AUTHOR=Haas Kalina T. , Fries Maximilian W. , Venkitaraman Ashok R. , Esposito Alessandro TITLE=Single-Cell Biochemical Multiplexing by Multidimensional Phasor Demixing and Spectral Fluorescence Lifetime Imaging Microscopy JOURNAL=Frontiers in Physics VOLUME=Volume 9 - 2021 YEAR=2021 URL=https://www.frontiersin.org/journals/physics/articles/10.3389/fphy.2021.637123 DOI=10.3389/fphy.2021.637123 ISSN=2296-424X ABSTRACT=

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