HyU: Hybrid Unmixing for longitudinal in vivo imaging of low signal-to-noise fluorescence

Hybrid Unmixing offers enhanced imaging of multiplexed fluorescence labels, enabling longitudinal imaging of multiple fluorescent signals with reduced illumination intensities. The expansion of fluorescence bioimaging toward more complex systems and geometries requires analytical tools capable of spanning widely varying timescales and length scales, cleanly separating multiple fluorescent labels and distinguishing these labels from background autofluorescence. Here we meet these challenging objectives for multispectral fluorescence microscopy, combining hyperspectral phasors and linear unmixing to create Hybrid Unmixing (HyU). HyU is efficient and robust, capable of quantitative signal separation even at low illumination levels. In dynamic imaging of developing zebrafish embryos and in mouse tissue, HyU was able to cleanly and efficiently unmix multiple fluorescent labels, even in demanding volumetric timelapse imaging settings. HyU permits high dynamic range imaging, allowing simultaneous imaging of bright exogenous labels and dim endogenous labels. This enables coincident studies of tagged components, cellular behaviors and cellular metabolism within the same specimen, providing more accurate insights into the orchestrated complexity of biological systems.

Automated summary

Hybrid Unmixing enables enhanced imaging of multiplexed fluorescence labels by reducing illumination intensities. It can cleanly separate and distinguish multiple fluorescent labels from background autofluorescence, enabling dynamic imaging in complex systems. HyU permits high dynamic range imaging, allowing simultaneous imaging of bright exogenous labels and dim endogenous labels, providing more accurate insights into the complexity of biological systems.