Fluorescent Probe For Nitric Oxide Research Articles

A fluorescent probe with an ultra-rapid response to nitric oxide.

Nitric oxide (NO) is a diatomic inorganic free radical ubiquitous in mammalian tissues and cells that plays a multifaceted role in a variety of physiological and pathophysiological processes. The strict dependence of the biological effects of NO on its concentration makes its real-time monitoring crucial. In view of the reactivity of NO with multiple bio-targets, the development of NO sensors that associate a fast response rate with selectivity and sensitivity is very challenging. Herein we report a fluorescent NO probe based on a BODIPY fluorogenic unit covalently linked to a trimethoxy aniline derivative through a flexible spacer. NO leads to effective nitrosation of the highly electron-rich amino active site of the probe through the secondary oxide N2O3, resulting in an increase of BODIPY fluorescence quantum yield from Φf = 0.06 to Φf = 0.55, accompanied by significant changes in the relative amplitude of the fluorescence lifetimes. In situ generation of NO, achieved by a tailored light-activatable NO releaser, allows the real-time detection of NO as a function of its concentration and permits demonstrating that the probe exhibits a very fast response time, being ≤0.1 s. This remarkable data combines with the high sensitivity of the probe to NO (LOD = 35 nM), responsiveness also to ONOO-, the other important secondary oxide of NO, independence from the fluorescence response within a wide pH range, good selectivity towards different analytes and small interference by typical physiological concentrations of glutathione. Validation of this probe in melanoma cell lines is also reported.

An electronic-tuning strategy to improve the responsiveness of nitric oxide probe

Nitric oxide (NO) plays a crucial role in a variety of pathophysiological processes. Although a large number of NO fluorescent probes have been developed, it remains barely unexplored how the electrical effect of the recognition site affects responsiveness. In this work, we demonstrate an electronic-tuning strategy to enhance the sensitivity and responsiveness of NO probes based on BODIPY by decorating various groups with different electronic capabilities in the recognition unit. Experimental results show that the electronic effect at the identification section has a significant impact on the signal response. BODIPY 5, containing the OH group at the para-position of aniline, exhibits the greatest fluorescence responsiveness with NO. The signal does not vary for the strong electron-drawing and electron-donating groups, in which electronic or charge transfer is always in the On or OFF state, as predicted by TD-DFT calculations. Additionally, the cellular assay demonstrated that the probe possesses the ability to visualize exogenous NO in lipid droplets of HeLa cells and the endogenous NO produced in Raw 264.7 cells. This study offers a useful NO imaging tool as well as a guideline for designing high-performance NO probes for practical application.

Development of a Facile Lysosome-Targeting Nitric Oxide Fluorescent Probe Based on a Novel Reaction Mechanism.

As a ubiquitous signal molecule in biosystems, nitric oxide (NO) plays an important role in many physiological and pathological processes. Therefore, it is of great significance to detect NO in organisms for the study of related diseases. Currently, a variety of NO fluorescent probes have been developed based on several types of reaction mechanisms. However, due to the inherent disadvantages of these reactions, like potential interference by biologically related species, there is a great need to develop NO probes based on the new reactions. Herein, we report our discovery of the unprecedented reaction between a widely used fluorophore of 4-(dicyanomethylene)-2-methyl-6-(p-(dimethylamino)styryl)-4H-pyran (DCM) and NO under mild conditions with fluorescence changes. By the analysis of the structure of the product, we proved that DCM undergoes a particular nitration process and proposed a mechanism for fluorescence changes due to the interruption of the intramolecular charge transfer (ICT) process of DCM by the nitrated product of DCM-NO2. Based on the understanding of this specific reaction, we then easily constructed our lysosomal-localized NO fluorescent probe LysoNO-DCM by linking DCM and a morpholine group, a lysosomal-targeting functional group. LysoNO-DCM exhibits excellent selectivity, sensitivity, pH stability, and outstanding lysosome localization ability with Pearson's colocalization coefficient of up to 0.92 and is successfully applied to the imaging of exogenous and endogenous NO in cells and zebrafish. Our studies expand design methods for NO fluorescence probes based on the novel reaction mechanism and will benefit the studies of this signaling molecule.

Thiophene and diaminobenzo- (1,2,5-thiadiazol)- based DAD-type near-infrared fluorescent probe for nitric oxide: A theoretical research.

