Real-Time Visualization of Cysteine Metabolism in Living Cells with

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Real-Time Visualization of Cysteine Metabolism in Living Cells with Ratiometric Fluorescence Probes Bingying Xu, Haibo Zhou, Qingsong Mei, Wei Tang, Yilun Sun, Mengping Gao, Cuilan Zhang, Shengsong Deng, and Yong Zhang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b04493 • Publication Date (Web): 24 Jan 2018 Downloaded from http://pubs.acs.org on January 24, 2018

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Analytical Chemistry

Real-Time Visualization of Cysteine Metabolism in Living Cells with Ratiometric Fluorescence Probes Bingying Xu,a‡ Haibo Zhou,c‡ Qingsong Mei,a,∗ Wei Tang,a Yilun Sun,a Mengping Gao,a Cuilan Zhang,a Shengsong Deng,a Yong Zhanga,b,∗ a

School of Biological and Medical Engineering, Hefei University of Technology, Hefei, Anhui 230009, China Department of Biomedical Engineering, Faculty of Engineering, National University of Singapore, Singapore 117575, Singapore c Institute of Pharmaceutical Analysis, College of Pharmacy, Jinan University, Guangzhou, Guangdong 510632, China Email: [email protected], [email protected]. b

ABSTRACT: Sulfite from cysteine metabolism in living cells plays a crucial role in improving the water solubility of metabolic xenobiotics for their easier excretion in urine or bile. However, unbalance of sulfite in vivo would lead to oxidative stress or agerelated diseases, and an effective strategy for real-time imaging of cysteine metabolism in living cells is still lacking due to its low metabolite concentration and rapid reaction kinetics. Herein, a cyanine moiety based ratiometric fluorescence probe was developed for highly selective and sensitive detection of sulfite in aqueous solution and living cells. The free probe exhibited an orange emission color, and the fluorescence color would gradually change to blue once sulfite anions selectively reacted with the unsaturated carbon double bonds in the probe molecule. This ratiometric fluorescence manner endowed the probe excellent sensitivity with detection limit of 0.78 nM, which was then explored to image the kinetic process of sulfite release in hepatic BRL cells after incubating with excess amount of cysteine. This strategy opens new opportunities for revealing thiol-containing species metabolism and even quantitatively tracking their distributions in live cells or organelles.

As an important intracellular mercapto-containing amino acid, cysteine in organisms plays pivotal roles in the oxidation resistance and elimination of free radicals through catalytic metabolism to glutathione (GSH). Excessive cysteine will be further metabolized to coenzyme A in the presence of pantothenic acid, ATP and Mg2+, or catabolize to taurine or inorganic sulfite through oxidative or desulfurization metabolism.1 It has been suggested that cysteine dioxygenase (CDO) activity in hepatic cells would dramatically increase after stimulation with excess cysteine.2 Intracellular cysteine is then catalyzed to cysteine sulfonate, further enzymatically converted to βsulfinylpyruvate by aspartate aminotransferase (AAT), and decomposed to pyruvate and sulfite (SO32-) or bisulfite (HSO3).3-5 This sulfition process generally would increase the water solubility of xenobiotics with the help of cytosolic sulfotransferases for easier excretion in urine or bile, thus acts as an important biotransformation pathway for xenobiotics.6 On the contrary, excess amounts of sulfite or bisulfite could lead to oxidative stress or age-related diseases including rheumatoid arthritis, Parkinson’s disease and Alzheimer’s disease.7-9 For example, it has reported that serum sulfite could be significantly increased in patients with acute pneumonia and renal failure. Additionally, sulfite can also regulate cardiac function and inhibit calcium channels in cardiomyocytes, protect myocardium against isoproterenol-induced injury, improve pulmonary vascular structural remodeling, and have an antiatherogenic effect, and so on.3 Therefore, it is of great importance to develop selective and sensitive methods for analysis of sulfite and bisulfite in biological fluids,10-14 imaging its

