Discriminating Live and Dead Cells in Dual-Color Mode with a Two

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Discriminating live and dead cells in dual-color mode with a two-photon fluorescent probe based on ESIPT mechanism Minggang Tian, Jie Sun, Yonghe Tang, Baoli Dong, and Weiying Lin Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b04252 • Publication Date (Web): 07 Dec 2017 Downloaded from http://pubs.acs.org on December 7, 2017

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Analytical Chemistry

Discriminating Live and Dead Cells in Dual-Color Mode with a Two-Photon Fluorescent Probe Based on ESIPT Mechanism Minggang Tian, Jie Sun, Yonghe Tang, Baoli Dong, Weiying Lin* Institute of Fluorescent Probes for Biological Imaging, School of Chemistry and Chemical Engineering, School of Materials Science and Engineering, University of Jinan, Jinan, Shandong 250022, P.R. China. ABSTRACT: Discrimination of live and dead cells is an important task in biological, pathological, medical, and pharmaceutical studies. In this work, we have developed a novel fluorescent probe DACA that can discriminate live and dead cells in a dual-color mode for the first time. DACA can stain dead cells with blue fluorescence peaked at 440 nm, while it can also label live cells with orange emission peaked at 570 nm. Compared with one-color fluorescent probes, such a dual-color probe can efficiently avoid false positive results from cellular autofluorescence and misleading signals brought by inhomogeneous staining, and thus can supply more accurate information in biological applications. By means of DACA, the health status of tumor cells pretreated by H2O2 and ultraviolet radiation has been successfully detected and imaged. Moreover, DACA and the hydrolyzed product exhibit excellent two-photon properties. Live and dead cells, as well as the zebrafishes, have been discriminated with dual emission colors under one- and two-photon microscope. These results demonstrate that DACA is a powerful tool for dual-color distinguishing live and dead cells in vitro and in vivo.

Discrimination of live and dead cells is crucial and necessary in the researches of biology, pathology, medicine, and pharmacology.1-4 For example, cell viability assay, dependent on the discrimination of live and dead cells, is indispensible in plenty of investigations of these areas. In pharmaceutical studies, cell viability test is an essential tool to evaluate the efficacy of drugs.1,5-7 Furthermore, for the discovery of drugs, dyes, and other biological reagents, cell viability assay is also always an important test to evaluate their cytotoxicity.8-12 Up to now, a reagent is needed to distinguish live and dead cells, 13,14 since it is greatly difficult to clearly understand cellular status just from cell morphology. Consequently, discovery of reagents for discrimination of live and dead cells is an essential and significant task. Up to now, the reagents used for discrimination between live and dead cells could be roughly classified into two kinds. The first kind is the colorimetric reagents.13-16 Amongst, 3(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) is a frequently used reagent in cytotoxicity and cell proliferation tests.17 MTT itself exhibits pale yellow color and short-wavelength absorption, and it can turn to deep purple formazan with long-wavelength absorption under the reduction of mitochondrial reductase. Therefore, cell viability can be obtained by measuring long-wavelength absorbance of the formazan. The second kind is the fluorescent probes. Compared with the colorimetric reagents, the fluorescent probes are more favorable tools due to its high sensitivity and capability of in situ imaging of cells and tissues.18,19 Propidium iodide (PI) and calcein acetoxymethyl (Calcein AM) are commercialized fluorescent probes to label dead and live cells, respectively. PI is impermeable to live cells but can penetrate into dead cells, bind to RNA and give strong fluorescence.20-22 On the other hand, Calcein AM is non-fluorescent in aqueous solutions as well as in dead cells, while it can be hydrolyzed to

