Optical Imaging Using Endogenous Contrast to Assess Metabolic State

Georgakoudi, Irene; Quinn, Kyle P.

doi:10.1146/annurev-bioeng-071811-150108

Cited by 289 publications

(329 citation statements)

References 70 publications

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“…Islet autofluorescence from NAD(P)H and flavin adenine dinucleotide (FAD) was monitored, and the NAD(P)H/ FAD redox ratio was calculated (17,18). The experimental setup was the same as described below for measurements of [Ca 2+ ] i , but without loading with any indicator.…”

Section: Mitochondrial Membrane Potential and Nad(p)h/flavin Adenine mentioning

confidence: 99%

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Defects in β-Cell Ca2+ Dynamics in Age-Induced Diabetes

Trifunović

Köhler

et al. 2014

Diabetes

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Little is known about the molecular mechanisms underlying age-dependent deterioration in β-cell function. We now demonstrate that age-dependent impairment in insulin release, and thereby glucose homeostasis, is associated with subtle changes in Ca2+ dynamics in mouse β-cells. We show that these changes are likely to be accounted for by impaired mitochondrial function and to involve phospholipase C/inositol 1,4,5-trisphosphate–mediated Ca2+ mobilization from intracellular stores as well as decreased β-cell Ca2+ influx over the plasma membrane. We use three mouse models, namely, a premature aging phenotype, a mature aging phenotype, and an aging-resistant phenotype. Premature aging is studied in a genetically modified mouse model with an age-dependent accumulation of mitochondrial DNA mutations. Mature aging is studied in the C57BL/6 mouse, whereas the 129 mouse represents a model that is more resistant to age-induced deterioration. Our data suggest that aging is associated with a progressive decline in β-cell mitochondrial function that negatively impacts on the fine tuning of Ca2+ dynamics. This is conceptually important since it emphasizes that even relatively modest changes in β-cell signal transduction over time lead to compromised insulin release and a diabetic phenotype.

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Section: Mitochondrial Membrane Potential and Nad(p)h/flavin Adenine mentioning

confidence: 99%

“…2A-C). We next measured glucose-stimulated changes in endogenous NAD(P)H/FAD autofluorescence, reflecting the islet cellular redox ratio (17,18,24). After glucose stimulation, islets responded with an initial rapid rise, followed by a sustained elevation in NAD(P)H/FAD ( Fig.…”

Section: Aging Impairs Mitochondrial Functionmentioning

confidence: 99%

Defects in β-Cell Ca2+ Dynamics in Age-Induced Diabetes

Trifunović

Köhler

et al. 2014

Diabetes

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“…The endpoints of OMI include the redox ratio, NAD(P)H fluorescence lifetime, FAD fluorescence lifetime, and a combination variable, the OMI index. The redox ratio is the intensity of NAD(P)H fluorescence relative to the intensity of FAD fluorescence and provides relative information on the global metabolism of the cell [7,8]. The redox ratio is sensitive to shifts in metabolic pathways [7,9].…”

Section: Introductionmentioning

confidence: 99%

“…The redox ratio is the intensity of NAD(P)H fluorescence relative to the intensity of FAD fluorescence and provides relative information on the global metabolism of the cell [7,8]. The redox ratio is sensitive to shifts in metabolic pathways [7,9]. The fluorescence lifetimes report changes in the microenvironment of NAD(P)H and FAD and are especially sensitive to the binding state of the fluorophore, as well as local temperature, pH, and proximity to quenchers such as molecular oxygen [10].…”

Section: Introductionmentioning

confidence: 99%

Optical metabolic imaging quantifies heterogeneous cell populations

Walsh¹,

Skala²

2015

Biomed. Opt. Express

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Abstract:The genetic and phenotypic heterogeneity of cancers can contribute to tumor aggressiveness, invasion, and resistance to therapy. Fluorescence imaging occupies a unique niche to investigate tumor heterogeneity due to its high resolution and molecular specificity. Here, heterogeneous populations are identified and quantified by combined optical metabolic imaging and subpopulation analysis (OMI-SPA). OMI probes the fluorescence intensities and lifetimes of metabolic enzymes in cells to provide images of cellular metabolism, and SPA models cell populations as mixed Gaussian distributions to identify cell subpopulations. In this study, OMI-SPA is characterized by simulation experiments and validated with cell experiments. To generate heterogeneous populations, two breast cancer cell lines, SKBr3 and MDA-MB-231, were co-cultured at varying proportions. OMI-SPA correctly identifies two populations with minimal mean and proportion error using the optical redox ratio (fluorescence intensity of NAD(P)H divided by the intensity of FAD), mean NAD(P)H fluorescence lifetime, and OMI index. Simulation experiments characterized the relationships between sample size, data standard deviation, and subpopulation mean separation distance required for OMI-SPA to identify subpopulations.

