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A lysosomal-targeted two-photon fluorescent probe to sense hypochlorous acid in live cells Beibei Zhang, Xiaopeng Yang, Rui Zhang, Yao Liu, Xueling Ren, Ming Xian, yong ye, and Yufen Zhao Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b02361 • Publication Date (Web): 04 Sep 2017 Downloaded from http://pubs.acs.org on September 4, 2017

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A lysosomal-targeted two-photon fluorescent probe to sense hypochlorous acid in live cells Beibei Zhang,a Xiaopeng Yang,a Rui Zhang,b Yao Liu,a Xueling Ren,b Ming Xian,c Yong Yea,d* and Yufen Zhaoa,d a

Phosphorus Chemical Engineering Research Center of Henan Province, The College of Chemistry and Molecular Engineering, Zhengzhou University, Zhengzhou 450001, China b School of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou 450001, Henan, China c Department of Chemistry, Washington State University, Pullman, WA 99164, USA. d The Key Laboratory for Chemical Biology of Fujian Province (Xiamen University), Xiamen 361005, Fujian, China ABSTRACT: A two-photon reversible fluorescent probe L1 was designed and synthesized. The fluorescence intensity of the probe solution was strong, while the fluorescence of the solution was obviously quenched and the color of the solution was changed after the addition of hypochlorous acid, indicating this is “naked-eye sensor” for the detection of HClO. The probe showed great selectivity for hypochlorous acid over other reactive oxygen species (ROS) and anions. Fluorescence titration experiments showed that the probe has a low detection limit of 0.674 uM. Due to a morpholine group introduced to the naphathalimide framework, probe L1 was successfully applied to detect intracellular HClO in lysosome.

Introduction Reactive oxygen species (ROS) have emerged as prevalent and important components of both pathological and physiological processes 1-3. Among ROS, hypochlorous acid (HClO) and its conjugate base hypochlorite (ClO-) are strong oxidants in our daily life 4. With a pKa of 7.463 at 35 oC, under physiological conditions, approximately half of HClO dissociates to hypochlorite anion (ClO-), which is one of the most powerful natural oxidants5. Evidences have shown that misplaced or excessive HClO is implicated in various human diseases, such as cardiovascular diseases, neurodegeneration, arthritis and even cancer 6-10. Moreover, HClO could give rise to the rupture of lysosome, then affecting the apoptosis of cells 11. Therefore, it is necessary to find suitable chemical tools to detect HClO in lysosome of living cells. Compared to other detection methods, the non-invasive fluorescent probes have advantages such as high sensitivity and selectivity, as well as the ability for real-time detection 12-14. Most importantly fluorescent imaging can be used for the observation functional events in living cells and monitoring the biomolecules at sub-cellular levels 15. Up to now, a number of fluorescent probes for HClO detection have been reported 16-40. For example, Lin et al. reported a ratiometric probe for imaging exogenous and endogenous HClO in live cells. 36 * Corresponding Author [email protected] (Yong Ye). Tel: +86-371-67767050. Fax: +86-371-67767051.

Wang’s group reported two cyanine-based NIR “on-off” fluorescent probes. The probes were oxidized to chlorohydrins and subsequent degradation by HClO 37-38. These probes have shown good selectivity to HClO. However, the performance of sensitivity and response speed are still to be improved. In addition, few probes have been developed for ratiometric monitoring HClO in lysosome 41-45. Two-photon microscopy (TPM) collocates with the long-wavelength excitation and has many advantages such as low phototoxicity, deeper tissue penetration, and three-dimensional (3D) imaging of living tissues 46−56. To the best of our knowledge, except for several mitochondria-located HClO probes, there is no report yet describing a two-photon reversible probe for HClO detection in lysosome. The above mentioned concerns encouraged us to develop novel lysosome-targetable two-photon fluorescent probes for the ultrasensitive and specific imaging of intracellular HClO. Naphathalimide is a well-known TPM fluorophore and has virtues such as large Stokes shift, high extinction coefficient, and good photo-stability 57-59. It is also reported morpholine group is lysosome targeting. Subsequently, by introducing (aminoethyl)morpholine into naphathalimide, we should get a novel lysosome-targetable two-photon fluorescent probe for HClO detection. To date, a few reversible fluorescence probes sensing redox process have been reported 60-61, but a reversible fluorescence probe for intracellular HClO/GSH redox cycle monitoring has yet to be established. To acquire a reversible fluorescent probe for the redox cycle between HClO and GSH, we choose a methyl thioether group