existence or distribution in living cells, and even tracking its origin from specific thiols metabolism.15 Fluorometry detection strategies recently have drawn more and more attentions in trace-amount targets analysis, especially in imaging intracellular molecular events due to its advantages of easy visualization, excellent sensitivity and selectivity.16-20 In general, selective detection of sulfite or bisulfite by fluorometry methods could be achieved through two typical avenues: the specific nucleophilic addition to aldehyde/ketone groups,21-24 or unsaturated carbon double bonds.25-29 For instance, Meng et al. have reported a novel twisted intramolecular charge transfer mechanism based two-photon fluorescent probe on the coumarin skeleton for selective detection of bisulfite through nucleophilic addition reacting with aldehydes, and successfully applied the probe to detect bisulfite anion in living cells under two-photon excitation.21 However, this kind of probe sometimes would suffer from the interferences of other thiols-containing compounds, such as hydrogen sulfide and cysteine. Cyanine-based dyes which contain C=C double bonds that can specifically react with sulfite are endearingly used to detect and image sulfite in living cells because of its excellent selectivity and rapid reaction kinetic. Various welldesigned cyanine-based molecular probe or cyanineconjugated nanosensors have been broadly reported,14, 24, 30-32 yet a viable strategy for identifying cellular metabolic sulfite is still lacking and needed to be explored for disclosing its physiological and pathological functions. Herein, we synthesized a novel cyanine-structured molecular probe based on 2,3,3-trimethylindolenine and indole-6carboxaldehyde through Aldol reaction mechanism (as

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Scheme S1 shown). The obtained product had excellent watersolubility and possessed an orange colored emission and eventually could turn to blue emission after reacting with sulfite. The ratiometric fluorescence variations were then successfully applied to image exogenous sulfite or endogenous generated sulfite from cysteine metabolism in living cells (Scheme 1). Scheme 1. Schematic illustration for visualizing the metabolism of cysteine in living cells by the ratiometric fluorescent probe.

SO32[β-sulfinylpyruvat] e AAT cysteinesulfinate CDO cysteine

EXPERIMENTAL SECTION 1. Materials and chemicals. All reagents and chemicals were purchased from commercial suppliers and used without any further purification. 2,3,3Trimethylindolenine, 8-bromooctanoic acid and indole-6carboxaldehyde were purchased from Aladdin. NaCl, Na2CO3, NaHCO3, NaHSO4, Na2SO4, Na2SO3, NaHSO3, Na2B4O7, Na2HPO4, NaH2PO4, NaNO2, NaNO3, ethanol, methylbenzene, dichloromethane and other commonly used solvents were obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Silica gel (200-300 mesh) used for column chromatography was supplied by Qingdao Haiyang Chemical Co., Ltd. Mass spectra were performed on a Waters Acquity UPLC-mass spectrometer in electrospray ionization mode. UV–vis absorption spectra and Fluorescence spectra were recorded by a UV-9000S spectrometer (Shanghai Metash instruments Co., Ltd.), and a Hitachi F-2700 spectrophotometer, respectively. 2. Synthesis of the molecular probe: (E)-2-(2-(1Hindol-6-yl)vinyl)-1-(7-carboxyheptyl)-3,3-dimethyl-3Hindol-1-ium. 8-Bromooctanoic acid (3 mmol, 0.6693 g) was first dissolved in 5 mL of methylbenzene in a 25 mL round bottom flask, and then 2,3,3-trimethylindolenine (2.7 mmol, 0.4334 mL) was dropwise added into above solution. The mixture was stirred and refluxed for 72 h. After cooling to room temperature, the solvent was evaporated under reduced pressure, and the residue was washed with diethyl ether anhydrous to colorless and then dried. At last, a red solid adduct was obtained. In the second step reaction, a mixture of the intermediate adduct (0.09070 g, 0.3 mmol), indole-6-carboxaldehyde (0.04360 g, 0.3 mmol) and sodium acetate anhydrous (0.04920 g, 0.6 mmol) were first dissolved in 10 mL of ethanol, and

