Bioimaging of Nitric Oxide

Nagano, Tetsuo; Yoshimura, Tetsuhiko

doi:10.1021/cr010152s

Cited by 383 publications

(314 citation statements)

References 337 publications

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“…Diaminofluoresceins (DAFs) are the most frequently used and best investigated NO imaging probes [30,31]. However, it is important to point out that these probes do not directly react with NO.…”

Section: Principlesmentioning

confidence: 99%

“…1. Initially, Nagano and colleagues proposed that one of the vicinal amino groups of the DAFs interacts with reactive nitrosating species derived from the reaction of NO with O 2 such as N 2 O 3 [15] or nitrous acid [32], to form an intermediary NͲnitrosamine that -following intramolecular reaction with the adjacent amino group Ͳ is subsequently converted to the highly fluorescent triazole derivative, DAFͲ 2T or DAFͲFMͲT [31]. Later, Wardman pointed out that N 2 O 3 is not likely to be formed efficiently in aqueous biological environments, except in lipid membranes [20].…”

Section: Principlesmentioning

confidence: 99%

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A multilevel analytical approach for detection and visualization of intracellular NO production and nitrosation events using diaminofluoresceins

Cortese‐Krott

Rodriguez-Mateos

Kuhnle

et al. 2012

Free Radical Biology and Medicine

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Diaminofluoresceins are widely used probes for detection and intracellular localization of NO formation in cultured/isolated cells and intact tissues. The fluorinated derivative, 4ͲaminoͲ5Ͳ methylaminoͲ2',7'Ͳdifluorofluorescein (DAFͲFM), has gained increasing popularity in recent years due to its improved NOͲsensitivity, pHͲstability, and resistance to photoͲbleaching compared to the firstͲ generation compound, DAFͲ2. Detection of NO production by either reagent relies on conversion of the parent compound into a fluorescent triazole, DAFͲFMͲT and DAFͲ2ͲT, respectively. While this reaction is specific for NO and/or reactive nitrosating species, it is also affected by the presence of oxidants/antioxidants. Moreover, the reaction with other molecules can lead to the formation of fluorescent products other than the expected triazole. Thus additional controls and structural confirmation of the reaction products are essential. Using human red blood cells as an exemplary cellular system we here describe robust protocols for the analysis of intracellular DAFͲFMͲT formation using an array of fluorescenceͲbased methods (laserͲscanning fluorescence microscopy, flow cytometry and fluorimetry) and analytical separation techniques (reversedͲphase HPLC and LCͲ MS/MS). When used in combination, these assays afford unequivocal identification of the fluorescent signal as being derived from NO and are applicable to most other cellular systems without or with only minor modifications.

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Section: Principlesmentioning

confidence: 99%

Section: Principlesmentioning

confidence: 99%

A multilevel analytical approach for detection and visualization of intracellular NO production and nitrosation events using diaminofluoresceins

Cortese‐Krott

Rodriguez-Mateos

Kuhnle

et al. 2012

Free Radical Biology and Medicine

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“…These indicators contain a benzoic group attached to a fluorophore that possesses a charge transfer state located energetically between the ground and excited states of the fluorophore moiety, which serves as an effective deactivation pathway for the highly fluorescent excited state [4,5]. When nitrosated by reactive nitrogen oxides such as N 2 O 3 at one of its vicinal amino moieties, an internal diazotation reaction takes place that leads to formation of the corresponding triazole.…”

Section: Introductionmentioning

confidence: 99%

Performance of diamino fluorophores for the localization of sources and targets of nitric oxide