calcein with strong fluorescence by the active esterase in live cells.23,24 Due to the importance, fluorescent probes for the discrimination of live and dead cells were also reported in recent works. A Cu2+-labeled dansyl compound was developed by Yu for selectively labeling dead cells.25 A special green fluorescent protein (GFP) variant was also devoted by Ozawa et al. for detection of cell apoptosis, by means of a special mitochondrial enzyme that would be released to cytoplasma during apoptosis.26 More recently, A near-infrared fluorescent probe for the imaging of necrotic cells was presented by Pecoraro et al., based on the fact that plasma membranes of dead cells are more permeable.27 Although plenty of elegant works have been devoted for the discrimination of live and dead cells, shortcomings are still existed. One of the drawbacks is that the currently available fluorescent probes can only label live or dead cells with one emission color. With this kind of probes, false positive resulted from intracellular endogenous fluorescent species may be obtained. Moreover, misleading signals could also be detected with these probes, due to the unavoidable inhomogeneous staining. To avoid possible misleading results and supply more accurate information in biological applications, a fluorescent probe that can label live and dead cells in dual colors is in great demand. With a dual-color probe, false positive signals and inhomogenous staining could be recognized by simultaneously detecting the fluorescence in two channels. However, such a probe has not been reported yet. In this work, we have developed a fluorescent probe for discriminating live and dead cells in a dual-color mode. The probe was synthesized by the acetylization of 3hydroxylflavone. The probe displays blue emission peaked at 440 nm in dead cells and orange one at 570 nm in live cells. Thus, live and dead cells can be clearly discriminated in two emission colors. With this probe, the unhealthy status of tumor

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cells induced by H2O2 and ultraviolet (UV) radiation has been successfully detected. Moreover, live and dead zebrafishes have been also distinguished under two-photon microscope.

EXPERIMENTAL SECTION Materials. All chemicals used are of analytical grade, 2hydroxybenzoleactone was purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). 4Diethylaminobenzaldehyde etc. was purchased from J&K Chemical (Beijing, China). The solvents used in the spectral measurement are of chromatographic grade. Spectroscopic measurements. The UV-visible-near-IR absorption spectra of dilute solutions were recorded on a U2910 spectrophotometer using a quartz cuvette having 1 cm path length. One-photon fluorescence spectra of dilute solutions were obtained on a HITACH F-2700 spectrofluorimeter equipped with a 450-W Xe lamp. Two-photon ones were measured on a SpectroPro300i and the pump laser beam came from a mode-locked Ti:sapphire laser system at the pulse duration of 200 fs, a repetition rate of 76 MHz (Coherent Mira900-D). PBS buffer solution: 10 mM, NaCl, NaHPO4·12H2O, NaH2PO4·2H2O, pH = 7.40. Cell culture and staining methods. HeLa cells were grown in H-DMEM (Dulbecco’s Modified Eagle’s Medium, High Glucose) supplemented with 10% FBS (Fetal Bovine Serum) in a 5% CO2 incubator at 37 °C. For living cells imaging experiment of the probes, the culture medium surrounding the cells were firstly removed, and the cells were washed with PBS twice. Then the cells were incubated in 1 mL of PBS. On the other hand, 1 mM stock solutions of the probe in DMSO were prepared. After that, 2 µL of stock solutions were mixed evenly with 1 mL PBS (pH 7.4) in a tube. The cells were incubated with the above mixed solutions at 37 °C. After rinsing with PBS three times, cells were imaged immediately. Fluorescent imaging methods. Confocal fluorescence and two-photon images were obtained with a Nikon A1R confocal laser scanning microscope.. In two-photon experiments, excitation wavelengths were 800 nm from a Ti:sapphire femtosecond laser source (Coherent Chamelon Ultra), and the incident power on samples was ~ 4 mW, modified by means of an attenuator and examined with Power Monitor (Coherent). A multiphoton emission filter (FF01–650) was used to block the IR laser. The differential interference contrast (DIC) images were taken with 488 nm Ar+ ion laser. H2O2 pretreatment. Stock solutions of 5 M H2O2 was firstly prepared in PBS buffer solutions. 0 µL, 1 µL, 2 µL, 8 µL, and 16 µL of the stock solutions was added into 1 mL of culture medium to obtain the culture medium with 0 mM, 0.5 mM, 1 mM, 4 mM, and 8 mM of H2O2, respectively. The culture medium with different concentration of H2O2 was used to incubate live HeLa cells for 2 h. After that, the culture medium was removed, and the cells were washed with PBS buffer solutions for three times. 2 µM of DACA in PBS buffer solutions was then used to stain these cells for another 40 min. After rinsing with PBS three times, cells were imaged immediately. UV irradiation. HeLa cells cultured in 1 mL of culture medium were placed under a UV lamp. UV light centered at 365 nm was used to irradiate the cells for different time (0 min, 10 min, 30 min, 40 min, 60 min, and 100 min). After that, the culture medium was removed, and the cells were washed with PBS for three times. 2 µM of DACA in PBS buffer solutions