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then stirred at room temperature under N2 atmosphere. After reacting for 24 h, the solvent was removed under reduced pressure. The crude product was purified by silica gel chromatography (CH2Cl2/MeOH =30:1, v/v) to give the pure product, i.e. the molecular probe. 3. Cytotoxicity evaluation by MTT assay. The cytotoxicity of the molecular probe was examined by MTT assays. HeLa cells were first seeded in 96-well plates (1×103 cells per well). After incubation for 24 h, the culture medium was replaced by Dulbecco’s modified Eagle’s (DMEM) basic medium dopant with different amounts of probe (0, 50, 100, 150, 200 µM). After further incubation for 12, 24, 36 h at 37 °C in a humidity incubator containing 5% CO2, the supernatant was removed and 20 µL of 3-(4,5dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT, 5 mg/mL) phosphate buffer solution was added into each well. Four hours later, 150 µL of dimethyl sulfoxide was added to dissolve the purple formazan crystals. The plate was then shaken for 10 min, and the optical density at 490 nm was taken by a microplate reader. Each of the experiments was performed three times. 4. Confocal fluorescence imaging. HeLa cells were seeded in confocal plates with a density of 1×105 cells per well, and incubated in DMEM medium with 10% fetal bovine serum with 5% CO2 at 37 °C for 24 h. To evaluate the practicability of the probe in living cells, HeLa cells were first treated with 100 µM of probe for 4 h, and then washed with PBS (pH =7.4). After that, HeLa cells were incubated with different amounts of sulfite (0, 10, 50, 100, 200 µM) for another four hours, followed by washing with PBS. The cell imaging was then carried out on a Nikon C2 laser confocal scanning microscope imaging system. To monitor cysteine metabolism, hepatic BRL cells were first seeded in confocal plates (1×105 cells per well) and incubated for 24 h in DMEM medium with 10% fetal bovine serum. BRL cells were then incubated with 100 µM of probe for 4 h, and washed with PBS. Subsequently, the cells were treated with different concentrations of cysteine (0, 1, 10, 100 mM) and 10 mM histidine or glutathione for another 4 h, respectively. The cell imaging was then collected by a Nikon C2 laser confocal scanning microscope at the interval of 0, 0.5, 1, 2 and 4 h, respectively.

RESULTS AND DISCUSSION Synthesis of the molecular probe and its spectral properties. An ideal fluorescent probe for selective and sensitive imaging intracellular sulfite should satisfy three prerequisites: high water solubility and biocompatibility, specific reacting sites for sulfite, and ratiometric signal output to reduce negative analysis results. To realize these requirements, the molecular probe reported herein was synthesized from 8bromooctanoic acid, 2,3,3-trimethylindolenine and indole-6carboxaldehyde via two step reactions through the Aldol reaction as shown in Scheme S1.33-34 Bromooctanoic acid was first reacted with trimethylindolenine to form an intermediate product with excellent water-solubility and membrane permeability. Additionally, the product exhibited a prominent absorption peak at 275 nm, and a maximum emission peak at 455 nm upon excitation with 365 nm (Figure 1a and 1b). The molecular ion peak at 302.21 in electrospray ionization mass spectra (ESI-MS) further verified the effective conjugation


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between bromooctanoic acid and trimethylindolenine (Figure 1c). The intermediate adduct was then reacted with indole-6carboxaldehyde to obtain the molecular probe with an unsaturated C=C double bond to offer specific recognized sites for sulfite anions. Along with increasing reaction time, the absorption peak of adducts red shifted to 465 nm and its aqueous solution exhibited an orange color. Meanwhile, the probe showed a yellow colored emission with a maximum peak at 575 nm upon excitation with 365 nm. The molecular ion peak at 429.25 in Figure 1c demonstrated the successful Aldol reaction, which could also be verified by the red shift of absorption and emission spectra. Additionally, the signals at 5.7 and 6.6 ppm in 1H NMR spectrum of molecular probe in DMSO-d6, which belong to the unsaturated C=C bond, also indicated the successful reaction (Figure S1). Interestingly, the nucleophilic addition with sulfite would obviously weaken the characteristic absorption of the probe, and blue shifted its emission peak to 470 nm, which should be ascribed to the disruption of the conjugation system of the probe that could be confirmed by the variation of molecular ion peak in Figure 1c.

significant fluorescent color change from orange to blue could be observed under a 365 nm UV lamp illumination (inset images of Figure 2b). This interesting colorimetric and ratiometric fluorescence variations could be ascribed to the 1,4addition reaction at the unsaturated bond of cyanine skeleton by sulfite that breaks the conjugation structure of the probe.

