It has been some years since super-resolution imaging first contributed to understanding macromolecular complex organization, a subject classically within structural biology. Borrowing particle-averaging methods from cryo-electron microscopy, researchers used single-molecule localization microscopy (SMLM) to map the locations of fluorophore-labeled proteins to the structure of the nuclear pore complex (NPC) (Science 341, 655–658, 2013).

Fluorescence can illuminate protein structure. Credit: Marina Corral Spence/Springer Nature

This work made use of the known symmetry of the pore. Although it was not strictly needed in that case, there remain few if any fluorescence-based structures that do not rely on prior knowledge. Several developments now raise the intriguing possibility that fluorescence may be used increasingly for structural studies.

First, super-resolution approaches are improving in resolution, down to the molecular scale. In the MinFlux method from the group of Stefan Hell, an intensity minimum is used for fluorophore localization, achieving resolution on the low-nanometer scale (Science 355, 606–612, 2017). The group of Vahid Sandogdhar reported Angstrom resolution with SMLM on proteins at cryogenic temperatures (Nat. Methods 14, 141–144, 2017).

Second, CRISPR-based gene editing is making it more possible to fluorescently label all endogenous copies of a protein. Together with brighter probes and smaller affinity reagents, this will help achieve the more complete labeling needed for fluorescence-based structural mapping.

Improved analytical methods to reconstruct structure from fluorescence data will probably also be needed. Methods that do not require structural templates and that are explicitly designed for fluorescence data may prove beneficial.

We predict that super-resolution fluorescence microscopy will continue to brighten and elucidate biological structure, even down to the molecular scale.