Rodríguez

Specian

Maloney

et al. 2005

Free Radical Biology and Medicine

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An emergent approach to the detection of nitric oxide (NO) in tissues relies on the use of fluorescence probes that are activated by products of NO autoxidation. Here we explore the performance of the widely-used NO probe 4,5-diaminofluorescein diacetate (DAF-2 DA) for the localization of sources of NO in rat aortic tissue, either from endogenous NO synthesis or from chemically or photolytically-released NO from targets of nitrosation/nitrosylation. Of importance toward understanding the performance of this probe in tissues is the finding that, with incubation conditions commonly used in the literature (10 µM DAF-2 DA), intracellular DAF-2 accumulates to concentrations that approach the millimolar range. Whereas such high probe concentrations do not interfere with NO release or signaling, they help to clarify why DAF-2 nitrosation is possible in the presence of endogenous nitrosation scavengers (e.g. ascorbate and glutathione). The gain attained with such elevated concentrations is, however, mitigated by associated high levels of background autofluorescence from the probe. This, together with tissue autofluorescence, limits the sensitivity of the probe to low-micromolar levels of accumulated DAF-2 triazole (DAF-2 T), the activated form of the probe, which is higher than the concentrations of endogenous nitrosation/nitrosylation products found in tissues. We further show that the compartmentalization of DAF-2 around elastic fibers further limits its potential to characterize the site of NO production at the subcellular level. Moreover, we find that reaction of DAF-2 with HgCl 2 and other commonly employed reagents are associated with spectral changes that may be misinterpreted as NO signals. Finally, UV illumination can lead to high levels of nitrosating species that interfere with NO detection from enzymatic sources. These findings indicate that while DAF-2 may still represent an important tool for the localization of NO synthesis, provided important pitfalls and limitations are taken into consideration, it is not suited for the detection of basally-generated nitrosation/nitrosylation products.Performance of DAF-2 for NO Imaging 3

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“…A series of fluorescent indicators for NO, developed beginning in 1998 (Kojima et al, 2001;Kojima et al, 1998;Nagano and Yoshimura, 2002), were based on 4,5-diaminofluorescein (DAF-2) and structurally similar dyes. These dyes are widely used for detection and imaging of NO because of their sensitivity and specificity (Kasim et al, 2001;Leikert et al, 2001;Strijdom et al, 2004).…”

Section: Introductionmentioning

confidence: 99%

Simultaneous nitric oxide and dehydroascorbic acid imaging by combining diaminofluoresceins and diaminorhodamines

Rubakhin

Sweedler

2008

Journal of Neuroscience Methods

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Spatial measurements of nitric oxide (NO) production are important to understand the function and metabolism of this molecule. The reagent, 4,5-diaminofluorescein (DAF-2) and several structurally similar probes are widely used for detection and imaging of NO. However, DAF-2 also reacts with dehydroascorbic acid (DHA) in biological samples, with both products having nearly indistinguishable fluorescence spectra. Measurements using fluorimetry and fluorescence microscopy cannot easily differentiate NO-related fluorescent signals from DHA-related signals. While DAFs and the structurally related diaminorhodamines (DARs) both react with NO and DHA, they do so to different extents. We report a multiderivatization method to image NO and DHA simultaneously by using both DAF and DAR. Specifically, DAF-2 and DAR-4M are used to image NO and DHA concentrations; after reaction, the solutions are excited, at 495 nm to measure fluorescence emission from DAF-2, and at 560 nm to measure fluorescence emission from DAR-4M. Using the appropriate calibrations, images are created that depend either on the relative NO or the relative DHA concentration, even though each probe reacts to both compounds. The method has been validated by imaging NO production in both undifferentiated and differentiated pheochromocytoma cells.

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Bioimaging of Nitric Oxide

Cited by 383 publications

References 337 publications

A multilevel analytical approach for detection and visualization of intracellular NO production and nitrosation events using diaminofluoresceins

A multilevel analytical approach for detection and visualization of intracellular NO production and nitrosation events using diaminofluoresceins

Performance of diamino fluorophores for the localization of sources and targets of nitric oxide

Simultaneous nitric oxide and dehydroascorbic acid imaging by combining diaminofluoresceins and diaminorhodamines

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