4.8 Article

Multi-Color, Bleaching-Resistant Super-Resolution Optical Fluctuation Imaging with Oligonucleotide-Based Exchangeable Fluorophores

期刊

ANGEWANDTE CHEMIE-INTERNATIONAL EDITION
卷 60, 期 12, 页码 6310-6313

出版社

WILEY-V C H VERLAG GMBH
DOI: 10.1002/anie.202013166

关键词

DNA-PAINT; fluorescence; photobleaching; SOFI; super-resolution microscopy

资金

  1. Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) [259130777, SFB 1177]
  2. Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germanys Excellence Strategy [EXC 2067/1-390729940]
  3. Projekt DEAL

向作者/读者索取更多资源

SOFI is a super-resolution microscopy technique that analyzes intensity fluctuations to overcome the diffraction limit, and can circumvent fluorophore photobleaching by using reversible and transient fluorophore labels that bind to a target.
Super-resolution optical fluctuation imaging (SOFI) is a super-resolution microscopy technique that overcomes the diffraction limit by analyzing intensity fluctuations of statistically independent emitters in a time series of images. The final images are background-free and show confocality and enhanced spatial resolution (super-resolution). Fluorophore photobleaching, however, is a key limitation for recording long time series of images that will allow for the calculation of higher order SOFI results with correspondingly increased resolution. Here, we demonstrate that photobleaching can be circumvented by using fluorophore labels that reversibly and transiently bind to a target, and which are being replenished from a buffer which serves as a reservoir. Using fluorophore-labeled short DNA oligonucleotides, we labeled cellular structures with target-specific antibodies that contain complementary DNA sequences and record the fluctuation events caused by transient emitter binding. We show that this concept bypasses extensive photobleaching and facilitates two-color imaging of cellular structures with SOFI.

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