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

Rozhin Penjweini; Branden Roarke; Greg Alspaugh; Anahit Gevorgyan; Alessio Andreoni; Alessandra Pasut; Dan L Sackett; Jay R Knutson

doi:10.1016/j.redox.2020.101549

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

Redox Biol. 2020 Jul:34:101549. doi: 10.1016/j.redox.2020.101549. Epub 2020 Apr 27.

Authors

Rozhin Penjweini¹, Branden Roarke¹, Greg Alspaugh¹, Anahit Gevorgyan¹, Alessio Andreoni², Alessandra Pasut³, Dan L Sackett⁴, Jay R Knutson⁵

Affiliations

¹ Laboratory of Advanced Microscopy and Biophotonics, National Heart, Lung, and Blood Institute (NHLBI), National Institutes of Health (NIH), Building 10, Room 5D14, Bethesda, MD, 20892-1412, USA.
² Laboratory of Advanced Microscopy and Biophotonics, National Heart, Lung, and Blood Institute (NHLBI), National Institutes of Health (NIH), Building 10, Room 5D14, Bethesda, MD, 20892-1412, USA; Laboratory of Optical Neurophysiology, Department of Biochemistry and Molecular Medicine, University of California Davis, Tupper Hall, Davis, CA, 95616, USA.
³ Laboratory of Angiogenesis and Vascular Metabolism, Center for Cancer Biology, VIB, Leuven Cancer Institute, KU Leuven, Leuven, 3000, Belgium.
⁴ Cytoskeletal Dynamics Group, Division of Basic and Translational Biophysics, Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD), National Institutes of Health (NIH), Building 9, Room 1E129, Bethesda, MD, 20892-0924, USA.
⁵ Laboratory of Advanced Microscopy and Biophotonics, National Heart, Lung, and Blood Institute (NHLBI), National Institutes of Health (NIH), Building 10, Room 5D14, Bethesda, MD, 20892-1412, USA. Electronic address: knutsonj@nhlbi.nih.gov.

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.

Keywords: FAD; FLIM; FLIRR; Free and bound NAD(P)H; Myo-mCherry; ORR.

Published by Elsevier B.V.

Publication types

Research Support, N.I.H., Intramural

MeSH terms

Animals
Flavin-Adenine Dinucleotide* / metabolism
NAD / metabolism
NADP / metabolism
Optical Imaging*
Oxidation-Reduction

Substances

NAD
Flavin-Adenine Dinucleotide
NADP