A near-infrared fluorescent probe (LS-NO) for the real-time detection of nitric oxide (NO) in inflammatory bowel disease (IBD) was developed recently. The probe used oligoglycol morpholine-functionalized thiophene as strong electron donors and diaminobenzene (1,2,5-thiadiazole) as a weak electron acceptor and NO trapping group. It could detect exogenous and endogenous NO in the lysosomes of living cells with high sensitivity and specificity. To further understand the fluorescent mechanism and character of the probes LS-NO and LS-TZ (after the reaction of the probe LS-NO with NO), the electron transfer in the excitation and emitting process within the model molecules DAD-NO and DAD-TZ was analyzed in detail under the density functional theory. The calculation results indicated the transformation from diaminobenzene (1,2,5-thiadiazole) as a weak electron acceptor to triazolo-benzo-(1,2,5-thiadiazole) as a strong electron acceptor made LS-NO an effective "off-on" near-infrared NO fluorescent probe.

A highly photostable and versatile two-photon fluorescent probe for the detection of a wide range of intracellular nitric oxide concentrations in macrophages and endothelial cells.

Nitric oxide (NO) is involved in many biological processes affecting the cardiovascular, nervous and immune systems. Intracellular NO can be monitored using fluorescent probes in combination with fluorescence imaging techniques. Most of the currently available NO fluorescent molecular probes are excited via one-photon excitation using UV or Vis light, which results in poor penetration and high photodamage to living tissues. Here, we report a two-photon fluorescent molecular probe, DANPY-NO, able to detect NO in live cells. The probe consists of an o-phenylenediamine linked to a naphthalimide core; and operates via photoinduced electron transfer. DANPY-NO exhibits good sensitivity (LOD of 77.8nM) and high selectivity towards NO, and is stable over a broad range of pHs. The probe targeted acidic organelles within macrophages and endothelial cells, and demonstrated enhanced photostability over a commercially available NO probe. DANPY-NO was used to selectively detect endogenous NO in RAW264.7ϒ NO- macrophages, THP-1 human leukemic cells, primary mouse (bone marrow-derived) macrophages and endothelial cells. The probe was also able to detect exogenous NO in endothelial cells and distinguish between increasing concentrations of NO. The NO detection was evidenced using confocal laser scanning and two-photon microscopies, and flow cytometry. Further evidence was obtained by recording the changes in the intracellular fluorescence emission spectrum of the probe. Importantly, the probe displayed negligible toxicity to the analysed biological samples. The excellent sensitivity, selectivity, stability and versatility of DANPY-NO confirm its potential for in vitro and in vivo imaging of NO.

Golgi-Targeted Fluorescent Probe for Imaging NO in Alzheimer's Disease.

Nitric oxide (NO) is a crucial neurotransmitter participating in many biological processes via nitrosylation reaction. NO produced in diverse subcellular regions also regulates the function of cells in different manners. A Golgi apparatus is rich in nitric oxide synthase and may serve as a potential therapeutic target for Alzheimer's disease (AD). However, due to the lack of an effective tool, it is difficult to reveal the relationship between Golgi-NO and AD. Herein, we report Golgi-NO as the first Golgi-targeted fluorescent probe for sensing and imaging NO in the Golgi apparatus. The probe is designed and synthesized by incorporating 4-sulfamoylphenylamide as a Golgi-targeted moiety to 6-carboxyrhodamine B, generating a fluorophore of Golgi-RhB with modifiable carboxyl, which is then combined with the NO recognition moiety of o-diaminobenzene. The probe shows superior analytical performance including accurate Golgi-targeted ability and high selectivity for NO. Moreover, using the probe, we disclose a significant increase of NO in Golgi apparatus in the AD model. This study provides a competent tool for studying the function and nitrosylation of NO in the Golgi apparatus in related diseases.