Absorbance (a.u.)

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Figure 1. The absorption spectra (a), fluorescence spectra (b) and mass spectra (c) of the intermediate product (red line), the probe before (blue line) and after (black line) reacting with sulfite.

Ratiometric and colorimetric detections of sulfite. As shown in Figure 2a, the probe exhibited an orange color under natural light, and showed a main absorption band at 465 nm with a shoulder peak at 275 nm. Upon addition of cumulative amounts of sulfite, the main absorption band at 465 nm gradually decreased, but the shoulder peak at 275 nm became more and more prominent (Figure 2a). Meanwhile, the color of solution gradually changed to brown and last turned to light pink color when the concentration of sulfite increased to 1 mM (inset images of Figure 2a). Along with the absorption variations, the probe also exhibited a ratiometric fluorescence response toward different amounts of sulfite. Figure 2b demonstrated that the free probe emission band at 575 nm gradually decreased, accompanying with a new emission band at 470 nm increased. The fluorescence intensity ratios of 470 nm to 575 nm (I470/I575) could be enhanced from 0.1 to 17 when the sulfite concentration increased to 1 mM (Figure S2). Therefore, a

Figure 2. The variations of absorption (a) and fluorescence (b) spectra of the probe after reacting with different amounts of SO32-. The concentrations of SO32- in Figure 2a and 2b from top to down are 0, 1×10-9, 1×10-8, 1×10-7, 1×10-6, 1×10-5, 1×10-4, 1×10-3 M, respectively. The inset images in Figure 2a and 2b are the color response of the probe towards different amounts SO32- under natural light and 365 nm UV lamp illuminations. The concentrations of SO32- are 0, 50, 100, 150, 200, 300 µM from left to right.

The pH effects on ratiometric fluorescence of the probe in the absence and presence of sulfite was investigated. Herein, we used enhancement factors of ratiometric fluorescence (R/R0) to evaluate the pH effects, where R and R0 referred to the fluorescence intensity ratios of 470 nm to 575 nm in the presence and absence of sulfite, respectively. As Figure S3 shown, the variations of pH values did not significantly affect the ratiometric fluorescence enhancement factors from pH 5 to 10. Therefore, it is appropriate to conduct the detection experiments in neutral conditions with pH 7.0 for its feasibility on intracellular detection and imaging. Figure 3a demonstrated the fluorescence enhancement kinetics with different amounts of sulfite addition. The results showed that the ratiometric fluorescence enhancement factors increased slowly in the period of 30 minutes after additions of low concentrations of


Analytical Chemistry sulfite, which should be due to the ionization balance of sulfite in aqueous solution. However, the enhancement factors increased quickly in the first 15 min, and changed very slowly in the next 15 min which only contribute less than 10% of the entire enhancement factors when high concentration of sulfite was added, indicating it is fast to reach equilibrium in the interaction between sulfite and the probe. Thus, the detection experiments were carried out in 15 min after analytes additions. 40 µM 80 µM 100 µM

