Multicolor Super-Resolution Imaging

Multicolor super-resolution imaging is usind the simultaneous acquisition of two spectral channels followed by spectral cross-cumulant analysis and unmixing. (Source: EPFL)

Multicolor fluores­cence micro­scopy is an important tool for the life sciences to study the relative arrange­ments of cellular structures or the inter­actions of different proteins. However, conventional micro­scopes, the workhorses for many bio­logical studies, can only resolve details on the order of the wavelength of light. In the past two decades, several super-resolution micro­scopy concepts helped researchers to overcome this diffraction limit and to make new discoveries. These new methods are only slowly finding their way into routine biological appli­cations. For some of the new techniques, this is due to complex micro­scope hardware, but also increased demands on sample pre­paration and fluores­cent labels pose significant hurdles. The require­ments for successful super-resolution imaging are even more challenging to meet for multicolor applications.

EPFL’s Labora­tory of Biomedical Optics headed by Theo Lasser has been exten­sively working on Super-resolution Optical Fluc­tuation Imaging (SOFI) to increase the spatial reso­lution and sampling in 2D and 3D. SOFI is an alter­native to Single-molecule Loca­lization Micro­scopy tech­niques such as STORM and PALM. It analyzes higher order spatio-temporal statistics of a time-series of blinking fluorophores and does not require the isolation of individual fluoro­phores′ emissions. SOFI is compatible with a wider range of labeling and imaging conditions, which simplifies fluoro­phore selection and experi­ments. Now, researchers around Theo Lasser and Alek­sandra Radenovic extended the statis­tical analysis into the spectral domain to pave the way towards a new approach for multi­color super-resolution imaging.

The idea behind the multi­color SOFI approach is the following: In classical multicolor imaging crosstalk between different spectral channels of the micro­scope should be avoided. Here, the researchers exploit the crosstalk for generating addi­tional color channels. They apply cross-cumulant analysis between multiple simul­taneously acquired spectral channels. The statis­tical analysis allows them to supplement the physical detection channels provided by the microscope with addi­tional virtual spectral channels. “Only the signals that are spatially and tempo­rally correlated in the different spectral channels will appear in the virtual channels. We are picking up exactly the crosstalk that everyone else wants to get rid of.” explains Kristin Grußmayer.

The additional computa­tionally generated spectral channels together with linear unmixing allow the imaging of more distinct fluoro­phore colors than recorded physical detection channels. The researchers provide the theory behind spectral cross-cumulant multi­color SOFI and include a framework to optimize the spectral channels of the microscope for a given combi­nation of fluoro­phores that should be imaged. Simulated datasets helped the team to verify that their new multicolor approach should work for a wide range of labels with different photo­physical properties, even for those with strongly over­lapping emission spectra.

“We could show that our approach works for three color imaging in fixed and in living cells for a variety of dyes and fluores­cent proteins. The imaging can be performed using commer­cial widefield setups with two-channel image splitting units that are widely available. In principle, we are not limited to 3 colors.” says Kristin Gruß­mayer. (Source: EPFL)

Reference: K. S. Grußmayer et al.: Spectral cross-cumulants for multicolor super-resolved SOFI imaging, Nat. Commun. 11, 3023 (2020); DOI: 10.1038/s41467-020-16841-1

Link: Laboratory of Nanoscale Biology, École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland

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