Acetaldehyde reacts with a fluorescent nitric oxide probe harboring an o-phenylenediamine structure that interferes with fluorometry

Nitric oxide (NO) is a ubiquitous signaling molecule, and thus a variety of methods have been developed for its detection and quantification. Fluorometric analyses using a fluorescent NO probe harboring an o-phenylenediamine (OPD) structure are widely used for NO analyses in various organisms, including yeast. Here, we discovered that an NO-independent fluorophore (UNK436) was generated from a fluorescent NO probe 4-amino-5-methylamino-2′,7′-difluorofluorescein (DAF-FM), which has an OPD structure, in yeast cells. The molecules responsible for this undesirable fluorescence and their reaction mechanisms were analyzed. Our mass spectrometric analysis showed that two carbon atoms from glucose were incorporated into UNK436. Subsequent analyses indicated that a non-proteinous small compound leads to the synthesis of UNK436 through an oxidative reaction. Furthermore, our LC/MS/MS analysis of the reaction mixture of DAF-FM with acetaldehyde in combination with stable isotope labeling demonstrated that acetaldehyde reacts with DAF-FM oxidatively, generating UNK436. Another NO probe with an OPD structure, diaminorhodamine-4M, reacted with acetaldehyde in the same way to emit fluorescence. Based on our findings, we recommend that in researches using OPD-based fluorescent NO probes, alternative analyses also be performed to identify the reaction products of the probes with NO to avoid false-positives.

Hemicyanine-based turn-off fluorescent probe for monitoring of nitric oxide in living cells

In situ imaging of nitric oxide (NO) is of great importance to study the critical roles of NO in various physiological and pathological processes. In most currently available fluorescent probes for NO, the o-phenylenediamine (OPD) based derivatives are the most greeted and studied, in which the OPD serves as the reactive site and fluorescence quencher to modulate a turn-on process via photoinduced electron transfer (PET). In this work, a unique turn-off probe containing OPD moiety, N-1, was designed and synthesized, which was radically different from the typical PET-based turn-on fluorescent probes. By theoretical calculation of molecular orbital, it was verified that not the widely used PET mechanism, but the intramolecular charge transfer (ICT) mechanism should be responsible for the turn-off process. Probe N-1 exhibited a remarkable performance with 7-folds fluorescence intensity decrease (at 490 nm), a low detection limit (20 nM), a fast response to NO within 12 min and a high selectivity for NO over other relevant analytes. Furthermore, probe N-1 was also applied to imaging NO in living cells. All results demonstrated that the new probe N-1 could be a practical and ideal NO indicator with good biological significance.

8‐oxoguanine glycosylase (OGG1) is a determinant of endothelial dysfunction via calcium and nitric oxide signaling

Diabetes mellitus is a chronic disease that hinders the body's ability to produce or respond to the hormone insulin, resulting in abnormal metabolism and elevated glucose levels. Type II diabetes has been characterized by a two-to-four-fold increase in cardiovascular disease, such as carotid artery stenosis, generally attributed to the adverse effects of hyperglycemia and oxidative stress on vascular biology. With it being a very common and significant health complication, understanding the pathology and the mechanisms by which diabetes-induced oxidative stress serves as a precursor to several notable vascular diseases is paramount, and is the overall objective of this study. Endothelial dysfunction (ED) is a vascular phenomenon attributed to a reduction in nitric oxide (NO) bioactivity and an increase in oxygen free radical formation with subsequent oxidative stress, thus leading to impaired vasodilation, and compromised vascular function. ED tends to be a consistent finding in diabetes. A major source of oxidative stress is the abnormal production of reactive oxygen species (ROS) in the context of diabetic inflammation. Increased ROS production leads to mitochondrial DNA (mt DNA) damage and aberrant cell signaling. Typically, mt DNA damage is repaired through the base excision repair (BER) pathway. However, during chronic inflammation in diabetes, this repairing capability is ineffective due to downregulation of key BER enzymes, namely 8-oxoguanine glycosylase (OGG1). Given this rational, we hypothesized that the BER pathway is an important determinant of endothelial dysfunction via impaired calcium and NO signaling. To test this hypothesis, we examined the effect of BER defects on endothelial calcium signaling and NO production using carotid arteries isolated from mice deficient in OGG1. Opened artery measurements of endothelial calcium and NO signaling were performed using a custom-designed parallel plate flow chamber. Briefly, segments of carotid arteries (~200 µm in diameter) were isolated from OGG1 deficient (KO) and wild type (WT) mice and cut open lengthwise to expose the endothelial surface. The isolated vessels were then stained with a loading solution composed of Pluronic acid (Fischer), Cal-520 (AAT bio) calcium indicator dye and DAR-4M (Calbiochem), a fluorescent probe for NO. Time lapse signal measurements were collected via confocal microscopy and were then analyzed and processed with S8, a custom automated solution to signal analysis based on noise filtering and object tracking, which computed dynamic amplitude, frequency, duration, and spatial spread. We discovered that although the number of calcium signals detected within the measurement interval were similar between KO and WT (10 ± 3 vs 9 ± 3, µ ± SEM, respectively), the spatial spread of signals within the KO (19.95 ± 3.78 µm2, µ ± SEM) was greater than WT (9.65 ± 2.7 µm2, p = 0.057 via Welch's unpaired t-test). These data suggest that OGG1 may be a determinant of normal endothelial calcium signaling and might serve as a therapeutic target for preventing the adverse effects of endothelial dysfunction present in diabetes.

