Mosaic-PICASSO: accurate crosstalk removal for multiplex fluorescence imaging

Ultra-multiplexed fluorescence imaging has revolutionized our understanding of biological systems, enabling the simultaneous visualization and quantification of multiple targets within biological specimens. A recent breakthrough in this field is PICASSO, a mutual-information-based technique capable of demixing up to 15 fluorophores without their spectra, thereby significantly simplifying the application of ultra-multiplexed fluorescence imaging. However, this study has identified a limitation of mutual information-based techniques. They do not differentiate between spatial colocalization and spectral mixing. Consequently, mutual information-based demixing may incorrectly interpret spatially co-localized targets as non-colocalized, leading to overcorrection. We found that selecting regions within a multiplex image with low spatial similarity for measuring spectroscopic mixing results in more accurate demixing. This method effectively minimizes overcorrections and promises to accelerate the broader adoption of ultra-multiplex imaging.