Fast live-cell conventional fluorophore nanoscopy with ImageJ through super-resolution radial fluctuations

TLDR

High‑speed live‑cell super‑resolution imaging is still constrained to specialized setups that require intense, phototoxic illumination. The authors introduce super‑resolution radial fluctuations (SRRF), a fast GPU‑enabled ImageJ plugin for live‑cell imaging. SRRF analyzes radial intensity fluctuations to reconstruct super‑resolution images, leveraging GPU acceleration within ImageJ. SRRF delivers sub‑150 nm resolution in low‑illumination GFP live‑cell data, matches PALM/STORM performance on high‑signal datasets, and enables continuous imaging over minutes to hours with widefield, confocal, or TIRF microscopes at illumination levels orders of magnitude lower than conventional methods.

Abstract

Despite significant progress, high-speed live-cell super-resolution studies remain limited to specialized optical setups, generally requiring intense phototoxic illumination. Here, we describe a new analytical approach, super-resolution radial fluctuations (SRRF), provided as a fast graphics processing unit-enabled ImageJ plugin. In the most challenging data sets for super-resolution, such as those obtained in low-illumination live-cell imaging with GFP, we show that SRRF is generally capable of achieving resolutions better than 150 nm. Meanwhile, for data sets similar to those obtained in PALM or STORM imaging, SRRF achieves resolutions approaching those of standard single-molecule localization analysis. The broad applicability of SRRF and its performance at low signal-to-noise ratios allows super-resolution using modern widefield, confocal or TIRF microscopes with illumination orders of magnitude lower than methods such as PALM, STORM or STED. We demonstrate this by super-resolution live-cell imaging over timescales ranging from minutes to hours.