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was then used to stain these cells for another 40 min. After rinsing with PBS three times, cells were imaged immediately. Photostability measurements. To measure the photostability of DACA, dead cells were used to avoid the hydrolysis. HeLa cells were firstly treated with 4% paraformaldehyde for 30 min to obtain dead cells. These cells were then incubated with 2 µM of DACA in PBS buffer solutions for 40 min. After rinsing with PBS three times, these cells were placed under the confocal microscope with ceaseless exposure to 405 nm or 800 nm laser. A series of fluorescent images was obtained during the exposure period, and the time-dependent intracellular emission intensity was used to evaluate the photostability of DACA. To measure the photostability of the hydrolyzed product, live HeLa cells were firstly incubated with 2 µM of compound 1 in PBS buffer solutions for 40 min. After rinsing with PBS three times, these cells were placed under the confocal microscope with ceaseless exposure to 405 nm or 800 nm laser. A series of fluorescent images was obtained during the exposure period, and the time-dependent intracellular emission intensity was used to evaluate the photostability. Synthesis and characterization of DACA Synthesis of 2-(4-(diethylamino)phenyl)-3-hydroxy-4Hchromen-4-one (1). 4-(N,N-diethylphenyl)-aldehyde (1.77g, 10 mmol) and 1-acetyl-2-hydroxybenzene (1.2 mL, 10mmol) were added to a round-bottom flask containing 20 mL ethanol. Then, NaOH (1.2g, 30 mmol) with 1mL of water was added into the flask under stirring. The system was stirred for 24 h at room temperature, and NaOH (0.4g, 10 mmol) with 1mL water and 10 mL of hydrogen peroxide solution (30%) were added sequentially under ice-water bath. After that, the flask was heated to 55 ℃ and stirred for 1 h. The mixture was cooled down to room temperature, and poured into ice-water under stirring. Diluted H2SO4 was added to tune the pH value to ~ 6, and 200 mL of dichloromethane was used to extract it twice. Dichloromethane was then dried with anhydrous MgSO4, filtered, and distilled in a rotary evaporator to obtain the crude product. Pure product was finally obtained by recrystallization in ethanol, presented as orange crystals, with a yield of ~ 50%. 1 H NMR (400 MHz, DMSO-d6) δ (ppm): 9.15 (s, 1H), 8.10 (t, J = 8.3 Hz, 3H), 7.86 – 7.62 (m, 3H), 7.51 – 7.38 (m, 1H), 6.81 (d, J = 9.2 Hz, 2H), 3.43 (q, J = 7.0 Hz, 4H), 1.14 (t, J = 7.0 Hz, 6H). Synthesis of 2-(4-(diethylamino)phenyl)-3-acetoxyl-4Hchromen-4-one (DACA). Compound 1 (0.6g, 2 mmol) was added into a flask containing 5 mL of acetic anhydride. The mixture was heated to 80 ℃ for 12 h and then cooled down to room temperature. The system was then poured into ice water under stirring. Dichloromethane was then dried with anhydrous MgSO4, filtered, and distilled in a rotary evaporator to obtain the crude product. Pure product was obtained by column chromatography, presented as yellow powder, with a yield of ~ 57%. 1H NMR (400 MHz, DMSO-d6) δ (ppm): 8.05 (dd, J = 7.9, 1.3 Hz, 1H), 7.84 (d, J = 9.2 Hz, 2H), 7.77 (d, J = 8.3 Hz, 1H), 7.51 (t, J = 7.4 Hz, 1H), 6.83 (d, J = 9.2 Hz, 1H), 3.45 (q, J = 7.0 Hz, 3H), 2.37 (s, 2H), 1.15 (t, J = 7.0 Hz, 4H). 13 C NMR (101 MHz, DMSO) δ (ppm): 170.94, 168.41, 156.44, 155.21, 150.13, 134.58, 131.47, 130.16, 125.74, 125.38, 123.23, 118.77, 114.85, 111.48, 44.26, 40.64, 40.43, 40.22, 40.01, 39.80, 39.59, 39.39, 20.92, 12.85. HR MS: m/z, calculated as 352.1543, found 352.1549.


