Multicolor Super-Resolution Imaging with Photo-Switchable Fluorescent Probes

TLDR

Far‑field optical nanoscopy now achieves 20–50 nm resolution, yet multicolor super‑resolution imaging remains difficult. We aim to develop photo‑switchable probes that enable multicolor STORM imaging. The probes pair a photo‑switchable reporter fluorophore with an activator; combinatorial reporter–activator combinations produce many colors, and iterative, color‑specific activation of sparse subsets allows nanometer‑accurate localization to construct STORM images. We achieved 20–30 nm resolution multicolor imaging of DNA models and mammalian cells, enabling direct visualization of molecular interactions at the nanometer scale.

Abstract

Recent advances in far-field optical nanoscopy have enabled fluorescence imaging with a spatial resolution of 20 to 50 nanometers. Multicolor super-resolution imaging, however, remains a challenging task. Here, we introduce a family of photo-switchable fluorescent probes and demonstrate multicolor stochastic optical reconstruction microscopy (STORM). Each probe consists of a photo-switchable "reporter" fluorophore that can be cycled between fluorescent and dark states, and an "activator" that facilitates photo-activation of the reporter. Combinatorial pairing of reporters and activators allows the creation of probes with many distinct colors. Iterative, color-specific activation of sparse subsets of these probes allows their localization with nanometer accuracy, enabling the construction of a super-resolution STORM image. Using this approach, we demonstrate multicolor imaging of DNA model samples and mammalian cells with 20- to 30-nanometer resolution. This technique will facilitate direct visualization of molecular interactions at the nanometer scale.