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A novel red light emissive two-photon fluorescent probe for hydrogen sulfide (H2S) in nucleolus region and its application for H2S detection in zebrafish and live mice
Accepted Manuscript Title: A novel red light emissive two-photon fluorescent probe for hydrogen sulfide (H2 S) in nucleolus region and its application for H2 S detection in zebrafish and live mice Authors: Keyin Liu, Chuang Liu, Huiming Shang, Mingguang Ren, Weiying Lin PII: DOI: Reference:
S0925-4005(17)31812-9 https://doi.org/10.1016/j.snb.2017.09.146 SNB 23237
To appear in:
Sensors and Actuators B
Received date: Revised date: Accepted date:
29-6-2017 20-9-2017 20-9-2017
Please cite this article as: Keyin Liu, Chuang Liu, Huiming Shang, Mingguang Ren, Weiying Lin, A novel red light emissive two-photon fluorescent probe for hydrogen sulfide (H2S) in nucleolus region and its application for H2S detection in zebrafish and live mice, Sensors and Actuators B: Chemical https://doi.org/10.1016/j.snb.2017.09.146 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
A novel red light emissive two-photon fluorescent probe for hydrogen sulfide (H2S) in nucleolus region and its application for H2S detection in zebrafish and live mice
Keyin Liu, Chuang Liu, Huiming Shang, Mingguang Ren, Weiying Lin ⃰
Institute of Fluorescent Probes for Biological Imaging, School of Materials Science and Engineering, School of Chemistry and Chemical Engineering, University of Jinan, Jinan, Shandong 250022, P.R. China.
Corresponding Author Professor Weiying Lin, Ph.D Institute of Fluorescent Probes for Biological Imaging, School of Materials Science and Engineering, School of Chemistry and Chemical Engineering, University of Jinan, Jinan, Shandong 250022, P.R. China. E-mail: [email protected];
Fax: (+) 86-531-82769031
Graphical Abstracts
Highlights:
The two-photon fluorescent probe was designed through an extension of the π-conjugated system of traditional dyes.
The fluorescence emission of the two-photon probe is over 630 nm.
The probe can penetrate nucleus membrane and detect H2S in nucleolus region effectively.
The probe was applied to detect H2S in cells and live animals
ABSTRACT Hydrogen sulfide (H2S) has been recognized as one of the most important gaseous signaling molecules and it is extensively present in cells. Detection of H2S in biological samples by fluorescent imaging techniques is advantageous due to the real-time, noninvasive nature of these techniques. However, owing to the protective obstacle of the cell nucleus membrane, it is hard for a fluorescent probe to detect H2S near the nucleolus region. Herein, we report the first example of a novel two-photon fluorescent probe 1-H2S for H2S with red light emission that can detect H2S near the nucleolus region. The probe was constructed through extending the conjugated system of naphthalene and coumarin analogue. 1-H2S showed obvious fluorescence enhancement in the presence of 50 equivalents Na2S (PBS, pH 7.4 buffer) and high selectively toward H2S in solution. In biological experiments, the fluorescent probe was found to aggregate in the nucleolus region of cells and can detect H2S near the nucleolus region. The probe was also applied for fluorescence imaging of H2S in zebrafish and live mice model successfully.
Keywords: Two-photon fluorescent probe, red light emission, H2S detection, nucleolus localization, live animals imaging
Introduction Hydrogen sulfide (H2S) has been recognized as one of the most important gaseous signaling molecules that plays important roles in physiological activities in recent years [1, 2]. For example, H2S is involved in aging and age ralated deseases and organs injury [3]. The genomic stability can be affected by H2S concentration and high concentration of H2S in nucleus and nucleolus regions may cause DNA damage [4]. Enzymatically generated endogenous H2S can be produced by enzymes such as 3-mercaptopyruvate sulfurtransferase, cystathionine β-synthase (CBS) and cystathionine gamma-lyase [5-7]. Investigations have demonstrated that higher concentration of H2S than its normal level in physiological environment is involved in lesion of cells and organs [8]. Inhalation of H2S may lead to respiratory depression and neural paralysis with intense neurotoxin and mucous membrane irritation [9-11]. Misregulation of H2S is related with diverse physiological diseases, including neurodegenerative disease, Down’s syndrome, angiocardiopathy, diabetes and liver cirrhosis [12-16]. Hence, detection of H2S in cells and live organisms is of great importance for exploring the biological role of H2S in molecular level [17].
Traditionally, many methods have been established for H2S detection [18, 19], such as gas chromatography (GC), gas chromatography/mass spectrometry (GC/MS), adsorption-desorption, electrochemical methods [20] and fluorescence detection. Among them, fluorescence detection is one of the most powerful methods for H2S analysis in biological samples, such as intracellular environment, tissue and live organisms for the noninvasive and high temporal-spatial resolution property [21-25]. Many fluorescent probes based on traditional dyes such as fluorescein, rhodamine, bodipy and naphthalene derivatives were developed and applied for H2S imaging in cellular and subcellular environment [26-33]. However, fluorescence detection of H2S in nucleolus region is still a big challenge due to the protective obstacle of nucleus membrane. Compared with one-photon excitation methods, two-photon fluorescence imaging technique owned many advantages such as long wavelength excitation [34], deep tissue penetration and three-dimensional imaging ability. Most of the two-photon fluorescent probes owns comparatively short emission wavelength, the maxima fluorescence signals is usually below 620 nm [35-37]. The shorter emission wavelength may lead to a series of problems in its biological application, such as tissue auto-fluorescence interference and weak tissue penetration ability of the emission light. Thus, development of two-photon probes with longer emission wavelength is urgent for improving the tissue penetration ability and extending the application in tissue imaging experiment
[38]. Herein, we report the first example of red light emissive two-photon probe for H2S in nucleolus region and its application in live animals imaging. The probe was constructed through extending the conjugated system of traditional two-photon platform such as naphthalene and coumarin (Fig. 1). The probe was designed (Fig. 1C) and the photophysical properties in the absence or presence of H2S in aqueous solution and the fluorescence imaging of H2S in cells and live animals were investigated. Interestingly, the fluorescent probe was found to aggregate in the nucleolus region of cells and can detect H2S near the nucleolus region. The probe was also applied for fluorescence imaging of H2S in zebrafish and live mice model.
