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.

该主题项目是利用NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子弹和调查员（PI）可能已经从其他NIH来源获得了主要资金，因此可以在其他清晰的条目中代表。列出的机构是针对该中心的，这不是调查人员的机构。在NCMIR，我们试图了解蛋白质如何将其映射到复杂的细胞微区域（如突触）上，以及这些结构域又如何与较高的结构阶相关。尽管未经染色的材料的冷冻描记术为原位研究天然分子结构提供了机会，但是对于细胞生物学中的许多最有趣的问题，尤其是在神经系统中，这种方法通常是不可行的。 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。当荧光化合物强烈兴奋时产生的活性氧，将二氨基苯胺（DAB）氧化驱动到不溶性的聚合物中，该聚合物可以通过用四氧化甲氧化剂处理来使电子致密。我们使用荧光团曙红（一种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。该方法具有将光学显微镜下的低分辨率分析与可在电子显微镜下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，光氧化生成的标记和定位的特异性是出色的，因为活性氧相对短暂寿命相对短，并且与生产部位相对较短，并且组织的广泛交叉链接可最大程度地减少该反应产物的扩散。我们正在应用选择性对比方法，包括四环素半胱氨酸（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专业化区域，并用电子层次摄影绘制三维布置。我们正在开发用于高压脆弱神经组织和光电转化样品的方法。使用我们的能量滤波显微镜和计算方法的能力来改善层析成像重建的对齐和分割，我们不断地推动分辨率的成像技术的分辨率，以弥合细胞动力学的光显微镜成像与直接可视化的光显微成像之间的差距。