Introduction
NO mediates both physiological and pathological processes in living organisms1, 2, 3, 4, 5, 6, 7. Direct in vivo detection of NO in real time is difficult, because it rapidly diffuses and reacts with cellular components. Although methods such as chemiluminescence8, EPR spectroscopy9 and amperometry10 have been applied for NO bioimaging, they suffer from low spatial resolution and in some cases require complicated instrumentation. An alternative means to image intracellular and extracellular NO is by light emission from fluorescent sensors in combination with microscopy9, 11. A protein-based intracellular NO sensor incorporating soluble guanylate cyclase was very recently reported, but the probe requires genetic encoding for its preparation and generates the biologically active molecule cyclic guanosine monophosphate (cGMP), which can induce other cellular responses1, 2, 3, 4, 5, 6, 7. The cGMP molecules bind both NO-associated and NO-free protein probes, resulting in an increase in fluorescence. Thus, the signal generated reflects the intracellular concentration of cGMP rather than that of NO12. Other small molecule–based fluorescent probes for NO, including o-diaminonaphthalene (DAN), o-diaminofluoresceins (DAFs) and o-diaminocyanines (DACs), have been documented9, 13, 14, but their changes in fluorescence require reactions with oxidized NO products and not with NO itself. Because NO has a lifetime of up to several minutes under certain conditions15, the fluorescence response of organic molecules containing an o-diamine functionality does not necessarily reflect real-time NO production. A previously described pyrene-nitronyl probe detects NO directly but requires high excitation energy16, which produces cellular autofluorescence, and the probe cannot provide spatiotemporal information.
In the present report we describe the synthesis and characterization of a Cu(II) fluorescein-based compound, CuFL (Compound 1
Compound 1), which reacts rapidly and specifically with NO over other potentially interfering reactive nitrogen species (RNS) to afford bright light emission with nanomolar sensitivity. We used detailed EPR, LC-MS, UV-visible (UV-vis), NMR and fluorescence spectroscopic experiments to reveal a sensing mechanism that involves sequential metal reduction and ligand nitrosation. This cell-permeable reagent images NO that is produced in live cells by cNOS and iNOS, and it can spatially distinguish which cells are producing NO in a mixed culture. Thus, CuFL is the first probe that can directly image NO production in living cells by turn-on fluorescence. Because this field has heretofore been the province solely of organic chemistry, these results also demonstrate the use of metal coordination compounds for biological imaging in the complex milieu of the cell.
Results
Preparation of FL and CuFL
Derivatized fluorescein molecules are excellent biosensors because they excite and emit in a region of the visible spectrum that is relatively free of interference. We prepared the fluorescein-based ligand FL (Compound 2
Compound 2, Scheme 1) by reacting 7'-chloro-4'-fluorescein-carboxaldehyde17 with 8-aminoquinaldine. We generated the Cu(II) fluorescein-based NO probe CuFL (Scheme 1) in situ by reacting FL with CuCl2 in a 1:1 ratio in buffered aqueous solution (50 mM PIPES, pH 7.0, 100 mM KCl). The Cu(II) species showed a blue-shifted 
max at 499 nm (
= 4.0 
104 M-1cm-1); the max of FL is 504 nm ( = 4.2 104 M-1cm-1). A Job's plot was constructed to evaluate the nature of the FL:Cu(II) complex by following the UV-vis absorption-spectral change at 512 nm in pH 7.0 buffered solution (Supplementary Fig. 1 online). A break at 0.5 indicated the formation of a 1:1 complex. The negative ion electrospray mass spectrum of this species had a peak with m/z of 632.0, which corresponds to that of [Cu(FL)Cl – H]- (calcd. m/z 632.0) (Supplementary Fig. 1). When we titrated FL with CuCl2 at 25 °C, the absorption changes could be fit to a one-step binding equation with an apparent dissociation constant (Kd) of 1.5 
0.3 
M for Cu(II) ion (Supplementary Fig. 1).