Experimental Materials and instruments All the reagents (purity over 99.9%) were purchased from commercial source (Sigma Aldrich, China) and used without further purification unless specified. The solvents were dried and purified by distillation according to standard procedures before use. All reactions were performed under nitrogen protection and monitored by thin-layer chromatography (TLC). The products were purified by silica gel (200-300 mesh) column chromatography. TLC plates and silica gels were purchased form Qingdao Ocean chemicals. All the intermediates were analyzed by 1H NMR, 13C NMR and high-resolution mass spectrometry (HRMS). The 1H NMR and 13C NMR were carried out on
AVANCE III 400 MHz Digital NMR Spectrometers (Bruker Daltonics Corp., USA), using tetramethylsilane (TMS) as an internal reference. HRMS spectra were recorded on Bruker apex-Ultra mass spectrometer in electrospray ionization (ESI) mode (Bruker Daltonics Corp., USA). The absorption spectra were taken on a Shimadzu UV-2700 spectrophotometer (Shimadzu Suzhou instruments Mfg. Co, Ltd). The fluorescence spectra were taken on a Hitachi F4600 spectrofluorimeter (Hitachi High-Tech Science) with a 10 mm quartz cuvette. Na2S stock solution (5 mM) was used as the source of H2S in the photophysical investigation of 1-H2S [39]. Imaging of cells, liver-tissue slices and zebrafish was performed on a Nikon A1 MP+ two-photon microscope (Nikon instruments Inc.) and live-animal imaging experiments were performed with PerkinElmer IVIS Lumina Series III Pre-clinical (PerkinElmer Inc.) in vivo imaging system.
Synthesis of two-photon fluorescent probe 1-H2S. The synthesis process of two-photon fluorescent probe was illustrated as scheme S1 in supporting information. The two-photon platform was synthesized by two steps. Briefly, salicylaldehyde and 6-amino-3,4-dihydronaphthalen-1(2H)-one were reacted in concentrated acid to yield the desired product 3-amino-10-(diethylamino)-5,6-dihydrobenzo[c]xanthen-12-ium (TP-1), then the amino group was converted to azide group to obtain the compound
3-azido-10-(diethylamino)-5,6-dihydrobenzo[c]xanthen-12-ium (1-H2S) with a high yield.
Cell culture and fluorescence imaging HeLa cells were obtained from Pharmaceutical College of Shandong University, cultured at 37 oC incubator with 5% CO2 and 95% air in modified Eagle’s medium supplemented with 10% calf bovine serum. For cell imaging experiments, the cells were seeded onto glass plates for 12 hours for adherence, then washed by PBS and incubated with the probe 1-H2S before fluorescence imaging. To evaluated the cytotoxicity of 1-H2S, the cell viability of 1-H2S was determined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The HeLa cells were seeded into a 96-well plate at the density of 105 cells per well and cultured for 12 hours. Then the cells were cultured with various concentrations (0, 1, 5, 10, 20, 30 μM) of 1-H2S for another 24 hours. 10 μL of MTT were added to each well and further incubated for 4 hours, followed by addition of 100 μL of DMSO. The plate was shaken for 30 min and the absorption at 570 nm was collected by a microplate reader made by Thermo Fisher Scientific. The cell viability was calculated by the absorption at 570 nm. Fluorescence imaging in cells by 1-H2S were performed on Nikon A1 MP+ two-photon microscope.
Live animal imaging experiments
All animal procedures for this study were approved by the Animal Ethical Experimentation Committee of Shandong University according to the requirements of the National Act on the use of experimental animals (China). Zebrafish were cultured for three days in clean water before use. The probe 1-H2S 10 μM was added and cultured for 30 min and imaged by two-photon microscope. For H2S detection, the zebrafish were first cultured with 1-H2S 10 μM for 30 min, the zebrafish were washed by clean water and then 20 μM of Na2S was added and further incubated for 30 min before fluorescence imaging by one-photon or two-photon light source. For living mice imaging experiments, Kunming mice were anesthetized by intraperitoneally injection of chloral hydrate 200 μL (4% in water). Then 20 μg of 1-H2S was injected into the mice intraperitoneally, followed by 60 μg of Na2S. The mice were imaged by the PerkinElmer IVIS Lumina Series III in vivo imaging system.
Result and discussion Photophysical properties of 1-H2S. The photophysical properties of 1-H2S in solution were explored. 1-H2S exhibits an intense absorption band at around 556 nm and a weak fluorescence peak at about 635 nm in PBS (pH 7.4) and DMSO (8:2) buffer (Fig. 2a and Fig. S1). As shown by Fig. S2, 1-H2S shows a stable fluorescence emission when irradiated by 580 nm light for 4 hours or heated the test solution to 80 oC, which indicated a good photo-stability and thermal-stability of 1-H2S in
solution. In absorption spectra (Fig. S1), in the presence of excess Na2S, the absorption band at 556 nm red shifted about 34 nm to 590 nm due to the reduction of azido group to amino group by Na2S, which leads to an enhanced intramolecular charge transfer (ICT) effect in the probe. To manifest the detection mechanism and the spectra change, the reaction process of 1-H2S with Na2S was also investigated by HRMS. As shown by HRMS analysis (Fig. S4, S5), the azido group of 1-H2S was reducted to amine by Na2S and both HRMS signals of 1-H2S and 1-TP were found in the HRMS spectra. The HRMS analysis experiment indicated that the spectra change is due to the generation of 1-TP. The HRMS analysis result and the spectra change behavior of 1-H2S in the presence of Na2S were also supported by literature [40, 41]. In emission spectra, the fluorescence peak at 635 nm increased sharply with the addition of Na2S. Compared with Na2S blank sample, about 7-fold fluorescence enhancement was observed in the presence of 30 eq. of Na2S (Fig 2a and Fig. S3). As shown by Fig. 2b, a linear growth of the fluorescence intensity with the Na2S-to-probe concentration ratio increase was found. The detection limit of 1-H2S to Na2S is about 4.7 × 10-5 M, as estimated by S / N = 3 method [42], which is lower than the concentration of endogenous H2S in physiological environment [2]. The reaction rate of 1-H2S with Na2S was tracked by fluorescent spectra (Fig. 2 c, d). It takes about 15 min to reach the maximum of fluorescence when 1-H2S reacted with 50 equivalents of Na2S at 37 oC. The rapid response toward Na2S is beneficial for detection of H2S in biological
samples (Table S1).
Selectivity experiment We tested the selectivity of 1-H2S toward Na2S and other biologically relevant reactive nitrogen, reactive oxygen and sulfur species including hydrogen peroxide (H2O2), superoxide anions (·O2-), tert-butylhydroperoxide (TBHP), nitric oxide (NO), peroxynitrite (ONOO-), sulfite (SO32-) and standard test amino acids such as cysteine (Cys), homocysteine (Hcy), glutathione (GSH). As shown by Fig. 3a, clearly, only Na2S turns on the fluorescence intensity of 1-H2S within 30 min, whereas other species produce an almost negligible fluorescence enhancement under the same condition. The fluorescence intensity at 635 nm showed a huge fluorescence enhancement only when Na2S was added to the solution (Fig. 3b). The high selectivity of 1-H2S toward Na2S could be used for Na2S detection of in physiological condition.
