Single cell-based fluorescence lifetime imaging of intracellular oxygenation and metabolism.

Abstract:

:Oxidation-reduction chemistry is fundamental to the metabolism of all living organisms, and hence quantifying the principal redox players is important for a comprehensive understanding of cell metabolism in normal and pathological states. In mammalian cells, this is accomplished by measuring oxygen partial pressure (pO2) in parallel with free and enzyme-bound reduced nicotinamide adenine dinucleotide (phosphate) [H] (NAD(P)H) and flavin adenine dinucleotide (FAD, a proxy for NAD+). Previous optical methods for these measurements had accompanying problems of cytotoxicity, slow speed, population averaging, and inability to measure all redox parameters simultaneously. Herein we present a Förster resonance energy transfer (FRET)-based oxygen sensor, Myoglobin-mCherry, compatible with fluorescence lifetime imaging (FLIM)-based measurement of nicotinamide coenzyme state. This offers a contemporaneous reading of metabolic activity through real-time, non-invasive, cell-by-cell intracellular pO2 and coenzyme status monitoring in living cells. Additionally, this method reveals intracellular spatial heterogeneity and cell-to-cell variation in oxygenation and coenzyme states.

journal_name

Redox Biol

journal_title

Redox biology

authors

Penjweini R,Roarke B,Alspaugh G,Gevorgyan A,Andreoni A,Pasut A,Sackett DL,Knutson JR

doi

10.1016/j.redox.2020.101549

subject

Has Abstract

pub_date

2020-07-01 00:00:00

pages

101549

issn

2213-2317

pii

S2213-2317(20)30347-5

journal_volume

34

pub_type

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