Spectral cross-cumulants for multicolor super-resolved SOFI imaging

Area of Science:

• Optical microscopy
• Super-resolution imaging
• Fluorescence spectroscopy

Background:

• Super-resolution optical fluctuation imaging (SOFI) surpasses the diffraction limit by analyzing fluorescence fluctuations using higher-order statistics.
• Current SOFI methods primarily focus on spatial resolution enhancement, with limited capacity for multicolor imaging.
• Distinguishing multiple fluorophores often requires a number of detection channels equal to the number of species.

Purpose of the Study:

• To extend cumulant analysis into the spectral domain for multicolor super-resolution.
• To develop a scheme for increasing spectral sampling and discriminating multiple fluorophore species.
• To enable easy-to-implement multicolor sub-diffraction imaging with standard microscopes.

Main Methods:

• Extension of spatio-temporal cross-cumulant analysis to the spectral domain.
• Simultaneous acquisition of two spectral channels.
• Spectral cross-cumulant analysis and unmixing for fluorophore identification.
• Eigenvalue/vector analysis for optimized spectral filter selection.

Main Results:

• Demonstrated spectral unmixing of three fluorophore species using two spectral channels in simulations.
• Validated the multicolor imaging approach experimentally in fixed and live cells.
• Achieved enhanced spectral sampling, overcoming the limitation of physical detection channels.

Conclusions:

• The proposed spectral cumulant analysis enables multicolor super-resolution imaging beyond the diffraction limit.
• This methodology allows for the discrimination of more fluorophore species than available detection channels.
• The technique is compatible with standard microscopy setups and preserves spatial super-resolution capabilities.