Cells imaging experiment H2S has been recognized as one of the most important gaseous signaling molecule that plays important roles in physiological activities. The above experiments have demonstrated that 1-H2S exhibited large fluorescence enhancement and high selectivity toward Na2S in solution environment. Thus the application of 1-H2S for Na2S detection in cells, tissue and live animals were investigated. Firstly, the toxicity of the probe was explored by MTT
method. As shown by the result (Fig. S6), the probe exhibited little toxicity to HeLa cells at the concentration measured (20 μM), over 80% cells survived after incubation with 10 μM 1-H2S of for 24 h. The cellular distribution of 1-H2S was then measured by colocalization analysis with traditional organelles labeling agents, such as mitochondria labeling agent Mito-Tracker Green and nucleus labeling agent Hoechst 33258. When the Hela cells were co-incubated with Mito-Tracker Green and 1-H2S, as shown by Fig. 4 (d - f), the probe can penetrate cell membrane easily and aggregated in mitochondria area at the first 10 min. As calculated from Fig. 4e, the colocalization coefficient is about 81% (Pearson correlation coefficient, calculated by software installation in Nikon A1 MP+ two-photon microscope). The fluorescence intensity analysis (Fig. 4f) along the arrow in a selected cell in Fig. 4d also demonstrated a high overlap of the two fluorescence signals. Furthermore, the HeLa cells were co-incubated with Hoechst 33258 and 1-H2S, as shown by Fig. 5 and Fig. 6. At the first 10 min, the probe mainly distributed in cytoplasm. When the incubation time was prolonged to about 40 min, 1-H2S tend to penetrate the cell nuclear membrane and retained in the nucleolus region (Fig. 5 f ̶ h; Fig. 6 c, d) [43, 44]. The result was further confirmed by fluorescence intensity analysis along the arrows in selected cells. As shown by Fig. 6d, the main fluorescence intensity enhancement of 1-H2S was observed in nucleolus region.
Fluorescence imaging of H2S in HeLa cells Encouraged by the large fluorescence enhancement of 1-H2S to Na2S in solution and the nucleolus localization ability, we tend to investigate the detection of 1-H2S to H2S in cells and tissues by fluorescence imaging. As shown by Fig. 7, when the HeLa cells were incubated with 1-H2S solely, only weak fluorescence were observed in TRITC channel when excited by 561 nm laser and two-photon excitation. However, a bright fluorescence of 1-H2S was observed in nucleolus region when the HeLa cells were pre-incubated with 10 μM of Na2S for 20 min then incubated with 1-H2S for 20 min. The experiment demonstrated that 1-H2S can penetrate the cell nuclear membrane and image H2S in nucleolus region successfully.
Tissue and zebrafish imaging Based on the result obtained on fluorescence detection of H2S in solution and cellular environment, we tried to apply the probe for H2S detection in tissues. The two-photon absorption cross-section spectra of 1-H2S and 1-TP were measured, 1-TP exhibits stronger absorption band at around 750 nm to 820 nm than 1-H2S, which indicated an effective tune function of azide group to the fluorescence (Fig. S7). Freshly prepared liver tissue slices (thickness: 400 μm) of mice were steeped in a solution of 10 μM 1-H2S for 30 min, then the tissue were washed by PBS and further incubated with 20 μM Na2S solution for another 30 min. Fluorescence imaging was carried out before and after the
treatment of the tissue slices by Na2S solution (Fig. 8). The liver tissue showed weak fluorescence when only incubated with 1-H2S. When the tissue was incubated with 1-H2S and further incubated with Na2S, an enhanced fluorescence was observed under the same imaging parameters. The liver tissue was analyzed by scan along the Z-axis and three-dimensional reconstruction, to test the tissue penetration ability of the probe. The fluorescent signal can be detected at a depth of 60 μm, the maxima fluorescent signal mainly emerged at a depth of about 40 μm. The experiment exhibited that the probe can be used for image Na2S in deep tissue by two-photon excitation mode. Compared with one-photon excitation mode, the two-photon excitation mode shows obvious advantage in reactive species detection in tissue, which facilitates the study of physiological activities in deep tissue. Thus, the probe has exhibited excellent tissue image property, and it was used to image H2S in live zebrafish. Zebrafish is a typical biological model for investigation of the physiological activity of vertebrate animals and usually used in evaluation of the function of small molecules and pharmaceutical analysis in live animals. The fluorescence detection of Na2S by 1-H2S was carried out in zebrafish model; one week old zebrafish was used. As shown by Fig. S8, when 10 μM of the probe was incubated with zebrafish for 30 min, weak fluorescence was observed in TRITC channel. However, when the zebrafish was pre-incubated with 20 μM Na2S and then incubated with 10 μM of the probe for 30 min, a strong fluorescent signal was observed. The same
fluorescence imaging result was obtained when excited by 760 nm laser under two-photon excitation mode (Fig. S9). The typical physiological levels of H2S are between 0.1- 300 μM in live animals, which is higher than the Na2S concentrations used in the above imaging experiments, thus the probe can potentially be used for detection physiological levels of H2S in live animals [2].
Fluorescence imaging of H2S in live animal We then tend to investigate the fluorescence imaging ability of H2S in live animals by 1-H2S. The four-week old female balb/c mice were kept in stainless steel cages that contain barriers for each mice for individual housing. They were fed commercial mice chow and left freely wandering in their cage for two weeks with 12 hours dark / light cycles for acclimatization before starting the experiment. The mouse was anesthetized by 200 μL trichloroacetaldehyde (4% wt), then 20 μg of the probe was subcutaneously injected into the abdomen of the mouse, Na2S was carefully injected to the same location. The mouse was placed into live animal imaging system. As shown by Fig. 9, the probe shows little fluorescence under 580 nm light excitation. When Na2S was added, the fluorescent signal increased steadily in a time range of 40 min. about 7-fold fluorescence enhancement was observed, when compared with the fluorescence of the probe itself. The experiment demonstrated that 1-H2S can be used to detect H2S in live animals successfully.
Conclusions Most of the two-photon fluorescent dyes have emission wavelength at comparatively short wavelength region. Development of two-photon dyes with emission in red light region is attractive for increasing their application area in biological imaging, which can eliminate interference from strong tissue auto-fluorescence and increase tissue penetration ability. Herein, a novel two-photon fluorescent probe 1-H2S for H2S with red light emission was constructed through extending the conjugated system of traditional two-photon platform naphthalene and coumarin. The photophyscial experiments demonstrate that 1-H2S can respond to H2S with large fluorescent enhancement at 635 nm, and shown high selectivity toward H2S. Colocalization analysis has shown that the probe can aggregate at the nucleolus region when the incubation time is prolonged. The probe can detect H2S successfully in nucleolus region of cells by one photon and two-photon excitation. Taking advantage of the emission in red light region, the probe was further applied to image H2S in zebra fish and living mice successfully.
Acknowledgements This work was financially supported by NSFC (61605060, 21672083, 21472067), Natural Science Foundation of Shandong Province (ZR2015PB016), Taishan Scholar Foundation (TS 201511041) and the startup fund of University of Jinan (309-10004, 160100137).
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Author Biographies
Dr. Keyin Liu obtained his Ph. D. degree from Fudan University in 2014. Currently, he is a lecturer in Institute of Fluorescent Probes for Biological Imaging in University of Jinan, Jinan, P.R. China. His research interests focus on the design and synthesis of novel functional fluorescent dyes/probes and their biological applications.
Chuang Liu obtained his B.S. degree at Langfang Teachers University in 2017. Currently, he is a graduate student under the supervision of Professor Weiying Lin in the Institute of Fluorescent Probes for Biological Imaging, University of Jinan. His research interests focus on the design and synthesis of fluorescent probes.