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“…Imaging of multiple endogenous fluorophores is usually performed by sequential excitation at different wavelengths using a tunable laser, leading to imaging rates decreasing with the number of excitation wavelengths, and difficulties in ensuring pixel-level registration between the channels in the case of a dynamic sample. For example, NADH and FAD are often sequentially excited at 750 nm and 880 nm 12,22,[29][30][31] . Strategies conceived so far to simultaneously excite NADH and FAD with one single wavelength 32 can compensate the difference in absorption spectra and concentrations with the payoff of low excitation efficiency.…”

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confidence: 99%

Multicolor Two-photon Imaging of Endogenous Fluorophores in Living Tissues by Wavelength Mixing

Stringari

Abdeladim

Mahou

et al. 2018

Biophotonics Congress: Biomedical Optics Congress 2018 (Microscopy/Translational/Brain/Ots)

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Two-photon imaging of endogenous fluorescence can provide physiological and metabolic information from intact tissues. However, simultaneous imaging of multiple intrinsic fluorophores, such as nicotinamide adenine dinucleotide(phosphate) (NAD(P)H), flavin adenine dinucleotide (FAD) and retinoids in living systems is generally hampered by sequential multi-wavelength excitation resulting in motion artifacts. Here, we report on efficient and simultaneous multicolor two-photon excitation of endogenous fluorophores with absorption spectra spanning the 750-1040 nm range, using wavelength mixing. By using two synchronized pulse trains at 760 and 1041 nm, an additional equivalent two-photon excitation wavelength at 879 nm is generated, and achieves simultaneous excitation of blue, green and red intrinsic fluorophores. This method permits an efficient simultaneous imaging of the metabolic coenzymes NADH and FAD to be implemented with perfect image coregistration, overcoming the difficulties associated with differences in absorption spectra and disparity in concentration. We demonstrate ratiometric redox imaging free of motion artifacts and simultaneous two-photon fluorescence lifetime imaging (FLIM) of NADH and FAD in living tissues. The lifetime gradients of NADH and FAD associated with different cellular metabolic and differentiation states in reconstructed human skin and in the germline of live C. Elegans are thus simultaneously measured. Finally, we present multicolor imaging of endogenous fluorophores and second harmonic generation (SHG) signals during the early stages of Zebrafish embryo development, evidencing fluorescence spectral changes associated with development.Multiphoton microscopy is a powerful tool for label-free and non-invasive functional imaging in small organisms and tissues 1, 2 . Pulsed near infrared excitation light allows in-depth imaging based on contrasts such as endogenous fluorescence 2 , second harmonic generation (SHG) 3 and third harmonic generation (THG) 4 . Endogenous fluorescence in living tissues arises from several intrinsic biomarkers that play important roles in physiological processes 2 . The primary intracellular sources are NAD(P)H and FAD, the two major cofactors of redox reactions in the cell and central regulators of energy production and metabolism 5,6 . Their fluorescence reports on the metabolic activity of cells allowing tissue physiology and processes such as stem cell differentiation, cancer development and neurodegenerative diseases to being non-invasively monitored [7][8][9][10][11][12] . The fluorescence lifetimes of NADH and FAD are different upon binding to the protein during the electron transport chain. FLIM provides sensitive measurements of the free and protein-bound NAD(P)H ratio and of the redox states (NADH/NAD + ) of cells, and can be used to distinguish glycolytic and oxidative phosphorylation metabolic states [13][14][15][16][17] . Monitoring lifetime of free and protein-bound FAD has also been exploited to quantify redox ratio FAD/FADH 2 , and used as a b...

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Optical Imaging Using Endogenous Contrast to Assess Metabolic State

Cited by 289 publications

References 70 publications

Defects in β-Cell Ca2+ Dynamics in Age-Induced Diabetes

Defects in β-Cell Ca2+ Dynamics in Age-Induced Diabetes

Optical metabolic imaging quantifies heterogeneous cell populations

Multicolor Two-photon Imaging of Endogenous Fluorophores in Living Tissues by Wavelength Mixing

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