Fluorescence and mechanistic studies of CuFL with NO
The fluorescence of a 1 M FL solution was diminished by 18 3% upon introduction of an equimolar quantity of CuCl2 at 37 °C. Addition of excess NO to a buffered aqueous CuFL solution led to an immediate 11.2 1.5–fold increase in fluorescence (Fig. 1). This result demonstrates rapid NO detection with substantial turn-on emission at a physiologically relevant pH. Fluorescence was also enhanced when we allowed CuFL to react with the NO-releasing chemical agent S-nitroso-N-acetyl-D,L-penicillamine (SNAP, 3) at pH 7.0 (50 mM PIPES, 100 mM KCl) for 30 min (Supplementary Fig. 2 online). The lower detection limit for NO is 5 nM. The fluorescence response of the CuFL probe detects NO specifically over other reactive species present in biological systems, including H2O2, HNO, NO2-, NO3- and ONOO- (Fig. 2).
Figure 1: The fluorescence response of CuFL to NO.
Fluorescence emission spectra (ex = 503 nm) of a deoxygenated 1 M solution of CuFL in buffered solution (50 mM PIPES, pH 7.0, 100 mM KCl) before (dashed line), immediately, 1, 2 and 5 min after (solid lines) admission of 1,300 equiv. of NO (g) at 37 °C.
Figure 2: Specificity of CuFL for NO over other reactive nitrogen and oxygen species.
We determined the fluorescence response of CuFL after addition of 100 equiv. of H2O2, HNO (Angeli's salt Na2N2O3), NO2-, NO3- and ONOO- for 2 h in buffered aqueous solution (50 mM PIPES, pH 7.0, 100 mM KCl). The excitation wavelength was 503 nm. All data (F) are normalized with respect to the emission of CuFL (FCuFL). [CuFL] = 1 M. Error bars indicate s.d.
We used a commercially available NO probe, DAF-2 (Compound 4
Compound 4), for comparison with CuFL. The fluorescence of DAF-2 is unchanged in the presence of NO and the absence of O2 over a period of 1 h. DAF-2 shows turn-on fluorescence only when O2 is present, indicating that it is incapable of direct NO detection. CuFL, however, shows an immediate fluorescence response under both anaerobic and aerobic conditions (Fig. 1 and Supplementary Fig. 2).
In a control experiment, a copper-free FL solution treated with excess NO showed only a small increase (1.5 0.2–fold) in fluorescence over 30 min (Supplementary Fig. 2). In addition, the fluorescence was only marginally increased (1.3 0.2–fold) upon addition of NO to the CuFL solution in the presence of excess EDTA, which chelates Cu(II) ion (Supplementary Fig. 2). Both results indicate that the binding of FL to Cu(II) is indispensable for fluorescence enhancement by NO.
NO detection by CuFL occurs, formally, by NO-induced reduction of Cu(II) to Cu(I), forming NO+, which nitrosates the ligand. Concomitant dissociation of FL-NO from copper induces fluorescence turn-on (Scheme 1). We investigated the mechanism of turn-on emission of CuFL by NO using EPR, UV-vis, NMR, fluorescence spectroscopy and LC-MS. The EPR experiment revealed a 2.9-fold decrease of the axial Cu(II) signal upon reaction of CuFL with 5 equiv. of NO in dimethylformamide (DMF) and thus confirmed the formation of a Cu(I) species (Supplementary Fig. 3 online). A mixture of FL and [Cu(CH3CN)4](BF4) had the same fluorescence intensity as FL and showed only a 1.3 0.1–fold increase in fluorescence over 30 min when treated with excess NO (Supplementary Fig. 3). This result indicates that Cu(I)FL is not the species responsible for the fluorescence increase in the NO reaction of CuFL.
