Self-blinking Dyes unlock High-order and Multi-plane Super-resolution Optical Fluctuation Imaging

TL;DR

This paper introduces a super-resolution imaging technique using self-blinking dyes combined with SOFI, enabling high-resolution, multi-plane, and live-cell compatible imaging with low laser power.

Contribution

The work demonstrates the integration of self-blinking dyes with SOFI for 2D and 3D super-resolution imaging, including volumetric and time-resolved applications, with improved suitability for live-cell studies.

Findings

Achieved 50-60 nm resolution in 2D imaging.

Extended SOFI to 4th order for multiplane volumetric imaging.

Enabled live-cell imaging with low laser power.

Abstract

Diffraction unlimited super-resolution imaging critically depends on the switching of fluorophores between at least two states, often induced using intense laser light and special buffers. The high illumination power or UV light required for appropriate blinking kinetics is currently hindering live-cell experiments. Recently, so-called self-blinking dyes that switch spontaneously between an open, fluorescent "on"-state and a closed colorless "off"-state were introduced. Here we exploit the synergy between super-resolution optical fluctuation imaging (SOFI) and spontaneously switching fluorophores for 2D functional and for volumetric imaging. SOFI tolerates high labeling densities, on-time ratios, and low signal-to-noise by analyzing higher-order statistics of a few hundred to thousand frames of stochastically blinking fluorophores. We demonstrate 2D imaging of fixed cells with a uniform resolution up to 50-60 nm in 6th order SOFI and characterize changing experimental conditions. We extend multiplane cross-correlation analysis to 4th order using biplane and 8-plane volumetric imaging achieving up to 29 (virtual) planes. The low laser excitation intensities needed for self-blinking SOFI are ideal for live-cell imaging. We show proof-of-principal time-resolved imaging by observing slow membrane movements in cells. Self-blinking SOFI provides a route for easy-to-use 2D and 3D high-resolution functional imaging that is robust against artefacts and suitable for live-cell imaging.