0 5 µM 20 µM

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the easy ionization of bisulfite to form sulfinic acid. Additionally, mercapto-containing species as cysteine and glutathione did not induce any fluorescence variations. The highly selective responses of the probe should be attributed to the strong nucleophilic addition of sulfite with unsaturated carbon double bonds, and that also make the probe suitable for imaging sulfite in aqueous solution or living cells, especially imaging cysteine metabolism in cells. The sensitivity study showed that the enhancement factors of ratiometric fluorescence were increased with a good linear relationship (R2=0.996) upon gradually addition of sulfite into the probe buffer solution (Figure S4). This quantitative analysis approach gave a good limit of detection (LOD) of sulfite at 0.78 nM (S/N=3), which is comparable or even better than previously reported LOD for sulfite. 10, 13, 30, 33, 35 To evaluate its applicability, the probe was further used to detect sulfite in real samples spiked with different concentrations, such as in tap water or lake water. Upon additions of these spiked sulfite samples, the fluorescence of probe showed a similar variation trends that also presented a ratiometric fluorescence enhancement between emission at 575 nm and 470. The spiked concentrations of sulfite in real water samples were calculated by the above linear equation according to the ratiometric fluorescence enhancement factors. The related standard derivatives (RSD) were obtained by repeating the identic experiments for three times at the same conditions. The satisfactory recovery and RSD results in Table S1 indicated that the reported molecular probe was suitable for sulfite detections in real samples.

R/R0

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40

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0 1 2 3 4 5 6 7 8 9 10 11 12 13

Figure 3. (a) The evolutions of enhancing factors of emission ratio upon additions of different SO32- concentrations during a period of 30 minutes. (b) Selectivity and interference research of the molecular probe toward different intracellular coexistence species with 1 mM concentrations. From 1 to 13, they are blank, cysteine, glutathione, Na2B4O7, Na2CO3, NaHCO3, Na2HPO4, NaH2PO4, Na2SO4, NaCl, NaNO2, NaNO3, NaHSO3, respectively. Gray and black bars represent the ratiometric florescence enhancement factors toward different species before and after additions of sulfite.

The selectivity and interference of the probe toward some co-existed anion ions in cells and some thiol-containing compounds were then evaluated. As Figure 3b shown, the responses of Na2B4O7, Na2CO3, NaHCO3, Na2HPO4, NaH2PO4, Na2SO4, NaCl, NaNO2 and NaNO3 except for NaHSO3 exhibited nearly no influence or interference on the enhancement of ratiometric fluorescence. We found that bisulfite could also react with the unsaturated carbon double bonds in the molecular probe, and induce a slightly weak enhancement of fluorescence intensity ratios relative to sulfite that may be ascribed to

Figure 4. Confocal images of hepatic BRL cells incubated with the probe and subsequently treated with 10 mM of cysteine for different time. From a to d, the time is 0, 1, 2 and 4 h, respectively.

Living cell imaging of sulfite. Inspired by the excellent performance of the probe toward sulfite detections in vitro, the abilities for intracellular detection and imaging were further investigated within HeLa cells. The cytotoxity research in Figure S5 showed that the cell viability was kept above 90%


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even the concentration of probe incubated in the cells was increased to 200 µM, indicating it is applicable to perform detection and imaging in living cells. HeLa cells were then incubated with 100 µM probe for about 4 h at 37 ºC. As shown in Figure S6, the probe in cells exhibited a remarkable red colored fluorescence, but not any observable fluorescence could be detected in blue channel. This phenomenon was consistent with the spectral detection results in aqueous solution. Along with increasing sulfite concentrations co-incubated in living cells, the fluorescence intensity in red channel gradually faded, and that in blue channel increased, making the merged fluorescence varied from red color to violet, and last to blue. The results in Figure S6 demonstrated that the probe is appropriate for detection of exogenous sulfite in living cells. Fluorescent imaging of cysteine metabolism in living hepatic cells. It has been widely suggested that CDO can catalytically add molecular oxygen to the thiol group of cysteine and convert it to cysteine sulfinate in the cytosol. Cysteine sulfinate is then transaminated by the mitochondria to produce sulfinopyruvate which immediately decomposes to sulfite (as Scheme 1 shown).4, 36 Previous literatures have reported that CDO activity in hepatic tissues is dramatically higher than other tissues after animals fed with a high protein diet, especially cysteine contained provisions.3, 5, 37 Based on these detailed researches, we assumed that incubation of hepatic BRL cells with the probe should successfully monitored the cysteine metabolism. As Figure 4 shown, after cultivating with 100 µM probe and 10 mM cysteine in hepatic BRL cells, the strong red fluorescence gradually quenched in the following 4 hours and the fluorescence intensity in blue channel increased, which demonstrated the endogeny of sulfite in cells and the concentrations gradually increased. Therefore, the intracellular fluorescence colors in merged confocal images gradually changed from red to purple and last to blue. However, as control experiments, without addition of cysteine in the culture medium or incubation with the same concentration of histidine in BRL cells at the same conditions did not induce identical fluorescence variations (Figure S7 and S8). Thus, the above ratiometric fluorescence changes in BRL cells should be ascribed to the generation of sulfite during cysteine metabolism. This novel cyanine skeleton based probe demonstrated an intriguing capability for real-time imaging the cysteine metabolism in living cells.