LC-MS analyses of the reaction product of NO with CuFL (pH 7.0 buffered aqueous solution) revealed a major peak (93 3%) in the chromatogram, with m/z = 564.7, 600.5 and 1,128.9—masses corresponding to those of [FL + NO – 2H]- (calcd. m/z 564.1), [FL + NO – H + Cl]- (calcd. m/z 600.1) and [2(FL + NO) – 3H]- (calcd. m/z 1,129.2), respectively (Supplementary Fig. 3). This species, which results from nitrosation of the FL ligand (FL-NO, 5), is stable for several days at pH 7.0, indicating the irreversibility of the NO reaction with CuFL. FL-NO was independently prepared by the reaction of FL with HNO2 and was analyzed by LC-MS, revealing only one LC peak with m/z = 564.6, 600.1 and 1,129.2 and appearing at the same retention time as that of the reaction product of CuFL with NO (Supplementary Fig. 3). The electrospray mass spectrum of FL-15NO obtained from a reaction of FL with H15NO2 showed the shifts in m/z (565.8, 601.3 and 1,131.2) expected for the 
m/z values of 14NO versus 15NO (Supplementary Fig. 3). ESI-MS/MS analysis of the major peaks with m/z = 564.6 or 565.8 indicated that a NO functionality is embedded in the final NO reaction product (Supplementary Fig. 3). We also obtained high-resolution MS of FL-14NO and FL-15NO that showed m/z = 600.0729 and 601.0736, respectively, corresponding to [FL + NO – H + Cl]- (calcd. m/z 600.0729 and 601.0700).
Upon addition of excess NO, the 499-nm peak in the UV-vis spectrum of CuFL (pH 7.0 buffered solution) red-shifted back to the max characteristic of FL or the synthetic FL-NO compound (504 nm). This max value differs from the 506 nm band formed in the reaction of FL with 1 equiv. of Cu(I) added as [Cu(CH3CN)4](BF4) (Supplementary Fig. 3). This result indicates that the nitrosation reaction may occur at the metal-binding site—which has one oxygen and two nitrogen donor atoms—and thereby release the nitrosated ligand from the copper center. The optical spectrum of the reaction of a pH 7.0 solution of CuFL with NO had the same features as that of the dianionic form of FL at pH 7.0. In addition, the spectrum of the NO reaction solution closely resembled that of the monoanionic fluorescein formed by lowering the pH from 7.0 to 5.0 (Supplementary Fig. 3). These spectroscopic observations are consistent with those for fluorescein, the properties of which vary with pH18, and they strongly imply that FL nitrosation does not occur at a hydroxyl group on the xanthene ring. To pinpoint the position of FL nitrosation, we recorded an 15N NMR spectrum of FL-15NO that revealed 15N chemical shifts at 167.33 and 169.61 p.p.m. versus CH3NO2 in a relative ratio of 7:3. These values are in the range previously reported for N-nitrosamines19, 20. Given that the 
of 2.29 is also similar to that of previously reported N-nitrosamines19, the two separate chemical shifts might arise from the presence of different isomers in solution19, 21. A 1H NMR spectrum of the isolated FL-NO molecule also revealed the presence of a 7:3 isomeric mixture. Both the 15N chemical-shift values and the existence of isomers in the 1H and 15N NMR spectra of FL-NO clearly demonstrate that FL is N-nitrosated at the secondary amine functionality21 (Scheme 1). The fragmentation of the molecule revealed in the MS/MS spectra for N-nitrosamines FL-14NO or FL-15NO is detailed in Supplementary Figure 3. Lastly, FL-NO is brighter than both FL and CuFL, the respective quantum yields being 
FL-NO = 0.58 0.02 and FL = 0.077 0.002.