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Analytical Chemistry

RESULTS AND DISCUSSION Design and synthesis of the probe. To achieve the goal of discriminating live and dead cells in a dual-color mode, we have conceived an idea to take advantages of excited-state intramolecular proton transfer (ESIPT) mechanism, based on the fact that active esterase would be deactivated in dead cells. As shown in Scheme 1a, the probe was constructed by the acetylation of a fluorescent dye with ESIPT property. The acetylation would block the ESIPT process. In live cells, the probe could be hydrolyzed by the active esterase with a product bearing long-wavelength emission, due to the recovery of ESIPT process. In dead cells, esterase would be deactivated, ESIPT process is still blocked, and the probe would give short-wavelength fluorescence. In this way, live and dead cells could be labeled with long and short-wavelength emission, respectively. In this work, 3hydroxylflavin (compound 1, molecular structure in Scheme 1b) was selected as the platform for the design of the probe, because it exhibits ESIPT properties. We constructed the probe DACA via the acetylation of compound 1, and DACA has been synthesized following the synthetic routine as shown in Scheme 1b. The synthetic details and characterization of DACA and compound 1 were displayed in the Experimental Section and Supporting Information. Scheme 1. The proposed sensing mechanism of a fluorescent probe based on ESIPT process (a), the synthetic routine of DACA (b), and the optimized structure of compound 1 (c).

large stokes shift up to 170 nm, suggesting the existence of ESIPT process. As shown in Scheme 1c, the chemical structure of 1 has been optimized with Gaussian 03 program package.28 The distance between Ha and Oa is ~2.0 Å, and a 5membered ring can be constructed with the hydrogen bond, suggesting that the ESIPT process could be existed.29 As shown in Figure S1d, in aprotic solvents, 1 displays a shorter emission peak peaked at 460 nm from the enol tautomer (E*), and a longer one peaked at 570 nm from the keto tautomer (K*). On the other hands, compound 1 shows only shortwavelength emission from E* in protic solvents such as ethanol, which may be caused by the formation of hydrogen bond between 1 and the surrounding solvents.30

Figure 1. Normalized absorption (solid line) and emission (dash line) spectra of DACA (black color) and compound 1 in 1,4dioxane. λex = 405 nm.

Optical properties of DACA and 1. The one-photon absorption and fluorescence spectra of probe DACA and its hydrolyzed product 1 were firstly measured, as shown in Figure 1 and S1. DACA displays absorption peak at 370 nm, while compound 1 shows a red-shifted absorption peaked at 400 nm. As shown in Figure S1, DACA displays emission peaked in the range of 440-500 nm, which is sensitive to environmental polarity. In comparison, compound 1 displays double emission peaks in aprotic solvents and a

