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
Affiliations
Rozhin Penjweini
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
Branden Roarke
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
Greg Alspaugh
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
Anahit Gevorgyan
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
Alessio Andreoni
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
Alessandra Pasut
Laboratory of Angiogenesis and Vascular Metabolism, Center for Cancer Biology, VIB, Leuven Cancer Institute, KU Leuven, Leuven, 3000, Belgium
Dan L. Sackett
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
Jay R. Knutson
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; Corresponding author.
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.
