Tech Development Core

技术新奇是三方面的。i）使用活细胞单颗粒成像、超分辨率单分子成像和冷冻ET成像建立相关的多模式成像系统推进了我们先前建立的相关活细胞和冷冻ET方法以确定HIV-i蛋白与宿主细胞因子的相互作用。在成像光谱的一端，特定目标在成像光谱中的动态行为可以被观察到。 可以用折衷的分辨率来表征活生物系统的背景，而在另一端，可以获得感兴趣区域的高分辨率结构快照。超分辨率分子成像将这些方法连接起来，并允许识别和定位与cryoET结构相关的特定靶蛋白。2)在玻璃体冷冻细胞中创建细胞板层，类似于yo-超薄切片，但没有切片伪影，是一种生成适当薄的玻璃体生物标本的新方法，并增强了cryoET用于天然细胞和组织的高分辨率3D结构分析的适用性。3)使用一个短的融合肽与金表面的高亲和力构成了一种新的方法，以克服一些困难，在开发一个绿色荧光蛋白等效的可标记的EM标签。最终，技术计划的创新依赖于我们开发的几种方法（相关光学显微镜/cryoEM成像）和其他方法（超分辨率成像）的整合。我们提出的研究建立在我们以前的工作和发展新的能力。

The technological novelty is three-fold, i) Establishing a correlative multi-mode imaging system using live cell single particle imaging, super-resolution single molecule imaging, and cryoET imaging advances our previously established correlative live-cell and cryoET methodology to determine HIV-i protein interactions with host cell factors. At one end of the imaging spectrum, the dynamic behavior of a specific target in the context of a living biological system can be characterized with compromised resolution, while at the other end, high resolution structural snapshots of regions of interest can be obtained. Super-resolution molecular imaging bridges these methods and allows identification and localization of specific target proteins associated with cryoET structures. 2) Creating cell lamella in vitreously frozen cells, similar to yo-ultramicrotomy, but without sectioning artifacts, is a novel approach to generate suitably thin vitreous biological specimens and enhances the applicability of cryoET for high resolution 3D structural analysis of native cells and tissues. 3) Using a short fusion peptide with high affinity to a gold surface constitutes a novel approach to overcome some of the difficulties in developing a GFP-equivalent taggable EM label. Ultimately the innovation of the technology program relies on the integration of several approaches developed by us (correlative light microscopy/cryoEM imaging) and by others (super resolution imaging). Our proposed studies build on our prior work and develop new capabilities.