Huiming Shang obtained his B.S. degree at Dezhou University for Nationalities in 2014. Currently, he is a graduate student under the supervision of Professor Weiying Lin in the Institute of Fluorescent Probes for Biological Imaging, University of Jinan. His research interests focus on the design and synthesis of fluorescent probes.
Dr. Mingguang Ren received his Ph.D. degree from University of Science and Technology of China in 2011. Now, he is an associate professor in Institute of Fluorescent Probes for Biological Imaging at University of Jinan, Jinan, P.R. China. His research interests focus on the design and synthesis of novel functional fluorescent dyes/probes and their biological applications
Professor Weiying Lin received his Ph.D. from the University of Kansas in 2000. After completing postdoctoral research at Massachusetts Institute of Technology, in 2005, he joined the faculty at Hunan University. Subsequently, he moved to the University of Jinan as the Dean and the Distinguished Professor of the Institute of Fluorescent Probes for Biological Imaging. His research interests cover the interdisciplinary areas of molecular recognition, photochemistry, materials chemistry, analytical chemistry, and chemical biology.
Figure Captions
Fig. 1. (A) Generation of fluorescence and (B) sketch of construction of two-photon probes with longer wavelength. (C) The red light emissive two-photon fluorescent probe designed and the sensing mechanism to H2S.
Fig. 2. The change of fluorescence spectra of 10 μM 1-H2S titrated with Na2S. (a) 10 μM of 1-H2S titrated with increasing concentrations of Na2S. (b) Linear fit of fluorescence intensity change at 635 nm with Na2S to probe ratio. (c) The fluorescence intensity change of 10 μM 1-H2S with time, in the presence of 50 equivalents Na2S. (d) The fluorescence intensity change at 635 nm with time, Excitation: λ = 580 nm.
Fig. 3. Selectivity of 1-H2S to Na2S. (a) Fluorescence spectra change of 10 μM 1-H2S in the presence of 50 equivalents of Na2S and other standard small test molecules. The probe was incubated with the molecules for 30 min. (b) Fluorescence intensity of 1-H2S at 635 nm after 30 min incubation with standard small test molecules. Number 1 – 13 along the horizontal axis represent samples of 1-H2S interacted with 1 blank, 2 H2O2, 3 ·O2-, 4 TBHP, 5 NO, 6 ONOO-, 7 SO32-, 8 Cys, 9 Hcy, 10 GSH, 11 S2O32-, 12 SO42-, 13 S2respectively.
Fig. 4. Colocalization experiment of 1-H2S with Mito-Tracker Green in HeLa cells. 10 μM 1-H2S and 1 μM of Mito-Tracker Green were coincubated with HeLa cells for 10 min washed by PBS before fluorescence imaging. (a – d) represent Bright field, Green channel, TRITC channel, and Merged image. (e) The colocalization coefficient analysis of TRITC and Green channels by software installation on Nikon microscope and (f) fluorescence intensity analysis of green and red channels along the arrow in selected cells in the white circle. Green channel: excitation: 488 nm, emission: 500 – 550 nm. (Parameters: laser wavelength: 488 nm, laser power: 0.1 mW; emission filter: 500 – 550 nm, PMT HV value: 90; pinhole radius: 26.8, scan speed: 0.5 second) TRITC channel: excitation: 561 nm, emission: 570 – 620 nm, pseudo color. (Parameters: laser wavelength: 561 nm, laser power: 0.4 mW; emission filter: 570 – 620 nm, PMT HV value: 100; pinhole radius: 26.8, scan speed: 0.5 second)
Fig. 5. Colocalization experiment of 1-H2S with Hoechst33258 in HeLa cells. 10 μM 1-H2S and 1 μM of Hoechst33258 were coincubated with HeLa cells for 10 min and 40 min respectively and washed by PBS before fluorescence imaging. (a, e) Bright field, (b, f) Blue channel, (c, g) TRITC channel, (d, h) Merged image. Blue channel: excitation: 405 nm, emission: 430 – 480 nm (Parameters: laser wavelength: 405 nm, laser power: 2.5 mW; emission filter: 425 – 475 nm, PMT HV value: 150; pinhole radius: 26.8, scan speed: 0.5 second). (c) TRITC channel: excitation: 561 nm, emission: 580 – 650 nm, pseudo color. (Parameters: laser wavelength: 561 nm, laser power: 0.2 mW; emission filter: 570 – 620 nm, PMT HV value: 100; pinhole radius: 26.8, scan speed: 0.5 second)
Fig. 6. Fluorescence intensity analysis of Hoechst33258 and 1-H2S in selected cells. 10 μM 1-H2S and 1 μM of Hoechst33258 were co-incubated with HeLa cells for 10 min [(a), (b)] and 40 min [(c), (d)], respectively. (a, c) Merged image of blue and TRITC channels of HeLa cells, (b, d) fluorescence intensity analysis along the arrows in (a, c) respectively.
Fig. 7. Fluorescence imaging of H2S in HeLa cells by one-photon and two-photon excitation. (a, b) HeLa cells were incubated with 5 μM 1-H2S for 20 min, (c, d) HeLa cells were pre-incubated with 10 μM Na2S for 20 min then incubated with 5 μM of 1-H2S for 20 min. (a, c) Single-photon image excited by 561 nm laser, collected at TRITC channel: 580 – 640 nm. (Parameters: laser wavelength: 561 nm, laser power: 0.1 mW; emission filter: 570 – 620 nm, PMT HV value: 100; pinhole radius: 26.8, scan speed: 0.5 second) (b, d) Two-photon image excited by 760 nm laser, collected at TRITC channel: 570 – 620 nm. (Parameters: laser wavelength: 760 nm, laser power: 30.0 mW; TRITC channel: emission filter: 570 – 620 nm, PMT HV value: 105; pinhole radius: 255.43, scan speed: 0.25 second) Scale bar: 20 μm.
Fig. 8. Fluorescence detection of H2S in liver tissue of mice. (a) liver tissue slice (thickness: 400 μm) incubated with 1-H2S for 30 min then washed by PBS (b) liver tissue incubated with 1-H2S for 30 min then incubated with 20 μM Na2S for another 30 min, (c) three-dimension reconstruction of liver tissue incubated with Na2S and 1-H2S image of by z-scan between a depth of form 0 to 60 μm. Excitation by 760 nm, emission collection: 570-620 nm. (Parameters: laser wavelength: 760 nm, laser power: 30.0 mW; TRITC channel: emission filter: 570 – 620 nm, PMT HV value: 105; pinhole radius: 255.43, scan speed: 0.25 second)
Fig. 9. Fluorescence imaging of H2S in living mouse by 1-H2S. (a) 20 μg of 1-H2S was subcutaneously injected only, (b - i) 20 μg of 1-H2S and 60 μg of Na2S were subcutaneous injected for 2, 5, 10, 15, 20, 25, 30, 40 min respectively. Excitation filter: 580 nm; emission filter: 600 - 650 nm. (The fluorescence imaging Parameters: Exposure time: 0.2 seconds; Binning factor: 2; F-stop: 2; Field of view: 12.5 cm; Object radius: 0.75; Subject size: 1.5; CCD Measure temperature: -90 oC).