Taken together, these results demonstrate that CuFL is capable of fluorescent NO detection via NO-induced metal reduction accompanied by the release of the nitrosated ligand from copper with concomitant fluorescence enhancement (Scheme 1). Formation of an N-nitrosamine has been previously observed in the reaction of NO with a Cu(II) complex containing two anthracene groups as light-emitting units in aqueous methanol solution with fluorescence turn-on over 46 min22. Other copper fluorophore complexes have been reported as fluorescent NO indicators and operate similarly, via reduction of Cu(II) to Cu(I) by NO23, 24.
CuFL detection of NO produced by cNOS
We investigated the ability of CuFL to detect NO that is produced in SK-N-SH human neuroblastoma cells under physiological conditions, because in this cell line cNOS can be activated by estrogen to produce NO25. Estrogen administration leads to an increase in the cytosolic Ca(II) concentration, which alters the structure of calmodulin, which in turn activates cNOS. The NO-dependent fluorescence response, which we monitored after simultaneous administration of 17
-estradiol (Compound 6
Compound 6, 100 nM) and CuFL (1 M) to the cells, was completed within 5 min with a 4.0 0.6–fold increase in fluorescence (Fig. 3a,b). We also demonstrated an increase in cytosolic Ca(II) levels following addition of 17-estradiol to SK-N-SH cells using the calcium dye fluo-4 AM (Compound 7
Compound 7), a result that is consistent with estrogen induction of Ca(II)-dependent NO production (Supplementary Fig. 4 online). We observed a notably weaker fluorescence response in the presence of the cNOS inhibitor NG-nitro-L-arginine (L-NNA, Compound 8
Compound 8), which pinpoints NO as being responsible for the fluorescence change (Fig. 3c). In a control experiment, stimulated SK-N-SH cells incubated with FL in the absence of Cu(II) ion showed no fluorescence increase over a period of 25 min (Supplementary Fig. 4). This result demonstrates that CuFL, but not FL, is responsible for the fluorescence change. As another control, we treated HeLa cells (a human cervical cancer cell line lacking the estrogen receptor) simultaneously with 17-estradiol (100 nM) and CuFL (1 M) (Supplementary Fig. 4). The absence of turn-on emission in these cells indicates that the fluorescence response of CuFL is not a consequence of its interaction with 17-estradiol.
Figure 3: CuFL detection of NO produced by cNOS.
(a) NO detection in SK-N-SH cells by CuFL. Left to right, 25-min incubation of CuFL (1 M) and 5, 10, 15 and 25 min after co-treatment of CuFL (1 M) and 17-estradiol (100 nM). Images were taken with a Nikon Eclipse TS100 microscope after removing the DMEM media and washing the cells with phosphate-buffered saline (PBS). Top, fluorescence images; bottom, phase-contrast images. (b) Fluorescence intensity (F(t)/FCuFL) from a plotted against incubation time. (c) NO production with or without L-NNA. Right, NO detection in cells after 10 min co-incubation of CuFL (1 M) and 17-estradiol (100 nM). Left, NO detection in cells pretreated with L-NNA for 1 h before addition of CuFL and 17-estradiol.
We further demonstrated the value of CuFL as a probe for NO-related research by comparing its ability to image NO in cells with that of the commercially available sensor o-diaminofluorescein diacetate (DAF-2 DA, 9). First, CuFL visualized NO in the estradiol-stimulated neuroblastoma cells with brighter fluorescence than DAF-2 DA (Supplementary Fig. 4). In addition, there was only a slight fluorescence increase in DAF-2 DA–treated cells 30 min after activation of cNOS, whereas CuFL provided complete fluorescence enhancement within 5 min (Fig. 3 and Supplementary Fig. 4). These results show that CuFL allows fast and direct visualization of NO in live cells.
