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

Abstract The expanded application of fluorescence imaging in biomedical and biological research towards more complex systems and geometries requires tools that can analyze a multitude of components at widely varying time- and length-scales. The major challenge in such complex imaging experiments is to cleanly separate multiple fluorescent labels with overlapping spectra from one another and background autofluorescence, without perturbing the sample with high levels of light. Thus, there is a requirement for efficient and robust analysis tools capable of quantitatively separating these signals. In response, we have combined multispectral fluorescence microscopy with hyperspectral phasors and linear unmixing to create Hybrid Unmixing (HyU). Here we demonstrate its capabilities in the dynamic imaging of multiple fluorescent labels in live, developing zebrafish embryos. HyU is more sensitive to low light levels of fluorescence compared to conventional linear unmixing approaches, permitting better multiplexed volumetric imaging over time, with less bleaching. HyU can also simultaneously image both bright exogenous and dim endogenous labels because of its high dynamic range. This allows studies of cellular behaviors, tagged components, and cell metabolism within the same specimen, offering a powerful window into the orchestrated complexity of biological systems.