S0925-4005(17)31812-9 https://doi.org/10.1016/j.snb.2017.09.146 SNB 23237
To appear in:
Sensors and Actuators B
Received date: Revised date: Accepted date:
29-6-2017 20-9-2017 20-9-2017
Please cite this article as: Keyin Liu, Chuang Liu, Huiming Shang, Mingguang Ren, Weiying Lin, A novel red light emissive two-photon fluorescent probe for hydrogen sulfide (H2S) in nucleolus region and its application for H2S detection in zebrafish and live mice, Sensors and Actuators B: Chemical https://doi.org/10.1016/j.snb.2017.09.146 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
A novel red light emissive two-photon fluorescent probe for hydrogen sulfide (H2S) in nucleolus region and its application for H2S detection in zebrafish and live mice
Keyin Liu, Chuang Liu, Huiming Shang, Mingguang Ren, Weiying Lin ⃰
Institute of Fluorescent Probes for Biological Imaging, School of Materials Science and Engineering, School of Chemistry and Chemical Engineering, University of Jinan, Jinan, Shandong 250022, P.R. China.
Corresponding Author Professor Weiying Lin, Ph.D Institute of Fluorescent Probes for Biological Imaging, School of Materials Science and Engineering, School of Chemistry and Chemical Engineering, University of Jinan, Jinan, Shandong 250022, P.R. China. E-mail: [email protected];
Fax: (+) 86-531-82769031
Graphical Abstracts
Highlights:
The two-photon fluorescent probe was designed through an extension of the π-conjugated system of traditional dyes.
The fluorescence emission of the two-photon probe is over 630 nm.
The probe can penetrate nucleus membrane and detect H2S in nucleolus region effectively.
The probe was applied to detect H2S in cells and live animals
ABSTRACT Hydrogen sulfide (H2S) has been recognized as one of the most important gaseous signaling molecules and it is extensively present in cells. Detection of H2S in biological samples by fluorescent imaging techniques is advantageous due to the real-time, noninvasive nature of these techniques. However, owing to the protective obstacle of the cell nucleus membrane, it is hard for a fluorescent probe to detect H2S near the nucleolus region. Herein, we report the first example of a novel two-photon fluorescent probe 1-H2S for H2S with red light emission that can detect H2S near the nucleolus region. The probe was constructed through extending the conjugated system of naphthalene and coumarin analogue. 1-H2S showed obvious fluorescence enhancement in the presence of 50 equivalents Na2S (PBS, pH 7.4 buffer) and high selectively toward H2S in solution. In biological experiments, the fluorescent probe was found to aggregate in the nucleolus region of cells and can detect H2S near the nucleolus region. The probe was also applied for fluorescence imaging of H2S in zebrafish and live mice model successfully.
Keywords: Two-photon fluorescent probe, red light emission, H2S detection, nucleolus localization, live animals imaging
Introduction Hydrogen sulfide (H2S) has been recognized as one of the most important gaseous signaling molecules that plays important roles in physiological activities in recent years [1, 2]. For example, H2S is involved in aging and age ralated deseases and organs injury [3]. The genomic stability can be affected by H2S concentration and high concentration of H2S in nucleus and nucleolus regions may cause DNA damage [4]. Enzymatically generated endogenous H2S can be produced by enzymes such as 3-mercaptopyruvate sulfurtransferase, cystathionine β-synthase (CBS) and cystathionine gamma-lyase [5-7]. Investigations have demonstrated that higher concentration of H2S than its normal level in physiological environment is involved in lesion of cells and organs [8]. Inhalation of H2S may lead to respiratory depression and neural paralysis with intense neurotoxin and mucous membrane irritation [9-11]. Misregulation of H2S is related with diverse physiological diseases, including neurodegenerative disease, Down’s syndrome, angiocardiopathy, diabetes and liver cirrhosis [12-16]. Hence, detection of H2S in cells and live organisms is of great importance for exploring the biological role of H2S in molecular level [17].
Traditionally, many methods have been established for H2S detection [18, 19], such as gas chromatography (GC), gas chromatography/mass spectrometry (GC/MS), adsorption-desorption, electrochemical methods [20] and fluorescence detection. Among them, fluorescence detection is one of the most powerful methods for H2S analysis in biological samples, such as intracellular environment, tissue and live organisms for the noninvasive and high temporal-spatial resolution property [21-25]. Many fluorescent probes based on traditional dyes such as fluorescein, rhodamine, bodipy and naphthalene derivatives were developed and applied for H2S imaging in cellular and subcellular environment [26-33]. However, fluorescence detection of H2S in nucleolus region is still a big challenge due to the protective obstacle of nucleus membrane. Compared with one-photon excitation methods, two-photon fluorescence imaging technique owned many advantages such as long wavelength excitation [34], deep tissue penetration and three-dimensional imaging ability. Most of the two-photon fluorescent probes owns comparatively short emission wavelength, the maxima fluorescence signals is usually below 620 nm [35-37]. The shorter emission wavelength may lead to a series of problems in its biological application, such as tissue auto-fluorescence interference and weak tissue penetration ability of the emission light. Thus, development of two-photon probes with longer emission wavelength is urgent for improving the tissue penetration ability and extending the application in tissue imaging experiment
[38]. Herein, we report the first example of red light emissive two-photon probe for H2S in nucleolus region and its application in live animals imaging. The probe was constructed through extending the conjugated system of traditional two-photon platform such as naphthalene and coumarin (Fig. 1). The probe was designed (Fig. 1C) and the photophysical properties in the absence or presence of H2S in aqueous solution and the fluorescence imaging of H2S in cells and live animals were investigated. Interestingly, the fluorescent probe was found to aggregate in the nucleolus region of cells and can detect H2S near the nucleolus region. The probe was also applied for fluorescence imaging of H2S in zebrafish and live mice model.
Experimental Materials and instruments All the reagents (purity over 99.9%) were purchased from commercial source (Sigma Aldrich, China) and used without further purification unless specified. The solvents were dried and purified by distillation according to standard procedures before use. All reactions were performed under nitrogen protection and monitored by thin-layer chromatography (TLC). The products were purified by silica gel (200-300 mesh) column chromatography. TLC plates and silica gels were purchased form Qingdao Ocean chemicals. All the intermediates were analyzed by 1H NMR, 13C NMR and high-resolution mass spectrometry (HRMS). The 1H NMR and 13C NMR were carried out on
AVANCE III 400 MHz Digital NMR Spectrometers (Bruker Daltonics Corp., USA), using tetramethylsilane (TMS) as an internal reference. HRMS spectra were recorded on Bruker apex-Ultra mass spectrometer in electrospray ionization (ESI) mode (Bruker Daltonics Corp., USA). The absorption spectra were taken on a Shimadzu UV-2700 spectrophotometer (Shimadzu Suzhou instruments Mfg. Co, Ltd). The fluorescence spectra were taken on a Hitachi F4600 spectrofluorimeter (Hitachi High-Tech Science) with a 10 mm quartz cuvette. Na2S stock solution (5 mM) was used as the source of H2S in the photophysical investigation of 1-H2S [39]. Imaging of cells, liver-tissue slices and zebrafish was performed on a Nikon A1 MP+ two-photon microscope (Nikon instruments Inc.) and live-animal imaging experiments were performed with PerkinElmer IVIS Lumina Series III Pre-clinical (PerkinElmer Inc.) in vivo imaging system.