CuFL detection of NO produced by iNOS
In macrophages, NO is produced by iNOS1, 6, 7, 26, 27. Time-dependent NO production by Raw 264.7 murine macrophage cells pretreated with bacterial lipopolysaccharide (LPS) and interferon-
(IFN-) has been previously demonstrated by using the Griess assay28. This method colorimetrically determines the concentration of NO2- resulting from NO oxidation in the extracellular space. Fluorescence detection of NO production by stimulated macrophage cells has also been achieved by incubating the extracellular fluid with DAN and DAFs29, 30, 31. These dyes improve upon the sensitivity of the Griess assay but do not reveal NO production inside cells with spatial and temporal fidelity31. The present CuFL construct, however, readily detects NO produced in activated Raw 264.7 cells by fluorescence turn-on. We incubated macrophage cells with LPS (500 ng ml-1) and IFN- (250 U ml-1) for 4 h and then applied 1 M CuFL. We monitored the fluorescence response at 2 h intervals by microscopy (Fig. 4a). The average fluorescence slowly increased over 12 h in almost every region of the treated cells.
Figure 4: CuFL detection of NO produced by iNOS.
(a) NO detection in Raw 264.7 macrophage cells by CuFL. Left to right, CuFL (1 M) incubation with cells for 12 h and for 2, 4, 6 and 8 h after addition of CuFL into cells that were prestimulated for 4 h with LPS (500 ng ml-1) and IFN- (250 U ml-1). The times depicted in the figure are the total incubation times with CuFL. Images were taken immediately after removing the media and washing the cells three times with PBS. The instrument used was a Zeiss Axiovert 200M inverted epifluorescence microscope with differential interference contrast (DIC). Top, fluorescence images; bottom, DIC images. (b) Silencing of iNOS by RNAi in Raw 264.7 cells. A plasmid expressing shRNA was constructed to target the mRNA of iNOS (Supplementary Scheme 1 online). It was transfected into cells. The plasmid vector without insert for RNAi was transfected to establish a control cell line. The expression of iNOS in these two cell lines after stimulation by LPS and IFN- for 12 h was investigated by western blotting of the whole cell extracts with antibodies to the protein and to actin, which served as loading control (lane 1: cells with control plasmid vector; lane 2: cells with plasmid expressing shRNA for iNOS). (c) NO detection in Raw 264.7 cells with iNOS silenced by RNAi. The two lines in b were treated with LPS and IFN- for 4 h before 8 h of incubation with CuFL.
We independently confirmed the production of NO in LPS- and IFN-–treated macrophages using the Griess assay (Supplementary Fig. 5 online), which revealed identical kinetics of NO formation inside and outside the cells over the 12 h period of the experiment. To investigate further the origin of fluorescence detected by CuFL, we silenced iNOS in Raw 264.7 cells using short hairpin RNA (shRNA)–induced RNA interference (RNAi; Fig. 4b). Upon stimulation by LPS and IFN-, the cells with attenuated iNOS showed a fluorescence response that was much weaker than that of cells harboring only the plasmid vector (Fig. 4c), a result that clearly demonstrates that the fluorescence enhancement is caused by NO production in Raw 264.7 cells. In addition, the fluorescence response for stimulated Raw 264.7 cells in the presence of NG-methyl-L-arginine (L-NMA, 11), a known inhibitor of iNOS that attenuates NO production, was notably weaker than in its absence (Supplementary Fig. 5). In control experiments, we did not observe turn-on fluorescence emission either for Raw 264.7 cells stimulated by LPS and IFN- followed by FL treatment without Cu(II) ion over the 12 h incubation period or for HeLa cells treated with LPS and IFN- before CuFL incubation (Supplementary Fig. 5).
NO imaging in a Raw 264.7 and SK-N-SH mixed culture
We also monitored the fluorescence response of a mixture of Raw 264.7 and SK-N-SH cells grown on the same plate and simultaneously treated with 17-estradiol (500 nM) and CuFL (1 M) for 10 min. We observed a fluorescence increase exclusively in the SK-N-SH cells following cNOS activation by 17-estradiol–triggered Ca(II) release into the cytosol (Fig. 5). This result demonstrates that CuFL might be used to provide information about which types of cells are producing NO in a heterogeneous tissue and possibly to identify the time and location of intercellular signaling events.