A highly specific rhodamine B based turn-on fluorescent probe for nitric oxide and application in living cells

Abstract The design and synthesis of two rhodamine derivatives with the same luminescent group, which have an interesting “turn-on” fluorescent response toward NO and can be readily prepared through one-step reaction in acetonitrile. Of the two compounds, rh-NO-1 showed high selectivity for NO among various reactive oxygen/nitrogen species, accompanied by a 24-fold fluorescent enhancement. With an increase of the amount of NO from 0 to 100 μM, the linear correlation between the fluorescent intensity of rh-NO-1 at 600 nm and the concentration of NO was found. The probe rh-NO-1 had good sensitivity and the detection limit was as low as 36.8 nM. Upon addition of NO, rh-NO-1 had a clear response within 2 min. This was a colour change from colorless to pink, which was visible to the naked eye. In addition, both rh-NO-1 and its derivative can be used to detect NO in living cells.

Dihydropyridine-coumarin-based fluorescent probe for imaging nitric oxide in living cells.

Nitric oxide (NO) is a messenger molecule in organisms, participating in the regulation of many biological processes. The abnormal expression of NO is often observed in a variety of diseases, including cerebral ischemia, atherosclerosis, and cancer. However, a suitable tool that can directly and sensitively detect NO in vitro and in vivo is important for understanding its various biological functions. In this report, a new fluorescent probe for nitric oxide, DHP-4, was prepared, based on dihydropyridine-coumarin. DHP-4 was able to greatly enhance the fluorescence of NO, but did not affect the fluorescence emissions of other reactive oxygen species and nitrogen species, demonstrating its highly selective and sensitive response to NO. The probe generated stable optical signals in a buffer solution at pH values ranging from 3 to 10. In addition, DHP-4 could detect NO directly, showed low cellular toxicity, and was successfully applied to determine NO in Raw 264.7 cells, indicating its great potential as a tool for investigating the biological roles of NO in vivo.

A rapid-response and ratiometric fluorescent probe for nitric oxide: From the mitochondria to the nucleus in live cells

Nitric oxide (NO) is a very important signal molecule implicated in numerous physiological and pathological processes, and its detection is the key to understand these processes. For this reason, various fluorescent probes have been developed for detection analysis of NO. However, few rapid-response (<1 min) and ratiometric fluorescent probe are reported for real-time detection of short-time NO in biological systems. In this work, we report a rapid-response (within several seconds) and ratiometric fluorescent probe, RatioTr, which displays selective and sensitive detection of NO in solutions, and detections of exo- and endogenous NO in live RAW 264.7 cells. Unexpectedly, the probe RatioTr and its sensing product (p-Nus) display different cellular localizations, the mitochondria and the nucleus, which were demonstrated by co-stained experiments. The sensing process of RatioTr toward NO from mitochondria to nucleus was observed in live cells by confocal fluorescence images. Furthermore, the subcellular localizations were demonstrated by measurements of pKa and interaction of p-Nus and DNA. In the presence of a natural DNA, calf thymus DNA, RatioTr is more sensitive to NO (LOD = 2.8 nM). Therefore, due to the nucleus localization together with a high fluorescence efficiency in the nucleus, p-Nus is a good candidate of cell-permeant nucleic acid stain or a fluorescent probe for the nucleus.

A nitric oxide induced “click” reaction to trigger the aggregation induced emission (AIE) phenomena of a tetraphenyl ethylene derivative: A new fluorescent probe for NO

An Aggregation Induced Emission (AIE) transduction mechanism has been used to detect nitric oxide (NO). A new tetraphenylethylene derivative functionalized with alkyne moieties has been prepared to work as a fluorescent probe. A “click” reaction was chosen because NO is able to generate the required catalytic species by reducing Cu(II) to Cu(I).