Synthesis of two-photon fluorescent probe 1-H2S. The synthesis process of two-photon fluorescent probe was illustrated as scheme S1 in supporting information. The two-photon platform was synthesized by two steps. Briefly, salicylaldehyde and 6-amino-3,4-dihydronaphthalen-1(2H)-one were reacted in concentrated acid to yield the desired product 3-amino-10-(diethylamino)-5,6-dihydrobenzo[c]xanthen-12-ium (TP-1), then the amino group was converted to azide group to obtain the compound
3-azido-10-(diethylamino)-5,6-dihydrobenzo[c]xanthen-12-ium (1-H2S) with a high yield.
Cell culture and fluorescence imaging HeLa cells were obtained from Pharmaceutical College of Shandong University, cultured at 37 oC incubator with 5% CO2 and 95% air in modified Eagle’s medium supplemented with 10% calf bovine serum. For cell imaging experiments, the cells were seeded onto glass plates for 12 hours for adherence, then washed by PBS and incubated with the probe 1-H2S before fluorescence imaging. To evaluated the cytotoxicity of 1-H2S, the cell viability of 1-H2S was determined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The HeLa cells were seeded into a 96-well plate at the density of 105 cells per well and cultured for 12 hours. Then the cells were cultured with various concentrations (0, 1, 5, 10, 20, 30 μM) of 1-H2S for another 24 hours. 10 μL of MTT were added to each well and further incubated for 4 hours, followed by addition of 100 μL of DMSO. The plate was shaken for 30 min and the absorption at 570 nm was collected by a microplate reader made by Thermo Fisher Scientific. The cell viability was calculated by the absorption at 570 nm. Fluorescence imaging in cells by 1-H2S were performed on Nikon A1 MP+ two-photon microscope.
Live animal imaging experiments
All animal procedures for this study were approved by the Animal Ethical Experimentation Committee of Shandong University according to the requirements of the National Act on the use of experimental animals (China). Zebrafish were cultured for three days in clean water before use. The probe 1-H2S 10 μM was added and cultured for 30 min and imaged by two-photon microscope. For H2S detection, the zebrafish were first cultured with 1-H2S 10 μM for 30 min, the zebrafish were washed by clean water and then 20 μM of Na2S was added and further incubated for 30 min before fluorescence imaging by one-photon or two-photon light source. For living mice imaging experiments, Kunming mice were anesthetized by intraperitoneally injection of chloral hydrate 200 μL (4% in water). Then 20 μg of 1-H2S was injected into the mice intraperitoneally, followed by 60 μg of Na2S. The mice were imaged by the PerkinElmer IVIS Lumina Series III in vivo imaging system.
Result and discussion Photophysical properties of 1-H2S. The photophysical properties of 1-H2S in solution were explored. 1-H2S exhibits an intense absorption band at around 556 nm and a weak fluorescence peak at about 635 nm in PBS (pH 7.4) and DMSO (8:2) buffer (Fig. 2a and Fig. S1). As shown by Fig. S2, 1-H2S shows a stable fluorescence emission when irradiated by 580 nm light for 4 hours or heated the test solution to 80 oC, which indicated a good photo-stability and thermal-stability of 1-H2S in
solution. In absorption spectra (Fig. S1), in the presence of excess Na2S, the absorption band at 556 nm red shifted about 34 nm to 590 nm due to the reduction of azido group to amino group by Na2S, which leads to an enhanced intramolecular charge transfer (ICT) effect in the probe. To manifest the detection mechanism and the spectra change, the reaction process of 1-H2S with Na2S was also investigated by HRMS. As shown by HRMS analysis (Fig. S4, S5), the azido group of 1-H2S was reducted to amine by Na2S and both HRMS signals of 1-H2S and 1-TP were found in the HRMS spectra. The HRMS analysis experiment indicated that the spectra change is due to the generation of 1-TP. The HRMS analysis result and the spectra change behavior of 1-H2S in the presence of Na2S were also supported by literature [40, 41]. In emission spectra, the fluorescence peak at 635 nm increased sharply with the addition of Na2S. Compared with Na2S blank sample, about 7-fold fluorescence enhancement was observed in the presence of 30 eq. of Na2S (Fig 2a and Fig. S3). As shown by Fig. 2b, a linear growth of the fluorescence intensity with the Na2S-to-probe concentration ratio increase was found. The detection limit of 1-H2S to Na2S is about 4.7 × 10-5 M, as estimated by S / N = 3 method [42], which is lower than the concentration of endogenous H2S in physiological environment [2]. The reaction rate of 1-H2S with Na2S was tracked by fluorescent spectra (Fig. 2 c, d). It takes about 15 min to reach the maximum of fluorescence when 1-H2S reacted with 50 equivalents of Na2S at 37 oC. The rapid response toward Na2S is beneficial for detection of H2S in biological
samples (Table S1).
Selectivity experiment We tested the selectivity of 1-H2S toward Na2S and other biologically relevant reactive nitrogen, reactive oxygen and sulfur species including hydrogen peroxide (H2O2), superoxide anions (·O2-), tert-butylhydroperoxide (TBHP), nitric oxide (NO), peroxynitrite (ONOO-), sulfite (SO32-) and standard test amino acids such as cysteine (Cys), homocysteine (Hcy), glutathione (GSH). As shown by Fig. 3a, clearly, only Na2S turns on the fluorescence intensity of 1-H2S within 30 min, whereas other species produce an almost negligible fluorescence enhancement under the same condition. The fluorescence intensity at 635 nm showed a huge fluorescence enhancement only when Na2S was added to the solution (Fig. 3b). The high selectivity of 1-H2S toward Na2S could be used for Na2S detection of in physiological condition.
