Ultra-bright Photoactivatable Fluorophores Created by Reductive Caging

Sub-diffraction-limit imaging can be achieved by sequential localization of photoactivatable fluorophores, where the image resolution depends on the number of photons detected per localization. Here, we report a strategy for fluorophore caging that creates photoactivatable probes with high photon yields. Upon photoactivation, these probes can provide 104–106 photons per localization and allow imaging of fixed samples with resolutions of several nanometers. This strategy can be applied to many fluorophores across the visible spectrum. Recent years have witnessed rapid development of super-resolution fluorescence imaging methods which substantially surpass the diffraction limit1,2. These include methods based on patterned illumination, such as stimulated emission depletion (STED and related RESOLFT) microscopy1,3 and saturated structured illumination microscopy (SSIM)4, as well as methods based on single-molecule switching and localization, such as stochastic optical reconstruction microscopy (STORM)5 and (fluorescence) photoactivated localization microscopy ((F)PALM)6,7. Resolutions of 10–100 nm have been achieved when imaging biological samples using these methods, allowing otherwise hidden details of subcellula