Meanwhile, the Log P of DACA and compound 1 is 3.1 and 2.8, respectively. Consequently, they are lipophilic reagents that would be mainly distributed in the hydrophobic regions in cells with relatively low polarity. Generally, 1,4-dioxane should be a suitable solvent to mimic the environments. As shown in Figure 1, in 1,4-dioxane, DACA shows blue emission peaked at 440 nm, while compound 1 displays orange emission peaked at 570 nm. Notably, the wavelength separation between DACA and 1 is up to 130 nm, which could be easily discriminated under fluorescence microscope. Therefore, DACA has the potential for dual-color imaging of live and dead cells, as long as it can be hydrolyzed to 1 in live cells. Moreover, two-photon excited fluorescence imaging, equipped with near-infrared excitation source, possesses unique advantages, including high penetrating depth, low background noises, low injury to live sample, and minimal photobleaching effects.31,32 Subsequently, we have also checked the two-photon performance of DACA and 1 in different solvents. As shown in Figure S2, both the two compounds possess intense fluorescence with the excitation of 800 nm, indicating that DACA is a suitable two-photon probe. Cytotoxicity test. Cytotoxicity is an important parameter for fluorescent probes. Therefore, before the application of DACA in cell imaging experiments, the cytotoxicity of DACA with different incubation time has been measured initially. As shown in Figure S3, the cell viability is above 95 % when incubated with DACA for 36 h, and above 98 % for 2 h. Consequently, the cytotoxicity of DACA is rather low to live cells. In 40 min, the influence of DACA on cell viability could be omitted.



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Figure 2. The real-color images and the corresponding in situ emission spectra of HeLa cells stained with 2 µM of DACA with different incubation time. Incubation time: (a) 2 min; (b) 14 min; (c) 26 min; (d) 38 min; (e) 41 min; (f) 50 min. λex = 405 nm; bar = 10 µm.

emission, and the corresponding in situ emission spectra are peaked at 440 nm. Moreover, it also displays a small peak around 570 nm, which may be attributed to the hydrolysis by the small amount of residual esterase in dead cells. Fortunately, this emission is weak enough that would bring little interference. In comparison, as shown in Figure 3b and 3d, live cells incubated with DACA give orange-color fluorescence peaked at 570 nm, which may indicate the product of compound 1. It displays a small peak at 450 nm in Figure 3d, which could be corresponding to E* state of 1 molecule. Similarly, this shortwavelength emission is very weak, which would not bring significant disturbance. Thus DACA can actually distinguish live and dead cells in orange and blue dual emission colors.

Dual-color imaging of live and dead cells.

Figure 3. The real-color images of dead (a) and live (b) cells and the corresponding in situ emission spectra (c, d). λex = 405 nm; bar = 10 µm.

In order to understand the kinetics of DACA in live cells and find the best incubation time, cell images were acquired with different incubation time of DACA. Moreover, Nikon A1R confocal microscope has a spectra imaging function, which can give images bearing the actual emission color (realcolor images) as well as the in situ emission spectra. Therefore, the dynamics of DACA in HeLa cells was investigated with the spectra imaging function. As shown in Figure 2, Figure S4, and Video S1, after the incubation with DACA for 2 min, Hela cells displayed blue emission peaked at 440 nm. With the extension of incubation time, the orange emission peaked around 570 nm gradually enhanced, and the blue emission decreased. As shown in Figure 2d-2f and S4, with the incubation time extended to 40 min, the cells showed strong orange emission peaked at 570 nm, while the emission peaked at 440 nm became rather weak. Therefore, 40 min should be a suitable time window to distinguish live and dead cells. Consequently, HeLa cells were fixed with paraformaldehyde to obtain dead cells, and then live and dead cells were stained with DACA for 40 min and imaged. As shown in Figure 3a and 3c, dead cells stained with DACA shows blue-color

Figure 4. The DIC and fluorescent images of dead (a-c) and live (d-f) HeLa cells stained with 2 µM of DACA for 40 min in two emission channels. λex = 405 nm; blue channel: λem = 425-475 nm; red channel: λem = 570-620 nm; bar = 10 µm.