Cells imaging experiment H2S has been recognized as one of the most important gaseous signaling molecule that plays important roles in physiological activities. The above experiments have demonstrated that 1-H2S exhibited large fluorescence enhancement and high selectivity toward Na2S in solution environment. Thus the application of 1-H2S for Na2S detection in cells, tissue and live animals were investigated. Firstly, the toxicity of the probe was explored by MTT
method. As shown by the result (Fig. S6), the probe exhibited little toxicity to HeLa cells at the concentration measured (20 μM), over 80% cells survived after incubation with 10 μM 1-H2S of for 24 h. The cellular distribution of 1-H2S was then measured by colocalization analysis with traditional organelles labeling agents, such as mitochondria labeling agent Mito-Tracker Green and nucleus labeling agent Hoechst 33258. When the Hela cells were co-incubated with Mito-Tracker Green and 1-H2S, as shown by Fig. 4 (d - f), the probe can penetrate cell membrane easily and aggregated in mitochondria area at the first 10 min. As calculated from Fig. 4e, the colocalization coefficient is about 81% (Pearson correlation coefficient, calculated by software installation in Nikon A1 MP+ two-photon microscope). The fluorescence intensity analysis (Fig. 4f) along the arrow in a selected cell in Fig. 4d also demonstrated a high overlap of the two fluorescence signals. Furthermore, the HeLa cells were co-incubated with Hoechst 33258 and 1-H2S, as shown by Fig. 5 and Fig. 6. At the first 10 min, the probe mainly distributed in cytoplasm. When the incubation time was prolonged to about 40 min, 1-H2S tend to penetrate the cell nuclear membrane and retained in the nucleolus region (Fig. 5 f ̶ h; Fig. 6 c, d) [43, 44]. The result was further confirmed by fluorescence intensity analysis along the arrows in selected cells. As shown by Fig. 6d, the main fluorescence intensity enhancement of 1-H2S was observed in nucleolus region.
Fluorescence imaging of H2S in HeLa cells Encouraged by the large fluorescence enhancement of 1-H2S to Na2S in solution and the nucleolus localization ability, we tend to investigate the detection of 1-H2S to H2S in cells and tissues by fluorescence imaging. As shown by Fig. 7, when the HeLa cells were incubated with 1-H2S solely, only weak fluorescence were observed in TRITC channel when excited by 561 nm laser and two-photon excitation. However, a bright fluorescence of 1-H2S was observed in nucleolus region when the HeLa cells were pre-incubated with 10 μM of Na2S for 20 min then incubated with 1-H2S for 20 min. The experiment demonstrated that 1-H2S can penetrate the cell nuclear membrane and image H2S in nucleolus region successfully.
Tissue and zebrafish imaging Based on the result obtained on fluorescence detection of H2S in solution and cellular environment, we tried to apply the probe for H2S detection in tissues. The two-photon absorption cross-section spectra of 1-H2S and 1-TP were measured, 1-TP exhibits stronger absorption band at around 750 nm to 820 nm than 1-H2S, which indicated an effective tune function of azide group to the fluorescence (Fig. S7). Freshly prepared liver tissue slices (thickness: 400 μm) of mice were steeped in a solution of 10 μM 1-H2S for 30 min, then the tissue were washed by PBS and further incubated with 20 μM Na2S solution for another 30 min. Fluorescence imaging was carried out before and after the
treatment of the tissue slices by Na2S solution (Fig. 8). The liver tissue showed weak fluorescence when only incubated with 1-H2S. When the tissue was incubated with 1-H2S and further incubated with Na2S, an enhanced fluorescence was observed under the same imaging parameters. The liver tissue was analyzed by scan along the Z-axis and three-dimensional reconstruction, to test the tissue penetration ability of the probe. The fluorescent signal can be detected at a depth of 60 μm, the maxima fluorescent signal mainly emerged at a depth of about 40 μm. The experiment exhibited that the probe can be used for image Na2S in deep tissue by two-photon excitation mode. Compared with one-photon excitation mode, the two-photon excitation mode shows obvious advantage in reactive species detection in tissue, which facilitates the study of physiological activities in deep tissue. Thus, the probe has exhibited excellent tissue image property, and it was used to image H2S in live zebrafish. Zebrafish is a typical biological model for investigation of the physiological activity of vertebrate animals and usually used in evaluation of the function of small molecules and pharmaceutical analysis in live animals. The fluorescence detection of Na2S by 1-H2S was carried out in zebrafish model; one week old zebrafish was used. As shown by Fig. S8, when 10 μM of the probe was incubated with zebrafish for 30 min, weak fluorescence was observed in TRITC channel. However, when the zebrafish was pre-incubated with 20 μM Na2S and then incubated with 10 μM of the probe for 30 min, a strong fluorescent signal was observed. The same
fluorescence imaging result was obtained when excited by 760 nm laser under two-photon excitation mode (Fig. S9). The typical physiological levels of H2S are between 0.1- 300 μM in live animals, which is higher than the Na2S concentrations used in the above imaging experiments, thus the probe can potentially be used for detection physiological levels of H2S in live animals [2].
Fluorescence imaging of H2S in live animal We then tend to investigate the fluorescence imaging ability of H2S in live animals by 1-H2S. The four-week old female balb/c mice were kept in stainless steel cages that contain barriers for each mice for individual housing. They were fed commercial mice chow and left freely wandering in their cage for two weeks with 12 hours dark / light cycles for acclimatization before starting the experiment. The mouse was anesthetized by 200 μL trichloroacetaldehyde (4% wt), then 20 μg of the probe was subcutaneously injected into the abdomen of the mouse, Na2S was carefully injected to the same location. The mouse was placed into live animal imaging system. As shown by Fig. 9, the probe shows little fluorescence under 580 nm light excitation. When Na2S was added, the fluorescent signal increased steadily in a time range of 40 min. about 7-fold fluorescence enhancement was observed, when compared with the fluorescence of the probe itself. The experiment demonstrated that 1-H2S can be used to detect H2S in live animals successfully.
Conclusions Most of the two-photon fluorescent dyes have emission wavelength at comparatively short wavelength region. Development of two-photon dyes with emission in red light region is attractive for increasing their application area in biological imaging, which can eliminate interference from strong tissue auto-fluorescence and increase tissue penetration ability. Herein, a novel two-photon fluorescent probe 1-H2S for H2S with red light emission was constructed through extending the conjugated system of traditional two-photon platform naphthalene and coumarin. The photophyscial experiments demonstrate that 1-H2S can respond to H2S with large fluorescent enhancement at 635 nm, and shown high selectivity toward H2S. Colocalization analysis has shown that the probe can aggregate at the nucleolus region when the incubation time is prolonged. The probe can detect H2S successfully in nucleolus region of cells by one photon and two-photon excitation. Taking advantage of the emission in red light region, the probe was further applied to image H2S in zebra fish and living mice successfully.
Acknowledgements This work was financially supported by NSFC (61605060, 21672083, 21472067), Natural Science Foundation of Shandong Province (ZR2015PB016), Taishan Scholar Foundation (TS 201511041) and the startup fund of University of Jinan (309-10004, 160100137).
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Author Biographies
Dr. Keyin Liu obtained his Ph. D. degree from Fudan University in 2014. Currently, he is a lecturer in Institute of Fluorescent Probes for Biological Imaging in University of Jinan, Jinan, P.R. China. His research interests focus on the design and synthesis of novel functional fluorescent dyes/probes and their biological applications.
Chuang Liu obtained his B.S. degree at Langfang Teachers University in 2017. Currently, he is a graduate student under the supervision of Professor Weiying Lin in the Institute of Fluorescent Probes for Biological Imaging, University of Jinan. His research interests focus on the design and synthesis of fluorescent probes.
