Spectral Cross-Cumulants for Multicolor Super-resolved SOFI Imaging

TL;DR

This paper introduces a spectral cross-cumulant analysis method for multicolor super-resolution imaging in SOFI, enabling simultaneous multi-fluorophore imaging with enhanced spectral sampling and resolution beyond the diffraction limit.

Contribution

The study extends SOFI cumulant analysis into the spectral domain, allowing for multicolor super-resolution imaging with spectral unmixing beyond the number of physical channels.

Findings

Successfully demonstrated spectral unmixing of three fluorophores.

Enhanced spectral sampling improves fluorophore discrimination.

Method compatible with standard microscopes for multicolor super-resolution.

Abstract

Super-resolution optical fluctuation imaging (SOFI) provides a resolution beyond the diffraction limit by analysing stochastic fluorescence fluctuations with higher-order statistics. Using nth order spatio-temporal cross-cumulants the spatial resolution as well as the sampling can be increased up to n-fold in all three spatial dimensions. In this study, we extend the cumulant analysis into the spectral domain and propose a novel multicolor super-resolution scheme. The simultaneous acquisition of two spectral channels followed by spectral cross-cumulant analysis and unmixing increase the spectral sampling. The number of discriminable fluorophore species is thus not limited to the number of physical detection channels. Using two color channels, we demonstrate spectral unmixing of three fluorophore species in simulations and multiple experiments with different cellular structures, fluorophores and filter sets. Based on an eigenvalue/ vector analysis we propose a scheme for an optimized spectral filter choice. Overall, our methodology provides a novel route for easy-to-implement multicolor sub-diffraction imaging using standard microscopes while conserving the spatial super-resolution property. This makes simultaneous multiplexed super-resolution fluorescence imaging widely accessible to the life science community interested to probe colocalization between two or more molecular species.