Considering that the spectra image function is not universally equipped in all confocal microscopes, we have also tried to identify live and dead cells in two emission channels with probe DACA. According to the in situ emission spectra in Figure 3c and 3d, dead cells incubated with DACA display strong intracellular emission in the wavelength range of 425475 nm and meanwhile rather weak fluorescence in 560-620 nm. On the other hand, live cells show weak emission in the range of 425-475 nm in live cells, and at the same time gives strong fluorescence in 560-620 nm. Consequently, we have selected a blue emission channel (425-475 nm) and a red emission channel (570-620 nm) for the discrimination of live and dead cells. As shown in Figure 4, with the excitation of 405 nm, the intracellular emission was collected in blue and


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Analytical Chemistry red channels. In dead cells, the emission in blue channel is rather intense, while the red channel only gives very weak fluorescent signals. By contrast, live cells show almost no fluorescence in the blue channel, while gives strong emission in the red channel. These results demonstrate that DACA can also discriminate live and dead cells in blue and red two emission channels. Strong red emission indicates that cells are rigorous, while intense blue signals imply unhealthy or death. Mechanism discussion.

Figure 5. The normalized emission spectra of DACA and compound 1 in 1,4-dioxane (Diox), dead cells, and live cells (a); The normalized emission spectra of DACA and 1 in dichloromethane (DCM) and DACA treated with no reagent (Non) and esterase for 30 min at 37 oC then extracted by DCM (b).

To understand the mechanism of DACA labeling live and dead cells in dual colors, the in situ emission spectra in dead and live cells were placed together for comparison. As shown in Figure 5a, the emission spectra of DACA in dead cells are peaked at 440 nm, which greatly resembles its emission in 1,4dioxane, indicating that the blue emission in dead cells is from DACA. On the other hand, the fluorescent spectra of DACA in live cells is in accordance with that of compound 1 in 1,4dioxane. Consequently, DACA should be hydrolyzed to com pound 1 in live cells. To get insight into the sensing mechanism, DACA was treated with active esterase and pure PBS buffer solution for 30 min at 37 ℃. Because both of DACA and compound 1 exhibit extremely weak fluorescence in aqueous conditions, the above system was extracted with dichloromethane and the fluorescence spectra were then measured for comparison. As shown in Figure 5b, the fluorescence of DACA treated with pure PBS is highly similar to that of DACA, indicating that DACA is stable in buffer solution. Meanwhile, the emission spectra of DACA treated with esterase solution resemble that of compound 1. Accordingly, the orange emission in live cells should be from the hydrolyzed product (1) of DACA by the intracellular active esterase.

Analysis of healthy status of cells pretreated with H2O2. Hydrogen peroxide is a metabolism by-product, which would block the oxidation respiratory chain in return, reduce mitochondria membrane potential, and induce the unhealthy status of cells. On the other hand, as shown in Figure S5, H2O2 induces no significant changes in the emission spectra of DACA and compound 1, indicating no interaction between H2O2 and the two compounds. Therefore, HeLa cells were treated with different amount of H2O2 then stained with DACA, to verify its ability detect cellular health status. As shown in Figure 6A, cells untreated with H2O2 shows almost no signals in blue channel, and intense emission in red channel, indicating that cells were in healthy state and intracellular esterase is highly active. As shown in Figure 6B-6E, with the addition of H2O2, the fluorescence in red channel turns weaker, and the signals in blue channel become more and more intense. These results demonstrate that intracellular active esterase is reduced, and cell activity gradually faded away. Accordingly, probe DACA can actually detect the cellular healthy state of cells. Analysis of cell unhealthy status induced by UV radiation. The radiation of UV light could bring damage to DNA, and thus induce the unhealthy status and death of cells. In order to testify the ability of DACA to analyze the health status of cells pretreated by UV radiation, we have pretreated HeLa cells with ceaseless exposure under a 365 nm UV lamp, and then stained them with probe DACA. As shown in Figure S6, in healthy HeLa cells untreated with UV radiation, there is almost no fluorescent signal in blue channel, while strong emission can be detected from red channel. With the extension of the exposure time under UV light, the emission from blue channel gradually enhanced and the fluorescence in red channel decreases, indicating the unhealthy status of HeLa cells. Consequently, probe DACA is capable of analyzing cellular health status induced by UV radiation. Two-photon applications. According to Figure S2, the two-photon excited fluorescence of DACA and compound 1 could also be excited by 800 nm. Therefore, live and dead HeLa cells were stained with DACA and imaged under two-photon microscope. As shown in Figure S7, under the excitation of 800 nm, dead cells give strong fluorescence in blue channel, and almost no emission in red channel. In comparison, live cells display nonfluorescence in blue channel, while shows intense emission signals in red channel. These results indicate that DACA could also distinguish live and dead cells under two-photon microscope. Consequently, DACA was used to stain dead and live zebrafish under two-photon microscope. As shown in Figure 7A, in dead zebrafish fixed by paraformaldehyde, DACA displays blue-color fluorescence. The zebrafish shows intense fluorescence in blue channel, and a small amount of fluorescence in red channels. This small amount of red fluorescence should be attributed the blood through the vessel near zebrafish heart. On the other hand, as shown in Figure 7B, live zebrafish was stained with orange-color emission. It displays almost no fluorescence in blue channel, while shows intense signals in red channel. These results demonstrate that probe DACA can be used to discriminate live and dead cells in live body such as the zebrafish, by means of two-photon microscopy.



