Photoswitching fingerprint analysis bypasses the 10-nm resolution barrier

Energy transfer between fluorophores is shown to impede SMLM at sub-10-nm spatial resolution. Time-resolved detection and photoswitching fingerprinting analysis are used to determine the number and separation of closely spaced fluorophores. Advances in super-resolution microscopy have demonstrated single-molecule localization precisions of a few nanometers. However, translation of such high localization precisions into sub-10-nm spatial resolution in biological samples remains challenging. Here we show that resonance energy transfer between fluorophores separated by less than 10 nm results in accelerated fluorescence blinking and consequently lower localization probabilities impeding sub-10-nm fluorescence imaging. We demonstrate that time-resolved fluorescence detection in combination with photoswitching fingerprint analysis can be used to determine the number and distance even of spatially unresolvable fluorophores in the sub-10-nm range. In combination with genetic code expansion with unnatural amino acids and bioorthogonal click labeling with small fluorophores, photoswitching fingerprint analysis can be used advantageously to reveal information about the number of fluorophores present and their distances in the sub-10-nm range in cells.

Automated summary

This study demonstrates the use of time-resolved detection and photoswitching fingerprinting analysis to determine the number and distance of closely spaced fluorophores within the sub-10-nm range. The study also reveals the impact of resonance energy transfer between fluorophores on the localization probabilities of sub-10-nm fluorescence imaging.