Bright and switchable fluorophores for highly multiplexed super-resolution microscopy towards molecular interaction imaging

明亮且可切换的荧光团，用于分子相互作用成像的高度多重超分辨率显微镜

• 批准号: 10195413
• 负责人: Yang Zhang
• 金额: $ 19.96万
• 依托单位: NORTHWESTERN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-01 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10195413
• 关键词: Architecture; Binding; Blinking; Budgets; Cell physiology; Cells; Cellular Structures; Color; Complex; DNA; Detection; Diffuse; Docking; Dyes; Engineering; Enhancers; Event; Family; Fluorescence; Fluorescent Dyes; Fluorescent Probes; Foundations; Goals; Hour; Image; Individual; Kinetics; Label; Lighting; Location; Microscopy; Molecular; Molecular Target; Morphology; Nuclear Pore Complex; Oligonucleotides; Optics; Pattern; Performance; Photochemistry; Photons; Process; Research Personnel; Resolution; Sampling; Science; Speed; System; Techniques; Technology; Time; Triplet Multiple Birth; absorption; base; biological research; design; ethylene glycol; fluorophore; image reconstruction; imager; imaging modality; microscopic imaging; molecular scale; multiplexed imaging; nanometer; nanoscale; optical imaging; optical spectra; single molecule; trafficking; water solubility

PROJECT SUMMARY Super-resolution imaging has been a revolutionary technique for biomedical sciences and provided invaluable information about cellular architecture and function at the molecular level with nanometer precision. However, the multiplexing capability and spatial resolution are mainly constrained by the performance of existing fluorescent labels. We propose to develop a palette of bright and switchable fluorescent dyes, referred to as SwitFluor, for multiplexing super-resolution imaging method to simultaneously resolve 6 molecular targets at 10-nm spatial resolution within 10 minutes. We aim to concurrently (1) achieve ultrahigh photon emission rate during the bright state of single-molecule switching process using a newly designed enhancer strand in DNA point accumulation for imaging in nanoscale topography (DNA-PAINT) dye system, and (2) simultaneously switch fluorescence of 6 specially designed spectrally-distinct SwitFluor dyes using orthogonal imager strands for simultaneous imaging with our recently developed spectroscopic single-molecule localization microscopy (sSMLM) optical system. The integration of bright SwitFluor (DNA-PAINT) dyes with sSMLM will collectively achieve the proposed goal. The proposed technology could make a profound impact on a wide range of biological research as we will be able to study complex cellular processes deep down to the molecular scale.

项目摘要 超分辨率成像一直是生物医学科学的革命性技术，并提供了无价的技术 纳米精度在分子水平上有关细胞结构和功能的信息。然而， 多路复用能力和空间分辨率主要受现有性能的限制 荧光标签。我们建议开发一个明亮可开关的荧光染料的调色板，称为 Switfluor，用于多路复用超分辨率成像方法，以同时解决6个分子靶标。 10 nm的空间分辨率在10分钟内。我们的目标是同时（1）实现超高光子排放率 在单分子切换过程的明亮状态下，使用新设计的增强剂链DNA中 纳米级地形（DNA-Paint）染料系统成像的点积累，以及（2）同时 使用正交成像器链的6种特殊设计的频谱旋转染料的开关荧光 用于与我们最近开发的光谱单分子定位显微镜同时成像 （SSMLM）光学系统。明亮旋流（DNA-Paint）染料与SSMLM的整合将集体 实现拟议的目标。拟议的技术可能会对多种 生物学研究将能够研究复杂的细胞过程，深入到分子量表。