Single-Cell Biochemical Multiplexing by Multidimensional Phasor Demixing and Spectral Fluorescence Lifetime Imaging Microscopy

Revealing mechanisms underpinning cell function requires understanding the relationship between different biochemical reactions in living cells. However, our capabilities to monitor more than two cells are limited. Therefore, development of methods for real-time multiplexing is fundamental importance. Here, we show that data acquired with multicolor (mcFLIM) or spectrally resolved (sFLIM) fluorescence lifetime imaging can be conveniently described multidimensional phasor transforms. We demonstrate a computational framework capable demixing three Forster resonance energy transfer (FRET) probes and quantifying multiplexed activities single provide comparison mcFLIM sFLIM suggesting might advantageous future heavily assays. mcFLIM—more readily available commercial systems—can applied concomitant monitoring enzymes without significant losses.