Three-Dimensional Angstrom Resolution In Fluorescence Microscopy: Insight Into Protein Structure

The achievements of super-resolution microscopy beyond the diffraction barrier were honored by the Nobel Prize in Chemistry in 2014. One of the common super-resolution approaches employs pinpointing the position of single fluorophores, via localizing the centers of the point-spread function from each individual label molecule. In this method, the fluorophore location can be determined with arbitrary precision depending on the attainable signal-to-noise ratio. Photobleaching limits the signal at room temperature so that typical localization precisions do not go beyond the order of ten nanometers. To improve on this, we exploit the enhanced photostability of molecules at cryogenic temperatures [1]. Two years ago, we demonstrated the feasibility of colocalization and cryogenic distance measurements by resolving two fluorophores on the backbone of a double-stranded DNA at a separation of several nanometers [2]. Here, we extend this technique to resolving the positions of multiple fluorophores attached to a single protein with Angstrom resolution [3].