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(MeS) as the modulator. The reversible detection was expected to be achieved depending on the redox chemistry of the versatile methyl sulfoxide. Experimental Apparatus, regents, and chemicals Fluorescence spectra measurements were performed on a F-4500 FL spectrophotometer, and the excitation and emission wavelength band passes were both set at 5.0 nm. Absorption spectra were measured on a Perkin Elmer UV-2102 double-beam UV/VIS spectrometer. NMR spectra were recorded on a Bruker DTX-400 spectrometer in CDCl3, using TMS as the internal standard. Mass spectral determination was carried on a HPLC Q-T of HR-MS. Unless otherwise noted, all starting materials and reagents were purchased from Tokyo Kasei Kogyo (TCI: Tokyo, Japan). AR grade or dry grade solvents were purchased from Alfa-Aesar and used without further purification. N-(Morpholinoethylamino)-4-Bromo-1,8-Naphthalimide (LA). 4-Bromo-1,8-naphthalic anhydride (3.2 g, 11.58 mmoL) and 4-(2-aminoethyl)-morpholine (3.0 g, 23.16 mmoL) were dissolved in 30 mL ethanol and the solution was refluxed for 8 hours. After cooling to room temperature, the yellowish sediments were collected by filtration. The crude products were purified by flash silica gel column chromatography (ethyl acetate) to afford the corresponding products LA as pale yellow solid (4.2 g, yield: 85%). Synthesis of probe L1 Compound LA (0.388 g, 1 mmol) was dissolved in 5 mL DMF. K2CO3 (138 mg) and CH3SNa (140 mg) was added into the solution in sequence. Then the mixture was heated to 80 ℃ for 4 h under N2. After the reaction was completed, the mixture was poured into iced-cold water to afford yellow solid. The solid was collected by filtration. The crude product was purified by silica gel chromatography (200-300 mesh) eluted with dichloromethane to give L1 as pale yellow solid (313 mg, yield 88%). mp: 158-159℃. 1H NMR (400 MHz, CDCl3, ppm): 2.68 (s, 4 H), 2.72 (s, 3 H), 2.78 (s, 2 H), 3.75 (s, 4 H), 4.39 (t, 2 H, J=7.0 Hz), 7.47 (d, 2 H, J=7.8 Hz), 7.76 (m, 1 H), 8.50 (m, 2 H), 8.63 (m, 1 H). 13C NMR (400 MHz, CDCl3, ppm) δ 14.87, 37.03, 53.75, 56.12, 66.90, 118.65, 120.98, 123.03, 126.56, 128.15, 128.97, 129.73, 130.73, 131.50, 146.54, 164.06. HR-MS: Calcd for [C19H21N2O3S]+: 357.1267, found: [M+H]+: 357.1271. N-(Morpholinoethylamino)-4-Methylsulfinyl-1,8-Naphtha limide (LB). To a solution of N-(morpholinoethylamino)-4methylthio-1,8-naphthalimide (L1) (100 mg, 0.28 mmol) in 15 mL anhydrous DCM, m-CPBA (96.6 mg, 0.56 mmol) was added gradually. The mixture was stirred for 6 hours at 0 ℃. When the reaction was completed, 5 mL saturated NaHCO3 solution was added into the mixture