Huiming Shang obtained his B.S. degree at Dezhou University for Nationalities in 2014. Currently, he is a graduate student under the supervision of Professor Weiying Lin in the Institute of Fluorescent Probes for Biological Imaging, University of Jinan. His research interests focus on the design and synthesis of fluorescent probes.
Dr. Mingguang Ren received his Ph.D. degree from University of Science and Technology of China in 2011. Now, he is an associate professor in Institute of Fluorescent Probes for Biological Imaging at University of Jinan, Jinan, P.R. China. His research interests focus on the design and synthesis of novel functional fluorescent dyes/probes and their biological applications
Professor Weiying Lin received his Ph.D. from the University of Kansas in 2000. After completing postdoctoral research at Massachusetts Institute of Technology, in 2005, he joined the faculty at Hunan University. Subsequently, he moved to the University of Jinan as the Dean and the Distinguished Professor of the Institute of Fluorescent Probes for Biological Imaging. His research interests cover the interdisciplinary areas of molecular recognition, photochemistry, materials chemistry, analytical chemistry, and chemical biology.
Figure Captions
Fig. 1. (A) Generation of fluorescence and (B) sketch of construction of two-photon probes with longer wavelength. (C) The red light emissive two-photon fluorescent probe designed and the sensing mechanism to H2S.
Fig. 2. The change of fluorescence spectra of 10 μM 1-H2S titrated with Na2S. (a) 10 μM of 1-H2S titrated with increasing concentrations of Na2S. (b) Linear fit of fluorescence intensity change at 635 nm with Na2S to probe ratio. (c) The fluorescence intensity change of 10 μM 1-H2S with time, in the presence of 50 equivalents Na2S. (d) The fluorescence intensity change at 635 nm with time, Excitation: λ = 580 nm.
Fig. 3. Selectivity of 1-H2S to Na2S. (a) Fluorescence spectra change of 10 μM 1-H2S in the presence of 50 equivalents of Na2S and other standard small test molecules. The probe was incubated with the molecules for 30 min. (b) Fluorescence intensity of 1-H2S at 635 nm after 30 min incubation with standard small test molecules. Number 1 – 13 along the horizontal axis represent samples of 1-H2S interacted with 1 blank, 2 H2O2, 3 ·O2-, 4 TBHP, 5 NO, 6 ONOO-, 7 SO32-, 8 Cys, 9 Hcy, 10 GSH, 11 S2O32-, 12 SO42-, 13 S2respectively.
Fig. 4. Colocalization experiment of 1-H2S with Mito-Tracker Green in HeLa cells. 10 μM 1-H2S and 1 μM of Mito-Tracker Green were coincubated with HeLa cells for 10 min washed by PBS before fluorescence imaging. (a – d) represent Bright field, Green channel, TRITC channel, and Merged image. (e) The colocalization coefficient analysis of TRITC and Green channels by software installation on Nikon microscope and (f) fluorescence intensity analysis of green and red channels along the arrow in selected cells in the white circle. Green channel: excitation: 488 nm, emission: 500 – 550 nm. (Parameters: laser wavelength: 488 nm, laser power: 0.1 mW; emission filter: 500 – 550 nm, PMT HV value: 90; pinhole radius: 26.8, scan speed: 0.5 second) TRITC channel: excitation: 561 nm, emission: 570 – 620 nm, pseudo color. (Parameters: laser wavelength: 561 nm, laser power: 0.4 mW; emission filter: 570 – 620 nm, PMT HV value: 100; pinhole radius: 26.8, scan speed: 0.5 second)
Fig. 5. Colocalization experiment of 1-H2S with Hoechst33258 in HeLa cells. 10 μM 1-H2S and 1 μM of Hoechst33258 were coincubated with HeLa cells for 10 min and 40 min respectively and washed by PBS before fluorescence imaging. (a, e) Bright field, (b, f) Blue channel, (c, g) TRITC channel, (d, h) Merged image. Blue channel: excitation: 405 nm, emission: 430 – 480 nm (Parameters: laser wavelength: 405 nm, laser power: 2.5 mW; emission filter: 425 – 475 nm, PMT HV value: 150; pinhole radius: 26.8, scan speed: 0.5 second). (c) TRITC channel: excitation: 561 nm, emission: 580 – 650 nm, pseudo color. (Parameters: laser wavelength: 561 nm, laser power: 0.2 mW; emission filter: 570 – 620 nm, PMT HV value: 100; pinhole radius: 26.8, scan speed: 0.5 second)
Fig. 6. Fluorescence intensity analysis of Hoechst33258 and 1-H2S in selected cells. 10 μM 1-H2S and 1 μM of Hoechst33258 were co-incubated with HeLa cells for 10 min [(a), (b)] and 40 min [(c), (d)], respectively. (a, c) Merged image of blue and TRITC channels of HeLa cells, (b, d) fluorescence intensity analysis along the arrows in (a, c) respectively.
Fig. 7. Fluorescence imaging of H2S in HeLa cells by one-photon and two-photon excitation. (a, b) HeLa cells were incubated with 5 μM 1-H2S for 20 min, (c, d) HeLa cells were pre-incubated with 10 μM Na2S for 20 min then incubated with 5 μM of 1-H2S for 20 min. (a, c) Single-photon image excited by 561 nm laser, collected at TRITC channel: 580 – 640 nm. (Parameters: laser wavelength: 561 nm, laser power: 0.1 mW; emission filter: 570 – 620 nm, PMT HV value: 100; pinhole radius: 26.8, scan speed: 0.5 second) (b, d) Two-photon image excited by 760 nm laser, collected at TRITC channel: 570 – 620 nm. (Parameters: laser wavelength: 760 nm, laser power: 30.0 mW; TRITC channel: emission filter: 570 – 620 nm, PMT HV value: 105; pinhole radius: 255.43, scan speed: 0.25 second) Scale bar: 20 μm.
Fig. 8. Fluorescence detection of H2S in liver tissue of mice. (a) liver tissue slice (thickness: 400 μm) incubated with 1-H2S for 30 min then washed by PBS (b) liver tissue incubated with 1-H2S for 30 min then incubated with 20 μM Na2S for another 30 min, (c) three-dimension reconstruction of liver tissue incubated with Na2S and 1-H2S image of by z-scan between a depth of form 0 to 60 μm. Excitation by 760 nm, emission collection: 570-620 nm. (Parameters: laser wavelength: 760 nm, laser power: 30.0 mW; TRITC channel: emission filter: 570 – 620 nm, PMT HV value: 105; pinhole radius: 255.43, scan speed: 0.25 second)
Fig. 9. Fluorescence imaging of H2S in living mouse by 1-H2S. (a) 20 μg of 1-H2S was subcutaneously injected only, (b - i) 20 μg of 1-H2S and 60 μg of Na2S were subcutaneous injected for 2, 5, 10, 15, 20, 25, 30, 40 min respectively. Excitation filter: 580 nm; emission filter: 600 - 650 nm. (The fluorescence imaging Parameters: Exposure time: 0.2 seconds; Binning factor: 2; F-stop: 2; Field of view: 12.5 cm; Object radius: 0.75; Subject size: 1.5; CCD Measure temperature: -90 oC).