Photon yield enhancement of red fluorophores at cryogenic temperatures

Single Molecule Localization Microscopy has become one of the most successful and widely applied methods of Super‐resolution Fluorescence Microscopy. Its achievable resolution strongly depends on the number of detectable photons from a single molecule until photobleaching. By cooling a sample from room temperature down to liquid nitrogen temperatures, the photostability of dyes can be enhanced by more than 100 fold, which results in an improvement in localization precision greater than 10 times. Here, we investigate a variety of fluorescent dyes in the red spectral region, and we find an average photon yield between 3.5 ⋅ 106 to 11 ⋅ 106 photons before bleaching at liquid nitrogen temperatures, corresponding to a theoretical localization precision around 0.1 nm.