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and it was stirred continuously for 2 hours at room temperature. The solvent was removed under reduced pressure. The crude product was purified by silica gel column chromatography (dichloromethane/methyl alcohol=20:1) to afford the compound (LB) as pale yellow solid (30 mg, yield 30%). 1H NMR (400 MHz, CDCl3) δ 8.76 (d, J = 7.64 Hz, 1H), 8.68 (d, J=6.76 Hz,1H), 8.43 (d, J =7.68 Hz, 1H), 8.30 (d, J =7.88 Hz, 1H), 7.89 (dd, J = 7.56, 7.68 Hz, 1H), 4.82 (dd, J = 6.12, 6.20 Hz, 2H), 4.48 (m, 2H), 3.82 (d, J = 12.2 Hz, 2H), 3.66 (t, J =6.92 Hz, 2H) ,3.41 (dd, J = 3.08, 1.28 Hz, 4H), 2.89 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 163.47, 163.35, 149.66, 132.05, 131.24, 128.28, 128.25, 127.63, 127.07, 124.43, 123.22, 123.03, 67.70, 64.88, 64.83, 61.57, 43.37, 34.23. HR-MS: Calcd. For C19H21N2O4S+: 373.1217, found: [M+H]+: 373.1220 Confocal microscopy imaging Cells were seeded on 24 well chambered cover glass at a density of 1x106 cells mL-1 for 24 h. Probes dissolved in DMSO were added to the cells medium (500 mL) at varying final concentrations. After incubating for 30 min, excess probes were removed by gentle rinsing with phosphate buffered saline (PBS, pH 7.4) three times. To observe the subcellular distributions of the probe, the cells were treated with a lysosome staining probe, Lyso-Tracker Red DND-99 (50 nM) for additional 30 min. The media was removed and the cells were washed three times with PBS buffer (pH 7.4). Fluorescence images were collected by sequentially line scanning with Leica TCS SP8 MP confocal laser-scanning microscope. Lyso-Tracker Red DND-99 was excited at 577 nm and its red emission was collected in the range of 590 nm; probes were excited at 400 nm and their green emissions were collected in the range of 500-510 nm. O

O

N

O

N

O

N

O

O

N

N

O

O

O

m-CPBA

MeSNa

yield 30%

K2CO3; DMF; N2 yield 88% Br

S

LA

N

L1

O

S

LB

Scheme 1: The synthesis of L1 and LB

Results and discussion To design highly selective fluorescent probes for HClO, the specific reaction between a recognition group and HClO is crucial. Alkyl thioether can be easily oxidized to sulfoxide. Sulfoxide is a redox sensitive functional group which can be reduced to thioether. Therefore, we envisioned that methyl thioether might be an ideal receptor for HClO over other biological active molecules. Furthermore, a preferable probe for HClO quantification in cells needs to have ratiometric fluorescent readouts and membrane permeability. In view of the intramolecular charge transfer (ICT) strategy 62-63,

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1,8-naphthalimide with a highly effective “push−pull” (sulfur anion-imide) structure can be used in the construction of ratiometric fluorescent probes which can provide built-in correction for environmental factors. Hence, we prepared a fluorescent probe L1 through a simple substitution between 4-bromine-1,8-naphthalimide and sodium thiomethoxide (Scheme 1, Experimental method shown in SI). The probe’s structure was fully characterized by 1H NMR, 13C NMR and HR-MS spectrometry (Supporting Information Figures S1-S3). Probe L1 contains an alkylmorpholine group which was used as the lysosomal targeting group. To develop a suitable solvent system for L1 to detect HClO, we tested many different solvents. It was interesting to find that only in deionized water and PBS buffer L1 could respond to HClO well. We recognized that pure buffer systems would be ideal for the uses in biological detections. As such, in the subsequent experiments, we selected PBS buffer as the testing system to investigate the responses of the probe to hypochlorite. The spectroscopic properties of L1 were studied under physiological conditions (PBS, 10 mM, pH 7.4). First, we investigated its selectivity for HClO vs other anions (AcO-, Br-, Cl-, CO32-, F-, H2PO4-, HCO3-, HPO42-,I-, NO3-, PO43-, SO42-) and ROS (H2O2, S2O82-, HClO4, NO. and O2-.) The absorption spectra of L1 in the presence of different species were shown in Figure 1. Upon the addition of ClO-, the color of the solution changed from yellow to colorless (Figure 1 inset). The absorption peak at 405 nm simultaneously decreased, and a new absorption peak at 350 nm increased with the addition of ClO- (10 equiv.). In contrast, the addition of other anions and ROS had almost no effects on the absorption spectra. These results indicated that L1 can be devoted to a “naked-eye sensor” for the detection of ClO-.