Figure 5: NO detection in SK-N-SH and Raw 264.7 cells by CuFL.
Cells were treated with CuFL (1 M) and 17-estradiol (500 nM) for 10 min. The media were subsequently removed, and the cells were washed with PBS. Images were taken with a Nikon Eclipse TS100 microscope.
Cytotoxicity of CuFL and FL-NO
To test the toxicity of CuFL, we performed an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (Compound 10
Compound 10) assay on SK-N-SH cells after 5 d of incubation with CuFL (1 M). The resulting cell survival rate of 80 9% (Supplementary Fig. 6 online) indicates that the Cu(II)-containing probe is not toxic to SK-N-SH cells under the conditions of NO imaging used herein.
The MTT assay also showed 90 3% survival of Raw 264.7 cells after incubation with CuFL (1 M) for 5 d (Supplementary Fig. 6), a result indicating that CuFL is not toxic to this cell line. Furthermore, CuFL does not affect the expression of iNOS in Raw 264.7 cells upon introduction of LPS and IFN- (Supplementary Fig. 6), which suggests that the concentration of CuFL used for imaging does not interrupt the biological pathways required for NO production via gene expression.
Lastly, we examined the toxicity of FL-NO, the product of the reaction of CuFL with NO, in SK-N-SH cells by the MTT assay, which showed 97 2% cell survival after 5 d (Supplementary Fig. 6). Thus, neither CuFL nor FL-NO is toxic under the conditions used here for bioimaging of NO.
Discussion
NO is a ubiquitous biological species. To elucidate its functions, it would be valuable to have a tool for determining the temporal and spatial distribution of NO in complex biological milieu. Fluorescent probes can provide this information, but the existing imaging agents are not capable of direct, immediate and selective NO detection—three highly desirable properties.
In the present study, we have devised an imaging agent to detect NO directly in vitro at neutral pH as well as in live cells. The probe is a Cu(II) complex, CuFL (Scheme 1), containing a fluorescein-based ligand that provides excitation and emission wavelengths as well as brightness suitable for NO bioimaging. CuFL directly captures NO in a reaction that generates Cu(I) and NO+. The latter reacts irreversibly with the fluorescein-based ligand FL, which is nitrosated and released from copper with substantial turn-on fluorescence (Fig. 1 and Scheme 1). This mechanism is supported by detailed spectroscopic and mass spectrometric measurements. A comparison of CuFL with DAF-2 (Fig. 1 and Supplementary Fig. 2) showed that only CuFL can directly detect NO with fluorescence turn-on in the absence of oxygen. The utility of CuFL for imaging NO specifically over other reactive nitrogen or oxygen species in living organisms (Fig. 2), such as HNO, NO2-, NO3-, ONOO- and H2O2, increases its value for a wide range of biological studies. To our knowledge, CuFL is the first probe capable of direct, fast and specific NO detection.
In mammalian cells, NO is produced by three isoforms of NO synthase (NOS)—neuronal NOS (nNOS), endothelial NOS (eNOS) and iNOS—the catalytic activities of which are well studied1, 5, 6, 7, 26, 27. Functionally, NOS can be categorized as cNOS or iNOS. cNOS, which includes nNOS and eNOS, is regulated by the cytosolic calcium concentration and produces physiological quantities of NO. The Ca(II)-independent iNOS that provides the pathophysiological concentrations of NO is controlled by gene transcription. We applied CuFL to image NO production in Raw 264.7 murine macrophage and SK-N-SH human neuroblastoma cells (Figs. 3 and 4). Our studies in both cell lines demonstrate that CuFL affords direct visual detection of NO production in a time- and concentration-dependent manner from both cNOS and iNOS in living cells with greater than four-fold fluorescence enhancement and spatial resolution at the cellular level. Cell type–specific fluorescent NO imaging in a mixed culture of the two cell lines revealed that CuFL should be capable of detecting a source of NO production in a complex and heterogeneous biological system (Fig. 5).
