMPLEXES; ANDCELLULAR

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.

该子项目是利用 NIH/NCRR 资助的中心拨款提供的资源的众多研究子项目之一，该子项目和研究者 (PI) 可能已从其他 NIH 来源获得主要资助，因此可以在其他 CRISP 机构中得到体现。列出的是该中心的信息，该中心不一定是研究人员的机构。在 NCMIR，我们寻求了解蛋白质如何映射到突触等复杂的细胞微结构域，以及这些结构域又如何与更高阶的结构相关。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的许多最有趣的问题通常并不可行，特别是在神经系统中，NCMIR 率先使用荧光光氧化作为一种​​方法。相关光和电子显微成像的染色技术。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，通过处理可以使其具有电子致密性。我们使用荧光团曙红（荧光素的溴化衍生物）使荧光光氧化与免疫标记和原位杂交兼容，该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获得的更精细的超微结构细节相关联。与酶相比，光氧化产生的标记和定位的特异性非常突出。基于方法，因为活性氧的寿命相对较短，不会扩散到远离生产部位的地方，并且组织的广泛交联最大限度地减少了反应产物的扩散，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，以突出和解剖细胞和组织中的大分子专业化区域，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神经组织的方法。利用我们的能量过滤显微镜的功能和计算方法来改进断层扫描重建的对齐和分割，我们不断提高成像技术的分辨率，以弥合细胞动力学的光学显微成像和大分子结构的直接可视化之间的差距。 。