CONCLUSION In summary, we have developed a novel ratiometric fluorescence probe for visual detection of sulfite in aqueous solution and living cells. The additions of sulfite would induce the orange emission at 575 nm quenched and a new emission peak at 470 nm emerged. The intensity ratios of orange to blue exhibited a prominent relationship with the concentrations of sulfite. Furthermore, due to the excellent selectivity toward thiol-containing compounds and many other coexistence intracellular anions, the probe has been successfully demonstrated for imaging cysteine metabolism in living hepatic BRL cells. Therefore, the probe is believed to provide a powerful strategy for understanding thiols species metabolism and their pathological roles. Metabolism of other thiols-containing compounds in living cells or even the distributions of sulfite in tissues are under researching in our future works.

ASSOCIATED CONTENT

Supporting Information Additional information as noted in the text. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected], [email protected].

Author Contributions ‡ These authors contributed equally.

ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (Grant Nos. 21675038, 21305143, 21505053), the Fundamental Research Funds for the Central Universities (Grant Nos. 2014HGCH0002 and JZ2016YYPY0042), and the Science and Technology Planning Project of Guangdong Province (2015A030401045, 2016A030310089).

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Table of Contents



Scheme 1. Schematic illustration for visualizing the metabolism of cysteine in living cells by the ratiometric fluorescent probe. 84x61mm (150 x 150 DPI)


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Figure 1. The absorption spectra (a), fluorescence spectra (b) and mass spectra (c) of the intermediate product (red line), the probe before (blue line) and after (black line) reacting with sulfite. 86x79mm (150 x 150 DPI)



Figure 2. The variations of absorption (a) and fluorescence (b) spectra of the probe after reacting with different amounts of SO32-. The concentrations of SO32- in Figure 2a and 2b from top to down are 0, 1×109, 1×10-8, 1×10-7, 1×10-6, 1×10-5, 1×10-4, 1×10-3 M, respectively. The inset images in Figure 2a and 2b are the color response of the probe towards different amounts SO32- under natu-ral light and 365 nm UV lamp illuminations. The concentrations of SO32- are 0, 50, 100, 150, 200, 300 µM from left to right. 71x120mm (150 x 150 DPI)


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Figure 3. (a) The evolutions of enhancing factors of emission ratio upon additions of different SO32concentrations during a period of 30 minutes. (b) Selectivity and interference research of the molecular probe toward different intracellular coexistence species with 1 mM concentrations. From 1 to 13, they are blank, cysteine, glutathione, Na2B4O7, Na2CO3, NaHCO3, Na2HPO4, NaH2PO4, Na2SO4, NaCl, NaNO2, NaNO3, NaHSO3, respectively. Gray and black bars represent the ratiometric florescence enhancement factors toward different species before and after additions of sulfite. 68x120mm (150 x 150 DPI)



Figure 4. Confocal images of hepatic BRL cells incubated with the probe and subsequently treated with 10 mM of cysteine for different time. From a to d, the time is 0, 1, 2 and 4 h, respectively. 82x82mm (150 x 150 DPI)


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Real-Time Visualization of Cysteine Metabolism in Living Cells with

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