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Figure 6. The fluorescent images of HeLa cells pretreated with different amounts of H2O2 for 2 h then incubated with 2 µM of DACA for 40 min. H2O2 concentration: (A) 0 mM; (B) 0.5 mM; (C) 1.0 mM; (D) 4.0 mM; (E) 8.0 mM. Optical parameters: λex = 405 nm; (a) blue channel: λem = 425-475 nm; (b) red channel: λem = 570-620 nm; (c) merged image. Bar = 10 µm. shown in Figure S8A, with the irradiation of 405 nm or 800 nm for 30 min, DACA still displayed bright intracellular emission in fixed HeLa cells, and the fluorescence intensity showed no significant change. These results indicate that DACA exhibits high photostability for cell imaging applications. On the other hand, compound 1 gave similar results, as shown in Figure S8B. Ceaseless laser exposure brought little change in its intracellular emission, which demonstrates its qualified photostability.

CONCLUSION

Figure 7. The DIC and two-photon fluorescent images of dead (IaIe) and live (IIa-IIe) zebrafish stained with 5 µM of DACA for 30 min. (a) DIC images; (b) real-color images, λex = 800 nm; (c) images from blue channel, λex = 800 nm, λem = 425-475 nm; (d) images from red channel, λex = 800 nm, λem = 570-620 nm; (e) merged images of c and d.

Photostability of DACA and compound 1. Photostability is an important property of fluorescent probes. Consequently, the one- and two-photon excited photostability of DACA and compound 1 in cells was investigated with ceaseless exposure in 405 nm and 800 nm laser sources. As

In summary, we have developed an unprecedented fluorescent probe DACA by esterification of 3-hydroxyflavone, which can discriminate live and dead cells in dual-color mode for the first time. In live cells, it can be hydrolyzed by active esterase into 3-hydroxyflavone with recovered ESIPT process and orange-color fluorescence peaked at 570 nm. In dead cells, esterase is deactivated, and thus the probe shows blue emission peaked at 440 nm with blocked ESIPT process. Consequently, probe DACA can discriminate dead and live cells in blue and orange dual emission colors. Compared with other probes that can only label live or dead cells in monocolor fluorescence, DACA can efficiently avoid false positive results and misleading signals, which can supply more accurate information in biological applications. Finally, we have successfully utilized DACA to image the cellular unhealthy status induced by H2O2 and UV radiation, and distinguish live and dead cells in zebrafish. These data demonstrate that DACA can discriminate live and dead cells in dual colors, indicating


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Analytical Chemistry that it may be employed as a powerful tool in wide areas including biology, pathology, and pharmacology.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Materials and methods, synthesis of the probes, absorption and fluorescence spectra, imaging assays, MTT assays, 1H NMR and 13C NMR spectra, etc. were placed in it.

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

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

ACKNOWLEDGMENT This work was financially supported by NSFC (21472067, 21672083), Taishan Scholar Foundation (TS201511041), and the startup fund of University of Jinan (309-10004).

REFERENCES

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