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Seeing what one cell component is up to in a tissue can be like trying to follow one person in a crowd of thousands. That’s why researchers turn to fluorescence microscopy where cell components are labelled with fluorescent tags to make them stand out. However, the more different fluorescent signals you have, the harder it is to separate them out. The natural fluorescence produced by unlabelled tissue (background autofluorescence) also muddies the waters. Researchers now present a solution: Hybrid Unmixing (HyU), which combines hardware, hyperspectral phasors, and a mathematical technique, linear unmixing. HyU separated out different fluorescent signals, even with little illumination, as shown in developing zebrafish genetically engineered with four different fluorescent tags (pictured, all merged top left). HyU also allowed simultaneous imaging of bright fluorescent tag signals and dim autofluorescence signals generated by cell metabolism. This highlights its usefulness in capturing a full spectrum of signals for complex investigations.

Written by Lux Fatimathas

Lux Fatimathas

Writer

Having pursued science through a career in research, communications and publishing, she is currently a writer and editor for a healthcare marketing agency and also juggles freelance science writing.

• Image from work by Hsiao Ju Chiang and Daniel E.S. Koo, and colleagues
• Translational Imaging Center, University of Southern California, Los Angeles, CA, USA
• Image originally published with a Creative Commons Attribution 4.0 International (CC BY 4.0)
• Published in Nature Methods, January 2023