Theoretical Design of a Two-Photon Fluorescent Probe for Nitric Oxide with Enhanced Emission Induced by Photoninduced Electron Transfer.

In the present work, we systematically investigate the sensing abilities of two recently literature-reported two-photon fluorescent NO probes, i.e., the o-phenylenediamine derivative of Nile Red and the p-phenylenediamine derivative of coumarin. The recognition mechanisms of these probes are studied by using the molecular orbital classifying method, which demonstrates the photoinduced electron transfer process. In addition, we have designed two new probes by swapping receptor units present on fluorophores, i.e., the p-phenylenediamine derivative of Nile Red and the o-phenylenediamine derivative of coumarin. However, it illustrates that only the latter has ability to function as off-on typed fluorescent probe for NO. More importantly, calculations on the two-photon absorption properties of the probes demonstrate that both receptor derivatives of coumarin possess larger TPA cross-sections than Nile Red derivatives, which makes a better two photon fluorescent probe. Our theoretical investigations reveal that the underlying mechanism satisfactorily explain the experimental results, providing a theoretical basis on the structure-property relationships which is beneficial to developing new two-photon fluorescent probes for NO.

Selective and Real-Time Detection of Nitric Oxide by a Two-Photon Fluorescent Probe in Live Cells and Tissue Slices.

Nitric oxide (NO) is an important signaling molecule involved in many physiological and pathological processes. To understand these NO-mediated processes, it is a key to develop rapid and specific detection methods for NO. In the past 2 decades, numerous excellent fluorescent probes for NO have been designed; however, it still remains limitations such as slow response, low selectivity, and short excitation wavelength (<600 nm). In this Article, a two-photon fluorescent probe, NO-QA5, has been developed with 3-dimethylaminophenyl linking at the 6-position of 5-aminoquinoline as both the active site and prefluorophore for detection of NO. The nonfluorescent NO-QA5 can fast react with NO via a diazonium intermediate to generate two azoic regioisomers, one of which exhibits intramolecular charge transfer (ICT) emission, and two-photon absorption behavior (δΦ = 57 GM), giving a turn-on fluorescence rapid response. The sensing reaction is pH-insensitive in the range of 6-11 and highly selective and well sensitive (LOD = 15 nM), possible undergoing the same intermediate diazonium with the reaction under diazotization condition (NaNO2/HCl). Also, as a nitrite fluorescent probe NO-QA5 exhibits highly sensitive (LOD = 7 nM). Therefore, NO-QA5 can serve as a dual functional fluorescent probe for NO and NO2-. Furthermore, NO-QA5 as a specific imaging agent has been demonstrated by achieving both exogenous and endogenous detections of NO in living cells under both one- and two-photon excitation and high resolution in tissue slices under two-photon excitation.

Development of a Silicon-Rhodamine Based Near-Infrared Emissive Two-Photon Fluorescent Probe for Nitric Oxide.

Two-photon (TP) fluorescent probes are potential candidates for near-infrared (NIR) imaging which holds great promise in biological research. However, currently, most TP probes emit at wavelength <600 nm, which impedes their practical applications. In this work, we explored the TP properties of a silicon-rhodamine (SiR) derivative and hence developed the first SiR scaffold based "NIR-to-NIR" TP probe (SiRNO) for nitric oxide (NO). SiRNO exhibited high sensitivity and specificity, as well as fast response for NO detection. It was able to track the subtle variation of intracellular NO content in live cells. Owing to the NIR excitation and emission, SiRNO enabled the detection of NO in situ in the xenograft tumor mouse model, revealing the NO generation during the tumor progression. This work indicates that SiR can be an ideal platform for the development of NIR emissive TP probe and may thus promote the advancement of NIR imaging.

