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.
Highlights
Despite significant progress, high-speed live-cell super-resolution studies remain limited to specialized optical setups, generally requiring intense phototoxic illumination
super-resolution radial fluctuations (SRRF) assumes the image is formed of point sources convolved with a point spread function (PSF) that displays a higher degree of local symmetry than the background
This enhancement is based on two concepts. (a) Noise-induced radiality peaks are uncorrelated in time, a pixel-wise temporal correlation at their location will approach zero as the number of imaged frames increases. (b) The highest degree of temporal correlation is located at the centre of the radiality peaks generated by fluorophore signal
Summary
High-speed live-cell super-resolution studies remain limited to specialized optical setups, generally requiring intense phototoxic illumination. 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. Recent years have seen considerable focus put on adapting PALM- and STORM-like approaches to allow live-cell nanoscopy These single-molecule localization microscopy (SMLM) approaches circumvent Abbe’s diffraction limit through the acquisition of a large sequence of frames (typically thousands), each containing a small population of transiently emitting non-overlapping fluorophores. We present a novel analytical approach, super-resolution radial fluctuations (SRRF), which follows similar principles to deconSTORM, 3B and SOFI, where analysis of a sequence of images acquired in a standard widefield or total internal reflection fluorescence (TIRF) microscope directly generates a superresolution reconstruction without fluorophore detection and localization. Super-resolution information at a temporal resolution of 1 s and a spatial resolution down to 60 nm can be extracted from live samples using conventional fluorophores, low-intensity illumination and as few as 100 raw frames
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