Journal
ANGEWANDTE CHEMIE-INTERNATIONAL EDITION
Volume 60, Issue 12, Pages 6310-6313Publisher
WILEY-V C H VERLAG GMBH
DOI: 10.1002/anie.202013166
Keywords
DNA-PAINT; fluorescence; photobleaching; SOFI; super-resolution microscopy
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Funding
- Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) [259130777, SFB 1177]
- Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germanys Excellence Strategy [EXC 2067/1-390729940]
- Projekt DEAL
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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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