Solution and live-cell studies (Figs. 1, 3 and 4 and Supplementary Figs. 2, 4 and 5) show that CuFL is the species responsible for NO detection with fluorescence turn-on. Because the Kd of CuFL is 1.5 M in 50 mM PIPES, pH 7.0, 100 mM KCl, however, the intensity of the observed signal might reflect both the degree of CuFL integrity and the amount of NO produced in the cells. Because the concentrations of FL, Cu(II) and CuFL inside cells have not been quantified, we cannot yet delineate the effects of these two variables on the intracellular fluorescence signal. A comparison of NO sensing between CuFL and the commonly used DAF-2 DA reagent (Supplementary Fig. 4), however, revealed that CuFL provides rapid NO detection with bright fluorescence signals in live cells, which is a substantial improvement over imaging with DAF-2 DA and other organic molecule–based NO probes.
Because the N-nitrosamine FL-NO generated by the chemistry of CuFL in NO detection is a member of a class of reactive molecules32, we investigated its potential cytotoxicity. An MTT assay indicated that it is not toxic at the concentration required for NO imaging in the present studies: 97 2% of SK-N-SH cells treated with FL-NO for 5 d survived (Supplementary Fig. 6). Another potential problem is that the copper ion in CuFL might damage cells before or following its reaction with NO. To address this possibility, we performed an MTT cytotoxicity assay on cells treated with 1 M CuFL; the assay indicated them to be largely (> 80%) viable after 5 d (Supplementary Fig. 6). Thus, under the conditions used for the present NO bioimaging experiments, the toxicity of CuFL is negligible.
The cytosol contains thiols that bind Cu(II) and possibly convert it to Cu(I), a species that might itself react with oxidized NO products such as NO+ or N2O3. Because NO+ is rapidly hydrolyzed to NO2- in water15, 33, 34, it will not interfere with NO imaging by CuFL. S-nitrosothiols, which are formed by reactions of thiols with NO in the presence of O2, react with both Cu(II)FL and Cu(I)FL to show turn-on fluorescence as demonstrated in experiments with SNAP (Supplementary Fig. 2). At present we cannot completely rule out the possibility that the fluorescence increase results from reaction of CuFL with S-nitrosothiols produced by NO in the stimulated cells. Their rate of formation from NO would have to be very rapid, however, because a control experiment indicated that the reaction of SNAP with CuFL is substantially slower than the NO reaction (Supplementary Fig. 2). Finally, reduction of Cu(II) by thiols may not alter the integrity or otherwise disrupt the NO-imaging ability of CuFL in cells. The binding of Cu(II) to FL is necessary for fluorescence turn-on by NO (Supplementary Fig. 2). Moreover, a mixture of FL and Cu(I) did not lead to greater fluorescence emission than FL alone either in the presence or in the absence of NO (Supplementary Fig. 3). These experiments strongly support the conclusions that the turn-on fluorescence in the stimulated cells results from the direct reaction of CuFL with NO and that intracellular thiols do not interfere with this chemistry.
In summary, we have synthesized a Cu(II)-based fluorescein compound, CuFL, for imaging NO based on redox chemistry. Reduction of CuFL by NO to Cu(I) with nitrosation of the FL ligand is accompanied by bright visible-light emission. The probe readily passes through cell membranes and can detect NO under physiological conditions. Studies of CuFL in pH 7.0 aqueous buffered solutions indicate that the NO response is direct, rapid and specific. Application of CuFL to cultures of macrophage and neuroblastoma cells reveals the time-dependent production of NO, which is measurable by fluorescence enhancement; this result also demonstrates the ability of the reagent to image NO over a wide range of concentrations. The power of CuFL is also manifest in its ability to select cells that emit NO from a background of those that do not, and to do so with spatiotemporal resolution at the cellular level.
