Ultra-stable, photon-efficient cryogenic super-resolution fluorescence imaging for visualizing vitrified biological samples with molecular-scale resolution

超稳定、光子效率高的低温超分辨率荧光成像，用于以分子级分辨率可视化玻璃化生物样品

• 批准号: 10510195
• 负责人: Alexandros Pertsinidis
• 金额: $ 21.58万
• 依托单位: SLOAN-KETTERING INST CAN RESEARCH
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-20 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10510195
• 关键词: 3-Dimensional; Area; Biochemistry; Biological; Biological Process; Biomedical Research; Cell physiology; Cells; Cellular biology; Characteristics; Collection; Complex; Complex Mixtures; Crowding; Cryo-electron tomography; Cryoelectron Microscopy; Data; Data Set; Detection; Development; Developmental Biology; Electron Microscopy; Environment; Fluorescence; Fluorescence Microscopy; Goals; Image; Imaging Techniques; Immunology; In Situ; Individual; Investigation; Ligands; Light; Maps; Mechanics; Methods; Microfilaments; Microscopy; Microtubules; Modality; Molecular; Molecular Analysis; Molecular Conformation; Molecular Structure; Molecular Target; Morphologic artifacts; Neurosciences; Noise; Nuclear Pore; Photons; Population; Process; Proteins; Resolution; Sampling; Shapes; Signal Transduction; Specificity; Specimen; Structure; Techniques; Temperature; Time; Work; base; cryogenics; cryostat; data acquisition; electron tomography; fluorescence imaging; imaging capabilities; imaging modality; imaging platform; instrument; interest; lens; light microscopy; macromolecular assembly; macromolecule; microscopic imaging; molecular scale; multicatalytic endopeptidase complex; nanometer; nanometer resolution; particle; prevent; prototype; single molecule; structural biology; vibration

ABSTRACT Cryogenic electron microscopy (CryoEM) and cryo-electron tomography (CryoET) are powerful techniques for visualizing macromolecular assemblies at a near-native state and in their functional context inside cells; however the undiscriminating/non-specific contrast of such techniques often complicates identifying regions of interest and specific molecular targets in high-resolution cryoEM/cryoET datasets. Super-resolution (SR) fluorescence methods have exquisite molecular specificity, but SR techniques have only achieved limited spatial resolutions (~20-60 nm FWHM) when imaging vitrified biological samples at cryogenic temperatures. The proposed project will create new ultra-stable, photon efficient cryogenic SR methods for imaging vitrified biological specimens with true molecular-scale resolution (<2-3 nm FWHM), closing the gap towards the (sub-)nanometer resolution of cryoEM/cryoET. The increased spatial resolution will be leveraged for further development of powerful correlative light-electron microscopy (CLEM) approaches that will ultimately enable elucidating the structural basis for many critical macromolecular processes inside cells.

抽象的 低温电子显微镜 (CryoEM) 和冷冻电子断层扫描 (CryoET) 功能强大 在接近天然状态下可视化大分子组装体的技术 细胞内的功能环境；然而，这种不加区别/非特定的对比 技术通常使识别感兴趣区域和特定分子靶标变得复杂 高分辨率 CryoEM/cryoET 数据集。超分辨率 (SR) 荧光方法具有 精细的分子特异性，但 SR 技术仅实现了有限的空间分辨率 (~20-60 nm FWHM) 在低温下对玻璃化生物样品进行成像时。这 拟议项目将创建新的超稳定、光子高效的低温 SR 成像方法 具有真正分子尺度分辨率（<2-3 nm FWHM）的玻璃化生物标本，关闭了 与cryoEM/cryoET 的（亚）纳米分辨率之间的差距。空间分辨率提高 将用于进一步开发强大的相关光电子显微镜 （CLEM）方法最终将能够阐明许多关键的结构基础 细胞内的大分子过程。