Cryogenic Colocalization Microscopy for Nanometer-Distance Measurements

The main limiting factor in spatial resolution of localization microscopy is the number of detected photons. Recently we showed that cryogenic measurements improve the photostability of fluorophores, giving access to Angstrom precision in localization of single molecules. Here, we extend this method to colocalize two fluorophores attached to well‐defined positions of a double‐stranded DNA. By measuring the separations of the fluorophore pairs prepared at different design positions, we verify the feasibility of cryogenic distance measurement with sub‐nanometer accuracy. We discuss the important challenges of our method as well as its potential for further improvement and various applications. With Angstrom accuracy: A cryogenic single‐molecule colocalization technique for identifying two fluorescent labels on the backbone of a double‐stranded DNA is demonstrated (see picture). By measuring the separations between fluorophore pairs placed at various design distances in the nanometer range, the authors verify the feasibility of their method for intramolecular distance measurements with Angstrom accuracy.