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

项目总结 超分辨率成像已经成为生物医学科学的一项革命性技术，并提供了无价的 关于分子水平上的细胞结构和功能的信息，具有纳米精度。然而， 多路复用能力和空间分辨率主要受现有网络性能的制约 荧光标签。我们建议开发一种明亮且可切换的荧光染料调色板，称为 SwitFluor，用于同时分辨6个分子靶的多路超分辨率成像方法 10分钟内的10纳米空间分辨率。我们的目标是同时(1)实现超高的光子发射率 利用DNA中新设计的增强子链在单分子开关过程中的亮态 用于纳米级形貌(DNA-PAINT)染料体系成像的点积累，以及(2)同时 用正交成像仪对6种特殊设计的光谱不同的开关荧光染料的研究 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。明亮的开关荧光(DNA-PAINT)染料与sSMLM的集成将共同完成 实现所提出的目标。这项拟议的技术可能会对广泛的 生物学研究，因为我们将能够研究深入到分子尺度的复杂细胞过程。

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.