Fluorescence lifetime imaging of endogenous biomarker of oxidative stress

Datta, Rupsa; Alfonso-García, Alba; Cinco, Rachel; Gratton, Enrico

doi:10.1038/srep09848

Cited by 114 publications

(144 citation statements)

References 35 publications

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“…Their levels reflect the varying balance of glycolysis and oxidative phosphorylation, depending on environmental signals (availability of oxygen, and/or lactate/pyruvate, the presence of inhibitors or uncouplers, etc) and genetic makeup of cells (Warburg effect) [43,44]. We emphasise that in this work, we interfere with the activity of the mitochondria by using FCCP at such high levels that they, eventually, become inactive leading to cell death (see cell viability data in Table 1), due to increased production of reactive oxygen species (ROS) and other effects [45,46]. A purely chemical effect of NADH/NADPH oxidation, which decreases NADH/NADPH fluorescence, as quantified in Fig.…”

Section: Hyperspectral Imaging and Unmixing Resultsmentioning

confidence: 99%

Fluorescence quenching of free and bound NADH in HeLa cells determined by hyperspectral imaging and unmixing of cell autofluorescence

Rehman¹,

Anwer²,

Gosnell³

et al. 2017

Biomed. Opt. Express

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Carbonyl cyanide-p-trifluoro methoxyphenylhydrazone (FCCP) is a well-known mitochondrial uncoupling agent. We examined FCCP-induced fluorescence quenching of reduced nicotinamide adenine dinucleotide / nicotinamide adenine dinucleotide phosphate (NAD(P)H) in solution and in cultured HeLa cells in a wide range of FCCP concentrations from 50 to 1000µM. A non-invasive label-free method of hyperspectral imaging of cell autofluorescence combined with unsupervised unmixing was used to separately isolate the emissions of free and bound NAD(P)H from cell autofluorescence. Hyperspectral image analysis of FCCP-treated HeLa cells confirms that this agent selectively quenches fluorescence of free and bound NAD(P)H in a broad range of concentrations. This is confirmed by the measurements of average NAD/NADH and NADP/NADPH content in cells. FCCP quenching of free NAD(P)H in cells and in solution is found to be similar, but quenching of bound NAD(P)H in cells is attenuated compared to solution quenching possibly due to a contribution from the metabolic and/or antioxidant response in cells. Chemical quenching of NAD(P)H fluorescence by FCCP validates the results of unsupervised unmixing of cell autofluorescence. Hidalgo, "Ultraviolet excitation fluorescence spectroscopy: a noninvasive method for the measurement of redox changes in ischemic myocutaneous flaps," Plast. Reconstr. Surg. 96(3), 673-680 (1995). 31. J. Eng, R. M. Lynch, and R. S. Balaban, "Nicotinamide adenine dinucleotide fluorescence spectroscopy and imaging of isolated cardiac myocytes," Biophys. J. 55(4), 621-630 (1989). 32. A. S. Galkin, V. G. Grivennikova, and A. D. Vinogradov, "→H+/2e-stoichiometry in NADH-quinone reductase reactions catalyzed by bovine heart submitochondrial particles," FEBS Lett. 451(2), 157-161 (1999). 33. J. P. Brennan, R. G. Berry, M. Baghai, M. R. Duchen, and M. J. Shattock, "FCCP is cardioprotective at concentrations that cause mitochondrial oxidation without detectable depolarisation," Cardiovasc.

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Section: Hyperspectral Imaging and Unmixing Resultsmentioning

confidence: 99%

Fluorescence quenching of free and bound NADH in HeLa cells determined by hyperspectral imaging and unmixing of cell autofluorescence

Rehman¹,

Anwer²,

Gosnell³

et al. 2017

Biomed. Opt. Express

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“…32 The two NADH states define the extremes of the so-called metabolic trajectory in the phasor plot. 33,34 In the cellular cytoplasm, we find both species at different ratios, which yield a lifetime distribution that falls along the metabolic trajectory [ Fig. 2(c), arrowhead].…”

Section: Fluorescence Lifetime Imaging Microscopy Of Free and Bound Nmentioning

confidence: 99%

Label-free identification of macrophage phenotype by fluorescence lifetime imaging microscopy

et al. 2016

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, "Label-free identification of macrophage phenotype by fluorescence lifetime imaging microscopy," J. Abstract. Macrophages adopt a variety of phenotypes that are a reflection of the many functions they perform as part of the immune system. In particular, metabolism is a phenotypic trait that differs between classically activated, proinflammatory macrophages, and alternatively activated, prohealing macrophages. Inflammatory macrophages have a metabolism based on glycolysis while alternatively activated macrophages generally rely on oxidative phosphorylation to generate chemical energy. We employ this shift in metabolism as an endogenous marker to identify the phenotype of individual macrophages via live-cell fluorescence lifetime imaging microscopy (FLIM). We demonstrate that polarized macrophages can be readily discriminated with the aid of a phasor approach to FLIM, which provides a fast and model-free method for analyzing fluorescence lifetime images.

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“…To overcome these limitations, we have applied fluorescence lifetime imaging microscopy (FLIM) (Datta et al, 2015; Lakowicz et al, 1992). FLIM is a non-invasive, high-sensitivity imaging technique that measures mitochondrial metabolism directly in living brain tissues of HdhQ(150/150) animals (Lin et al, 2001).…”

Section: Introductionmentioning

confidence: 99%

Metabolic Reprogramming in Astrocytes Distinguishes Region-Specific Neuronal Susceptibility in Huntington Mice

et al. 2019

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SUMMARY The basis for region-specific neuronal toxicity in Huntington disease is unknown. Here, we show that region-specific neuronal vulnerability is a substrate-driven response in astrocytes. Glucose is low in HdhQ(150/150) animals, and astrocytes in each brain region adapt by metabolically reprogramming their mitochondria to use endogenous, non-glycolytic metabolites as an alternative fuel. Each region is characterized by distinct metabolic pools, and astrocytes adapt accordingly. The vulnerable striatum is enriched in fatty acids, and mitochondria reprogram by oxidizing them as an energy source but at the cost of escalating reactive oxygen species (ROS)-induced damage. The cerebellum is replete with amino acids, which are precursors for glucose regeneration through the pentose phosphate shunt or gluconeogenesis pathways. ROS is not elevated, and this region sustains little damage. While mhtt expression imposes disease stress throughout the brain, sensitivity or resistance arises from an adaptive stress response, which is inherently region specific. Metabolic reprogramming may have relevance to other diseases.

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Fluorescence lifetime imaging of endogenous biomarker of oxidative stress

Cited by 114 publications

References 35 publications

Fluorescence quenching of free and bound NADH in HeLa cells determined by hyperspectral imaging and unmixing of cell autofluorescence

Fluorescence quenching of free and bound NADH in HeLa cells determined by hyperspectral imaging and unmixing of cell autofluorescence

Label-free identification of macrophage phenotype by fluorescence lifetime imaging microscopy

Metabolic Reprogramming in Astrocytes Distinguishes Region-Specific Neuronal Susceptibility in Huntington Mice

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