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 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.
Redox Biol. 2020 Jul;34:101549. doi: 10.1016/j.redox.2020.101549. Epub 2020 Apr 27.
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 (pO) 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 pO and coenzyme status monitoring in living cells. Additionally, this method reveals intracellular spatial heterogeneity and cell-to-cell variation in oxygenation and coenzyme states.
氧化还原化学是所有生物体代谢的基础，因此定量分析主要的氧化还原物质对于全面了解正常和病理状态下的细胞代谢非常重要。在哺乳动物细胞中，这可以通过同时测量氧分压（pO）、游离和酶结合的还原型烟酰胺腺嘌呤二核苷酸（磷酸）[H]（NAD（P）H）以及黄素腺嘌呤二核苷酸（FAD，NAD 的替代物）来实现。以前用于这些测量的光学方法存在细胞毒性、速度慢、群体平均和无法同时测量所有氧化还原参数等问题。本文介绍了一种基于Förster 共振能量转移（FRET）的氧传感器 Myoglobin-mCherry，它与基于荧光寿命成像（FLIM）的烟酰胺辅酶状态测量兼容。这为实时、非侵入性、逐个细胞的胞内 pO 和辅酶状态监测提供了代谢活性的实时、非侵入性、逐个细胞的胞内 pO 和辅酶状态监测，同时还揭示了细胞内氧合和辅酶状态的空间异质性和细胞间变异性。