Figure 2. UV-vis spectra of L1 (10.0 µM) in the presence of ClO- (0.1-10 equiv.) in PBS buffer (0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0). We further checked concentration-dependent colorimetric responses of L1 to ClO-. The absorption spectra of probe L1 in the absence and presence of different amounts of ClO- were shown in Figure 2. With the increase of ClO- concentrations, absorption peaks of L1 at 405 nm gradually decreased, and a new absorption peak at 350 nm appeared with a distinct isosbestic point at 366 nm. These results indicated that L1 can be used for ratiometric detection of hypochlorite. We then examined the specificity of the probe’s fluorescence responses toward ClO-. L1 showed strong fluorescence in the absence of ClO- at the excitation of 405 nm (one-photon excitation wavelength) and 800 nm (two-photon excitation wavelength), which was attributed to the highly effective ICT structure. As shown in Figure 3, L1 did not display obvious fluorescence spectra changes in the presence of other ROS, RNS and anions including H2O2, HClO4, O2-, NO., S2O82-, F-, Cl-, Br-, I-, PO43-, HPO42-, H2PO4-, NO3-, CO32-, HCO3-, AcO- and SO42-. Interestingly, the introduction of ClO- into the solution of L1 resulted in an intensive fluorescence quenching at 505 nm. We can also found green fluorescence upon excitation at 365 nm employing a hand-hold UV lamp (Figure 3 inset). After the addition of ClO- (10 equiv.), the green fluorescence was quenched quickly. Other biologically relevant cations such as Al3+, Cu2+, Hg2+, Pb2+ and Zn2+ did not lead to any notable changes under the same test conditions (Figures S4 and S5). Furthermore, the competitive experiments of the background species to the selectivity of L1 for ClOshown that they had no interference with the detection of ClO- (Figure 4).

Figure 1. UV/vis spectra of L1 (10 µM) in PBS with different anions (100 µM). Inset: the photo of L1 (left) and L1+ ClO- (10 equiv.) (right).

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L1 at 505 nm had no change in the absence of ClOduring the experimental period (1 h). The fluorescence was quenched immediately after the addition of 10 eq. ClO- (Figure S6) and the reaction were almost completed within 2.5 min. This indicates that L1 has a fast response to ClO-.

Figure 3. Fluorescence spectra of L1 (10 µM) in the presence of 100 µM different anions in PBS (λex =405 nm, slit = 5 nm)

Figure 4. Fluorescence intensity (at 505 nm) of L1 (10 µM) upon the addition of 100 µM ClO- in the presence of 100 µM background anions (1-18: F-, Cl-, Br-, I-, PO43-, HPO42-, H2PO4-, NO3-, CO32-, HCO3-, AcO-, SO42-, ClO-, H2O2, HClO4, S2O82-, NO., O2-) in PBS (pH=7.4, 10 mM) solution. (λex = 405 nm, slit = 5 nm). Next titration experiments were carried out to understand the probe’s concentration-dependent fluorescence response to ClO-. As shown in Figure 5a, the probe itself showed a strong fluorescence band at 505 nm. Upon the addition of ClO-, the fluorescence peak at 505 nm decreased gradually because of the oxidation of thioether. In addition, a good linear relationship between the fluorescence intensities at 505 nm and ClOconcentrations (3-150 µM) was obtained. The detection limit (3σ/slope) of probe L1 for the determination of ClOwas estimated to be 0.674 uM (Detail method shown in SI). The regression equation was Y = 196.51584 -39.56486X with a linear coefficient R2 = 0.99376, which indicated that our probe L1 could detect ClOquantitatively under physiological conditions. Reaction time is another important parameter to evaluate the feasibility of a probe in real time detection. Therefore, time-dependent response of L1 to ClO- was evaluated. In PBS (pH=7.4, 10 mM), the fluorescence of

Figure 5. (a) Fluorescence spectra of L1 (10 µM) with gradual addition of various amounts of ClO- (from bottom 0 to 7 equiv.) in PBS (pH=7.4, 10 mM). (b) The fluorescence intensity (at 505 nm) of L1 (10 µM) as a function of the ClO- concentration (3-150 µM) in PBS (pH=7.4, 10 mM) (λex =405 nm, slit = 5 nm).

