Fast Super-Resolution Imaging with Ultra-High Labeling Density Achieved by Joint Tagging Super-Resolution Optical Fluctuation Imaging (JT-SOFI)

TL;DR

This paper introduces JT-SOFI, a super-resolution imaging method that uses joint tagging with multiple fluorescent dyes to achieve ultra-high labeling density, enabling fast, high-fidelity imaging of microtubules at sub-diffraction resolution.

Contribution

The paper presents a novel joint tagging super-resolution technique combining spectral separation and fluorescence fluctuation analysis to improve imaging speed and fidelity.

Findings

Achieved high-density super-resolution imaging of microtubules.

Reduced frame number needed for super-resolution imaging.

Enhanced contrast and continuity of microtubule structures.

Abstract

Previous stochastic localization-based super-resolution techniques are largely limited by the labeling density and the fidelity to the morphology of specimen. We report on an optical super-resolution imaging scheme implementing joint tagging using multiple fluorescent blinking dyes associated with super-resolution optical fluctuation imaging (JT-SOFI), achieving ultra-high labeling density super-resolution imaging. To demonstrate the feasibility of JT-SOFI, quantum dots with different emission spectra were jointly labeled to the tubulin in COS7 cells, creating ultra-high density labeling. After analyzing and combining the fluorescence intermittency images emanating from spectrally resolved quantum dots, the microtubule networks are capable of being investigated with high fidelity and remarkably enhanced contrast at sub-diffraction resolution. The spectral separation also significantly decreased the frame number required for SOFI, enabling fast super-resolution microscopy through simultaneous data acquisition. As the joint-tagging scheme can decrease the labeling density in each spectral channel, we can faithfully reflect the continuous microtubule structure with high resolution through collection of only 100 frames per channel. The improved continuity of the microtubule structure is quantitatively validated with image skeletonization, thus demonstrating the advantage of JT-SOFI over other localization-based super-resolution methods.