These results will encourage the use of CuFL as a direct NO probe for investigating NO biology in a variety of contexts.
Methods
Synthesis of 2-{2-chloro-6-hydroxy-5-[(2-methyl-quinolin-8-
ylamino)-methyl]-3-oxo-3H-xanthen-9-yl}-benzoic acid (FL).
To 2 ml of EtOAc we added 7'-chloro-4'-fluorescein-carboxaldehyde (12, 30 mg, 0.076 mmol)17 and 8-aminoquinaldine (13, 12 mg, 0.076 mmol). After stirring the reaction overnight at room temperature (
22 °C), we removed the solvent under reduced pressure. We dissolved the residue in 2 ml of MeOH and cooled the reaction solution to 0 °C. We added a portion of NaBH4 (14 mg, 0.38 mmol) and stirred the reaction solution at 0 °C for 1 h before allowing it to come slowly to room temperature while stirring overnight. We removed the solvent under reduced pressure and purified the crude material using preparative TLC on silica gel (CH2Cl2:MeOH, 20:1 v/v, RF = 0.34), affording the FL product as a magenta solid (9 mg, 0.017 mmol, 22%). Details about the characterization of FL are provided in Supplementary Methods online.
Synthesis of 2-{2-chloro-6-hydroxy-5-[((2-methylquinolin-8-yl)
(nitroso)amino)-methyl]-3-oxo-3H-xanthen-9-yl}-benzoic acid, FL-NO.
We added sodium nitrite (Na14NO2 or Na15NO2, 5 mg, 72 mol, in 100 l distilled, deionized H2O) to a mixture of FL (1.5 mg, 2.8 mol, in 200 l CH3OH) and 0.3 M NaOH (aqueous, 100 l) on ice. We slowly introduced hydrochloric acid (100 l, 6 M aqueous) to the reaction solution on ice, affording a reddish precipitate. After centrifuging the solution, we redissolved both the supernatant and the precipitate in MeOH/H2O and performed LC-MS analyses. The precipitate revealed a mixture of FL-NO and FL (data not shown). The supernatant mostly contained the expected product FL-NO (Supplementary Fig. 3). We removed excess sodium nitrite from the supernatant by dialysis using a Spectra/Pro CE (Spectrum) membrane (MW cutoff 500). We obtained an orange solid sample (0.6 mg, 1.1 mol, 39%) of FL-NO by lyophilization and characterized it without further purification. TLC (silica, 1:9 CH3OH:CH2Cl2) showed only one component, RF = 0.6. Further characterization of this material is provided in Supplementary Methods.
Cell cultures and assays.
We purchased Raw 264.7, SK-N-SH and HeLa cells from the American Type Culture Collection (ATCC). We maintained all three cell lines in Dulbecco's modified Eagles' media (DMEM) (Gibco-BRL) containing 10% (v/v) heat-inactivated FBS (HyClone), 1 mM sodium pyruvate (Sigma), 100 U ml-1 penicillin, 100 g ml-1 streptomycin (Invitrogen) and 0.1 mM nonessential amino acid solution for minimal essential media (Sigma). We grew all cells at 37 °C in a humidified atmosphere of 10% CO2. We performed a nitrite assay with Griess reagents (Promega) on Raw 264.7 cells grown in DMEM that was free of phenol red. We purchased calcium sensor fluo-4 AM from Invitrogen. The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (Sigma-Aldrich) assay is described in Supplementary Methods. We analyzed the expression of iNOS in Raw 264.7 cells by western blotting on the extracts of cells stimulated by LPS and IFN-. We silenced the protein by shRNA-induced RNAi (Supplementary Scheme 1), and we used the resulting cell lines in fluorescence imaging by CuFL. The details of these experimental procedures are described in Supplementary Methods.
Other methods.
Additional details are provided in Supplementary Methods.
Note: Supplementary information is available on the Nature Chemical Biology website.
