Current challenges in cryogenic super-resolution fluorescence microscopy in the context of cryogenic electron microscopy/tomography

Correlating fluorescence information with cryogenic electron microscopy/tomography (cryo-EM/ET) allows directly linking functional and structural information. The development of cryogenic super-resolution fluorescence microscopy (cryo-SR-FM) has elevated this method to a new level by bridging the large resolution gap between conventional cryogenic light and electron microscopy. However, many methodological challenges still have not been fully overcome. In this review article, we will discuss the current state of cryo-SR-FM in the context of correlative workflows with cryo-EM/ET. We focus on recent developments in the field of cryogenic single molecule localization microscopy as this methodology currently shows the largest potential in utilizing fluorescent signals as annotations in cryo-EM/ET. Specifically, we discuss the areas of avoiding devitrification of the specimen, better understanding of single molecule cryo-photophysics and the application of cryo-SR-FM in the single-particle imaging field.