In order to obtain the optimal working pH range, the fluorescence intensity of L1 and L1+ClO- under different pH was tested (Figure S7). The fluorescence intensity of L1 at 505 nm showed no obvious changes from pH 4.0 to pH 10.0, indicating that the probe was stable in this pH range. The fluorescence was quenched efficiently after the addition of ClO- at the above pH range. Thus the probe has the potential to be used to detect ClO- with high sensitivity in lysosome and in complicated surroundings. To date, many fluorescent probes have been synthesized and applied to detect HClO and GSH, separately. The redox cycle between HClO and GSH is a complicated process. The development of reversible fluorescent probes for probing HClO/GSH concentration changes would be very helpful for understanding

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intracellular HClO/GSH redox cycle. This is still an unmet need in the field. We hypothesized that the oxidized product of L1 upon treated with ClO- could be converted back to L1 by certain reductants, thus achieving the regeneration of the probe. This process was then tested with a verity of reductants. (Figures 6 and S8). We found that GSH was the most powerful reductant and it could resume the fluorescence intensity to almost the original level. Other reductants, such as Cys, Hcy, H2S, HSO3-, Fe2+, S2O42and Vc, could not regenerate the probe’s fluorescence. So the quench of L1 by ClO- is reversible by GSH. The redox cycles could be repeated at least five times (Figure S9). These results suggested that the probe L1 could be applied to monitor the redox cycles between ClO- and GSH continuously.

intracellular fluorescence was observed (Figure 7d). A bright field transmission image of cells with L1 and L1 with NaClO confirmed that the cells were viable throughout the imaging experiments (Figures 7a and 7c). Therefore, these results demonstrated that probe L1 was cell membrane permeable and capable of fluorescence imaging of ClO- in cells.

Figure 7. Fluorescence images of HClO in MCF-7 cells with 10 µM solution of L1, bright-field transmission images (a, c) and fluorescence images (b, d) of MCF-7 cells incubated with 0 µM , 100 µM of NaClO for 5 min, respectively.

Figure 6. Fluorescence recovery of L1 (1 uM) in PBS (pH=7.4, 10 mM) (λex =405 nm, slit = 5 nm). (1) Black line: L1 only; (2) red line: L1 with 5 equiv of ClO-; (3) green line: L1+ ClO- and 5 mins later treated with GSH. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article). According to the experimental results and previous reports, we speculated that the thioether group of L1 was oxidized to sulfoxide by ClO- and then the fluorescence was turned off. To verify the proposed mechanism, the mixture of L1 and ClO- was checked by high-resolution-mass spectrometry (HR-MS). As shown in Figure S10, ESI-MS spectra showed the presence of a dominant peak at m/z 373.1216 and two noticeable isotopic peaks at 374.1248 and 375.1213, which correspond to the characteristic MS peaks of the oxidation product LB (calcd m/z 373.1222, 374.1256 and 375.1180). The authentic sample of LB was prepared by the reaction of mCPBA with L1, whose structure was confirmed by 1H NMR, 13C NMR and HR-MS (Figures S11-13). We further investigated the fluorescence imaging of L1 in biological systems 64-66. Live MCF-7 cells were loaded with 10 µM L1 and showed green fluorescence (Figure 7b). When the cells were incubated with 100 µM ClO- for another 5 min, a significant quenching in the

Figure 8. One-photon fluorescence images of live 4T1 cells: (a, d) bright-field transmission images of 4T1 cells, (b) 4T1 pretreated with L1 (10.0 µM) for 30 min, (e) pretreated with NaClO (100 µM) for another 30 min after 4T1 cells preincubation with L1 for 30 min, (c, f) overlap images of a with b, d with e, respectively. Fluorescence images were obtained using a 405 nm light source. Fluorescence emission windows: 470-650 nm. Scale bar: 5 um.

