Tech Development Core

技术开发核心

基本信息

批准号：
9129693
负责人：
Peijun Zhang
金额：
$ 27.88万
依托单位：
UNIVERSITY OF PITTSBURGH AT PITTSBURGH
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/9129693
关键词：
Affinity Antibody Affinity Area Behavior Biological Biology Capsid Cell Communication Cells Cellular Membrane Crowding Cryoelectron Microscopy Cytoplasm Cytoskeleton Development Docking Electron Beam Electrons Event Freezing Funding Gold HIV Hybrids Image Imaging technology Infection Ions Label Life Life Cycle Stages Mammalian Cell Membrane Fusion Methodology Methods Microscopic Microscopy Molecular Morphologic artifacts Nuclear Nuclear Import Optics Organelles Peptides Process Proteins Resolution Role Specimen Staging Structure Surface Technology Thick Tissues Tomogram Ultramicrotomy Viral Virus Diseases Work biological systems cellular imaging chemical fixation electron tomography image processing imaging system innovation interest light microscopy live cell imaging live cell microscopy molecular imaging nanometer nanoscale novel novel strategies particle practical application programs protein complex reconstruction single molecule spectrograph tomography tool trafficking

项目摘要

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.

技术新颖性是三倍，i）使用活细胞单个粒子成像，超分辨率的单分子成像以及冷冻成像建立相关的多模式成像系统，而我们先前确定的相关活细胞和冷冻方法可以确定HIV-I蛋白质与宿主细胞因子的相互作用。在成像谱的一端，特定目标在生物学系统的背景可以通过折衷的分辨率来表征，而在另一端，可以获得高分辨率的结构快照。超分辨率分子成像桥接这些方法，并允许鉴定和定位与冷冻结构相关的特定靶蛋白。 2）在玻璃体中产生细胞薄片，类似于YO-粉丝术，但没有切除伪影，是一种新型的方法，是一种新的方法，可以生成适当的薄玻璃体生物学标本，并增强冷冻物在高分辨率的高分辨率3D结构分析的天然细胞和组织中的适用性。 3）使用对金表面高亲和力的短融合肽构成了一种新的方法，以克服开发GFP等效标记EM标签的某些困难。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/冷冻成像）和其他方法的整合（超级分辨率成像）。我们提出的研究以我们先前的工作为基础，并发展了新的能力。