Lead-induced stress, which triggers the production of nitric oxide (NO) and superoxide anion (O2·-) in Arabidopsis peroxisomes, affects catalase activity

Lead (Pb) contamination has a toxic effect on plant metabolisms, leading to a decrease in biomass production. The free radical nitric oxide (NO) is involved in the mechanism of response to a wide range of abiotic stresses. However, little is known about the interplay between Pb-induced stress and NO metabolism. Peroxisomes are sub-cellular compartments involved in multiple cellular metabolic pathways which are characterized by an active nitro-oxidative metabolism. Thus, Arabidopsis thaliana mutants expressing cyan fluorescent protein (CFP) through the addition of peroxisomal targeting signal 1 (PTS1), which enables peroxisomes to be visualized in vivo by confocal laser scanning microscopy (CLSM) combined with fluorescent probes for nitric oxide (NO), superoxide anion (O2·-) and peroxynitrite (ONOO−), were used to evaluate the potential involvement of these organelles in the mechanism of response to 150 μM lead-induced stress. Both NO and O2·- radicals, and consequently ONOO−, were overproduced under Pb-stress. Additionally, biochemical and gene expression analyses of peroxisomal enzymes, including the antioxidant catalase (CAT) and two photorespiration enzymes, such as glycolate oxidase (GOX) and hydroxypyruvate reductase (HPR), show that, under Pb-stress, only the catalase was negatively affected, while the two photorespiration enzymes remained unaffected. These results corroborate the involvement of plant peroxisomal metabolisms in the mechanism of response to lead contamination and highlight the importance of the peroxisomal NO metabolism.

Conjugating S-nitrosothiols with fluorescent nanofibers for the controlled release and real-time detection of nitric oxide

In this work, a novel hybrid fibrous vehicle was developed for the release and real-time detection of nitric oxide (NO). The vehicle consisted of poly(L-lactide)/chitosan (PLLACS), cadmium tellurium quantum dots (CdTe QDs), and conjugating S-nitrosothiols. CdTe QDs were placed on the surface of PLLACS fibers as NO fluorescent probes through coaxial electrospinning; in this method, the PLLACS was used as filament material. Water-dispersible CdTe QDs were synthesized using mercaptoacetic acid as a stabilizer. The amino groups of chitosan served as reactive sites where chitosanbased S-nitrosothiols were formed. The fibrous PLLACS-QDs-NO composite was found to release NO under UV. The released NO could be detected in real time on the basis of the fluorescence quenching mechanism of QDs. These PLLACSQDs-NO nanofibers provide insights into novel designs of biocompatible NO delivery systems.

Effect of fluorophore on sensing NO for newly synthesized PET-based two-photon fluorescent probes

Density functional theory in combination with polarizable continuum model is used to investigate one- and two-photon absorption (TPA) as well as emission properties of newly synthesized photoinduced electron transfer (PET)-based two-photon fluorescent probes QNO and LNO in the absence and presence of nitric oxide (NO). Both fluorescent intensity and TPA are enhanced when QNO (LNO) reacts with NO, which theoretically proves that the compounds QNO and LNO are promising two-photon fluorescent probes for NO. Moreover, QNO is demonstrated to be preferable because of its superior fluorophore. Analysis of molecular orbitals is presented to explore responsive mechanism of QNO (LNO) for NO.

Fast-response and highly selective fluorescent probes for biological signaling molecule NO based on N-nitrosation of electron-rich aromatic secondary amines

Nitric oxide (NO) is a ubiquitous biological messenger molecule, and plays the active roles in the regulation of various physiological processes. Although numerous NO fluorescent probes have also been successfully developed in the past ten years, it still remains challenging to increase the response rate for NO while having the high selectivity and sensitivity. In this work, a simple N-nitrosation reaction of the electron-rich aromatic secondary amine with NO under aerobic condition has been utilized for the first time to construct fluorescent probe for NO. The resulting probe 1, containing a N-benzyl-4-hydroxyaniline moiety as reaction group and a BODIPY dye as fluorescence reporter, could detect NO with the fast fluorescence off-on response (within seconds), high sensitivity (nM level), and excellent selectivity over various reactive oxygen species (ROS) as well as dehydroascorbic acid (DHA), ascorbic acid (AA), and methylglyoxal (MGO). Even in the presence of glutathione (GSH, a high reactive biothiol for NO), the probe still works well for NO. Further, a mitochondria-targetable probe 2 was exploited by introducing a targeted triphenylphosphonium cation into probe 1 scaffold. It's excellent NO sensing performance as well as its ability to specifically target mitochondria and image NO there have been nicely demonstrated. With the two probes, the basal and stimulation-induced NO in RAW264.7 murine macrophages as well as the endogenous NO in endothelial cells after oxygen–glucose deprivation (OGD) have been successfully visualized.