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Single-Cell Biochemical Multiplexing by Multidimensional Phasor Demixing and Spectral Fluorescence Lifetime Imaging Microscopy
Authors
A Esposito
MW Fries
KT Haas
AR Venkitaraman
Publication date
27 May 2021
Publisher
'Frontiers Media SA'
Doi
Abstract
Data Availability Statement: The original contributions presented in the study are included in the article/Supplementary Material; further inquiries can be directed to the corresponding author.Copyright © 2021 Haas, Fries, Venkitaraman and Esposito. 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.Gates Foundation studentship; Medical Research Council core grants (MC_UU_12022/1 and MC_UU_12022/8); Wellcome Trust (090340/Z/09/Z); Cancer Research UK (C54674/A27487)
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Last time updated on 05/11/2022