Spectral cross-cumulants for multicolor super-resolved SOFI imaging

M Leutenegger,A Radenovic,K S Grußmayer,T Lasser,T Lukes,S Geissbuehler,A Descloux

doi:10.1038/s41467-020-16841-1

Abstract

Super-resolution optical fluctuation imaging 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 and the sampling can be increased up to n-fold in all spatial dimensions. In this study, we extend the cumulant analysis into the spectral domain and propose a multicolor super-resolution scheme. The simultaneous acquisition of two spectral channels followed by spectral cross-cumulant analysis and unmixing increases 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 experiments in fixed and live cells. Based on an eigenvalue/vector analysis, we propose a scheme for an optimized spectral filter choice. Overall, our methodology provides a route for easy-to-implement multicolor sub-diffraction imaging using standard microscopes while conserving the spatial super-resolution property.

Highlights

Super-resolution optical fluctuation imaging provides a resolution beyond the diffraction limit by analysing stochastic fluorescence fluctuations with higher-order statistics
Appropriate blinking for Super-resolution optical fluctuation imaging (SOFI) processing was achieved using a buffer with thiols and oxygen scavengers; the fluorophores were excited with three different lasers at 488, 561 and 635 nm wavelength and moderate illumination intensities
Spectral cross-correlation leads to the known optical sectioning and background reduction inherent to SOFI analysis[15] and generates the third virtual channel κ2,room temperature (RT) that is dominated by the wheat-germ agglutinin (WGA) signal (Fig. 3b, blue and Supplementary Fig. 11)

Summary

Introduction

Super-resolution optical fluctuation imaging provides a resolution beyond the diffraction limit by analysing stochastic fluorescence fluctuations with higher-order statistics. Superresolution microscopes are only slowly finding their way into routine biological application due to complexity in instrument and sample preparation Overcoming these hurdles with novel schemes may increase the adoption of advanced microscopy techniques. The difficulty in obtaining optimal fluorophore behavior across the spectrum compatible with the constraints imposed by SMLM limits multicolor camera-based nanoscopy at present, i.e. it is problematic to identify suitable fluorophore multiplets Workarounds such as spectrally resolved STORM14 allow the use of several far-red-emitting fluorophores, albeit at the cost of muchincreased hardware and analysis complexity. Super-resolution optical fluctuation imaging (SOFI)[15,16] provides an elegant way of overcoming the diffraction limit in all spatial dimensions[17]. By imaging multiple spectral channels step by step, correlations cannot be used, i.e. the cross-cumulation in between detection channels is not exploited

Methods

Results

Conclusion