Base interactions in the triplet states of NAD+ and NADH

Jb, Ross; Kw, Rousslang; Ag, Motten; Al, Kwiram

doi:10.1021/bi00576a027

Cited by 19 publications

(13 citation statements)

References 19 publications

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“…We omit a triplet state because one associated with the nicotinamide ring of NADH has yet to be observed, although a weak phosphorescence from the adenine moiety has been reported (37). Excited‐state spectroscopy of NADH in PBS with picosecond time resolution has been performed by Rotomskis et al (38).…”

Section: Double‐biphotonic Photobleaching Of Nadhmentioning

confidence: 99%

Photobleaching of Reduced Nicotinamide Adenine Dinucleotide and the Development of Highly Fluorescent Lesions in Rat Basophilic Leukemia Cells During Multiphoton Microscopy

Tiede

Nichols

2006

Photochem & Photobiology

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Endogenous reduced nicotinamide adenine dinucleotide (NADH) fluorescence provides an intrinsic indicator of the cellular metabolic state, but prolonged monitoring is limited by photobleaching and/or phototoxicity. Multiphoton excitation of NADH by ultrashort, 740-nm laser pulses provides a significant improvement over UV excitation by eliminating peripheral photobleaching; however, molecules within the subfemtoliter excitation volume remain susceptible. We have investigated the photophysical mechanisms responsible for multiphoton photobleaching of NADH in living cells to permit the imaging technique to be optimized. The loss of fluorescence because of multiphoton photobleaching was measured by repetitively imaging individual planes within rat basophilic leukemia cells. The photobleaching rate was proportional to the fourth power of the laser intensity. Based on these measurements, we propose a double-biphotonic, four-photon photobleaching mechanism and estimate the quantum yield of photobleaching of intracellular NADH to be 0.0073 +/- 0.0002 by this mechanism. In addition to photobleaching, the development of bright, punctate fluorescent lesions can also be observed. The frequency of lesion formation also increased approximately as the fourth power of the laser intensity after an intensity-dependent threshold number of images had been exceeded. The consequences for two-photon metabolic imaging are discussed.

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Section: Double‐biphotonic Photobleaching Of Nadhmentioning

confidence: 99%

Photobleaching of Reduced Nicotinamide Adenine Dinucleotide and the Development of Highly Fluorescent Lesions in Rat Basophilic Leukemia Cells During Multiphoton Microscopy

Tiede

Nichols

2006

Photochem & Photobiology

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“…1A,B, are well in line with previous photophysical studies of NADH. With optical detection of magnetic resonance (ODMR) and photoluminescence studies, no 34 , or very minor 35 evidence of triplet state formation in NADH could be observed upon excitation at 313 nm or longer wavelengths. In contrast, transient state absorption 26,27,29 and ESR 28,36 studies show that radical cations of NADH are readily formed upon light excitation.…”

Section: Resultsmentioning

confidence: 99%

“…Based on the effects of excitation intensity and oxygen concentration observed with TRAST (Fig. 1A,B), and taking these previous studies of NADH photophysics 26–29,34–36 into account, we constructed a photophysical model for NADH (Fig. 2).…”

Section: Resultsmentioning

confidence: 99%

Local redox conditions in cells imaged via non-fluorescent transient states of NAD(P)H

Tornmalm

Sandberg

Rabasović

et al. 2019

Sci Rep

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The autofluorescent coenzyme nicotinamide adenine dinucleotide (NADH) and its phosphorylated form (NADPH) are major determinants of cellular redox balance. Both their fluorescence intensities and lifetimes are extensively used as label-free readouts in cellular metabolic imaging studies. Here, we introduce fluorescence blinking of NAD(P)H, as an additional, orthogonal readout in such studies. Blinking of fluorophores and their underlying dark state transitions are specifically sensitive to redox conditions and oxygenation, parameters of particular relevance in cellular metabolic studies. We show that such dark state transitions in NAD(P)H can be quantified via the average fluorescence intensity recorded upon modulated one-photon excitation, so-called transient state (TRAST) monitoring. Thereby, transitions in NAD(P)H, previously only accessible from elaborate spectroscopic cuvette measurements, can be imaged at subcellular resolution in live cells. We then demonstrate that these transitions can be imaged with a standard laser-scanning confocal microscope and two-photon excitation, in parallel with regular fluorescence lifetime imaging (FLIM). TRAST imaging of NAD(P)H was found to provide additional, orthogonal information to FLIM and allows altered oxidative environments in cells treated with a mitochondrial un-coupler or cyanide to be clearly distinguished. We propose TRAST imaging as a straightforward and widely applicable modality, extending the range of information obtainable from cellular metabolic imaging of NAD(P)H fluorescence.

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“…Regions in grey indicate parameters that do not support lasing. Dotted lines denote τ t / τ isc at physiological conditions (~2 × 10 3 for flavins 26 , 34 and ~3 × 10 −5 for NAD(P)H 30 , 35 ). …”

Section: Resultsmentioning

confidence: 99%

Lasing with cell-endogenous fluorophores: parameters and conditions

Yong

Ding

2017

Sci Rep

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The notion of lasing with biologics has recently been realized and has rapidly developed with the collective objective of creating lasers in vivo. One major limitation of achieving this is the requirement of exogenous dyes and fluorescent materials. We thus investigate for the first time the possibility of lasing unlabelled cells, using just cell-endogenous fluorophores - the source of cell autofluorescence. In this work, we theoretically studied the lasing potential and efficiency of flavins and reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) using a dye lasing model based on coupled rate equations. Analytical solutions for one- and two-photon pumped system were used in multi-parameter studies. We found that at physiological conditions, the more abundant NAD(P)H can be lased with a cavity quality factor of 105. We then recommended the tuning of intersystem crossing to make the lasing of flavins feasible even at their low physiological concentrations. Under conditions of reduced intersystem crossing, we concluded that it is more practical to lase unlabelled cells using flavins, because lasing thresholds and cavity quality factors were both at least an order lower. We also note the higher threshold requirements and lower efficiencies of two-photon pumping, but recognize its potential for realizing lasing in vivo.

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Base interactions in the triplet states of NAD+ and NADH

Cited by 19 publications

References 19 publications

Photobleaching of Reduced Nicotinamide Adenine Dinucleotide and the Development of Highly Fluorescent Lesions in Rat Basophilic Leukemia Cells During Multiphoton Microscopy

Photobleaching of Reduced Nicotinamide Adenine Dinucleotide and the Development of Highly Fluorescent Lesions in Rat Basophilic Leukemia Cells During Multiphoton Microscopy

Local redox conditions in cells imaged via non-fluorescent transient states of NAD(P)H

Lasing with cell-endogenous fluorophores: parameters and conditions

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