To improve the accuracy of intracellular ClOdetection and demonstrate that TPM possesses higher sensitivity than one-photon microscopy in the bioimaging applications, one-photon and two-photon fluorescence imaging techniques were adopted, respectively. As shown in Figures 8b and 9b, the strong fluorescence was

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observed from the control 4T1 cells. Interestingly, a fluorescence quenching of 4T1 cells preincubation with L1, then pretreated with NaClO (100 µM) for 30 min was observed (Figures 8e and 9e), indicating that L1 was sensitive enough for ClO- detection in 4T1 cells. Notably, the two-photon imaging of native with higher resolution was successfully obtained because TPM effectively reduced the background fluorescence by using the long-wavelength excitation. Encouraged by the excellent ClO- sensing performance of L1, we exploited intracellular location of the fluorescent probe L1 inside cells. Firstly, L1 and Lyso Tracker Red DND-99 (a commercially available lysosomal marker) were coincubated with live 4T1 cells, which was then pretreated with NaOCl (10.0 µM) for 30 min. As shown in Figure 10, the images of green channel and red channel merged well, and it demonstrated that probe L1 mainly stained in the lysosome of live cells. Co-localization experiments by co-staining the 4T1 cell with Lyso Tracker Red DND-99 and the probe L1 gave the Pearson's and overlap coefficients of 0.8789 and 0.8937, respectively. Above results suggested that probe L1 could image ClO- in lysosomes very well.

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for Lyso-Tracker fluorescence; (d) merged image from panels (b) and (c); (e) Intensity profile of linear region of interest across the 4T1 cell costained with Lyso-Tracker Red and probe L1; (f) intensity correlation plot of L1 and Lyso-Tracker Red. Pearson’s Correlation: 0.8789 Overlap Coefficient: 0.8937, Scale bar = 5µm. Conclusions In summary, we have successfully developed an intramolecular charge transfer (ICT)-based two-photon fluorescent probe L1 for the selective detection of hypochlorous acid (HOCl). L1 exhibited good selectivity to HOCl over other ROS, RNS, metal ions, which might be ascribed to the selective oxidation of thioether. The probe has a very good application potential because it exhibited excellent selectivity, sensitivity and naked eye detection of HOCl. Experiments on bioimaging applications demonstrate that L1 can specifically track the intrinsic HOCl levels of lysosome in living cells. The present study offers an easy-to-synthesize specific two-photon fluorescent probe for monitoring HOCl, as well as an excellent tool for exploring physiological and pathological functions of HOCl in living cells.

ACKNOWLEDGMENT This work was financially supported by NSFC (No. 21572209) and Program for Innovative Research Team (in Science and Technology) in University of Henan Province (No. 17IRTSTHN002).

Supporting Information Figure 9. Two-photon fluorescence images of live 4T1 cells: (a, d) bright-field transmission images of 4T1 cells, (b) 4T1 pretreated with L1 (10.0 µM) for 30 min, (e) pretreated with NaClO (100 µM) for another 30 min after 4T1 cells preincubation with L1 for 30 min, (c, f) overlay of a with b, d with e, respectively. Fluorescence images were obtained using an 800 nm light source.

The Supporting Information is available free of charge on the ACS Publications website at DOI: 1 H NMR, 13C NMR, and HR-MS spectra; fluorescent spectroscopic data (PDF)

Notes The authors declare no competing financial interest.

REFERENCES (1) AutrEaux, B.D.; Toledano, M.B. Nat. Rev. Mol. Cell Biol., 2007, 8, 813. (2) (3) (4) (5) (6) (7) (8)

Figure 10. Confocal fluorescence images of live 4T1 cells pretreated with L1 (10.0 µM) and Lyso-Tracker Red DND-99 (500.0 nM) for 20 min: (a) bright-field image; (b) green channel for probe fluorescence; (c) red channel

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