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

项目摘要 超分辨率成像是生物医学领域的一项革命性技术， 在分子水平上以纳米级的精度提供有关细胞结构和功能的信息。然而，在这方面， 复用能力和空间分辨率主要受现有的 荧光标记。我们建议开发一种明亮且可切换的荧光染料调色板，称为 SwitchFluor，用于多路超分辨率成像方法，以同时分辨6个分子靶点， 10分钟内达到10 nm的空间分辨率。我们的目标是同时（1）实现光子发射率 在使用DNA中新设计的增强子链的单分子转换过程的亮态期间， 用于在纳米级形貌（DNA-PAINT）染料系统中成像的点累积，以及（2）同时 使用正交成像链切换6种特别设计光谱不同的SwitchFluor染料的荧光 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。明亮的SwitFluor（DNA-PAINT）染料与sSMLM的整合将共同 实现提出的目标。拟议中的技术可能会对广泛的 生物学研究，因为我们将能够深入到分子尺度研究复杂的细胞过程。

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.