Photoswitching Fingerprint Analysis Bypasses the 10-nm Resolution Barrier

Overview

Journal Nat Methods

Specialties Biomedical Engineering
Pathology

Date 2022 Aug 1

PMID 35915194

Authors

Dominic A Helmerich

Gerti Beliu

Danush Taban

Mara Meub

Marcel Streit

Alexander Kuhlemann

Soren Doose

Markus Sauer

Affiliations

Soon will be listed here.

Abstract

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.

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