Reductive Caging enables Ultra-Bright Photoactivatable Fluorophores for Superresolution Imaging

Abstract

From GFP to intracellular sensor dyes, developments in fluorescent probes have been major driving forces in imaging-based biological research. Recently, photoswitchable fluorophores have enabled super-resolution imaging based on the sequential localization of individual fluorescent molecules (STORM, PALM, etc.), such that researchers may now use widely available commercial instrumentation to study biological structures at ∼20-50 nm distance scales.We report a chemical strategy for fluorophore caging by reduction that creates photoactivatable fluorescent probes with ultrahigh photon yields. The caging process is achieved simply and rapidly by treating a labeled sample with an aqueous reducing agent to convert the fluorophores to a long-lived reduced and nonfluorescent form. Upon photoactivation, these probes can provide up to 1,700,000 detected photons per photoswitching event and allow localization precision as high as 1-2 nm. The photon yield is 2-3 orders of magnitude higher than previously available photoswitchable/photoactivable probes. The improved image resolution allows biomolecular structures previously unobservable by super-resolution fluorescence microscopy to be resolved now. The novel reductive caging and photoactivation method is broadly applicable to many fluorophores spanning the visible spectrum.