Photostable Ratiometric Two-photon Fluorescent ... - ACS Publications

Subscriber access provided by UNIV AUTONOMA DE COAHUILA UADEC

Article

Photostable Ratiometric Two-photon Fluorescent Probe for Visualizing Hydrogen Polysulfide in Mitochondria and Its application Qingxin Han, Jiaxi Ru, Xuechuan Wang, Zhe Dong, Li Wang, Huie Jiang, and Weisheng Liu ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.9b00044 • Publication Date (Web): 05 Apr 2019 Downloaded from http://pubs.acs.org on April 5, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Bio Materials

Photostable Ratiometric Two-photon Fluorescent Probe for Visualizing Hydrogen Polysulfide in Mitochondria and Its application Qingxin Hana,b, Jiaxi Ruc*, Xuechuan Wangb*, Zhe Donga, Li Wanga, Huie Jiangb and Weisheng Liua*

a

Key Laboratory of Nonferrous Metal Chemistry and Resources Utilization of Gansu

Province and State Key Laboratory of Applied Organic Chemistry, College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou, 730000, China. b

Institute for Biomass and Function Materials, College of Bioresources Chemistry

and Materials Engineering, Shaanxi University of Science and Technology, Xi’an 710021, China. c

State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary

Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, 730046, China. ABSTRACT Hydrogen polysulfide (H2Sn) has currently attracted much research interest because it not only plays important physiological function in many biological and health-related events, but also considered as a newfound potent signal transducer. Small-molecule

ACS Paragon Plus Environment

ACS Applied Bio Materials 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

based ratiometric fluorescent probes have advantages in sensitivity and bio-detections but such approaches that intentionally developed for H2Sn detection expected to be mitochondria-accessible are still lacking. In this work, due to that triphenylphosphine group introduced into the molecular scaffold of naphthalimide derivative, Mito-NRT-HP was successfully applied to visualize intracellular H2Sn in mitochondria with excellent aqueous solubility, super photobleaching resistance, favorable cellular membrane permeability and good biocompatibility. This one- and two-photon fluorescent probe with high selectivity and sensitivity (LOD = 0.01 μM) evinced 70-fold enhancement of fluorescence ratio (I546 nm/I478 nm) in the presence of H2Sn over other reactive sulfur species (RSS). The experimental results also give Mito-NRT-HP the potential for mapping the H2Sn distribution in mitochondria and evaluating the H2Sn roles in more biological processes and demonstrated the practical application possibility of Mito-NRT-HP in early diagnosis of LPS-induced acute organ injury. Keywords: ratiometric; mitochondria; two-photon; fluorescent probe; hydrogen polysulfide 1. INTRODUCTION The chemical biology of reactive sulfur species (RSS), mainly including biothiols and other sulfur- containing molecules such as hydrogen sulfide (H2S), hydrogen polysulfide (H2Sn), and S-modified cysteine adducts, has become the ever-increasing focus of research.1-4 Thereinto H2S was supposed to be a gaseous transmitter known after carbon monoxide (CO) and nitric oxide (NO), and it exert protective effects in


Page 2 of 50

Page 3 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


regulating the intracellular redox status and other fundamental signaling processes involved in human health5-9. However, increasing evidences suggest that some H2S-involved signal transduction and physiological effects are mainly done by H2Sn10-12, the oxidized form of H2S and generated from enzymatic H2S biosynthesis13-16. Recent studies indicate that H2Sn is more favourable than H2S to react with bisulphide and cysteine residues in S-sulfhydration which is responsible for the activity changes of some protein enzymes.17-18 For example, the efficiency of H2Sn in activating transient receptor potential (TRP) channels and inducing Ca2+ influx in astrocytes via S-sulfhydration is 320 times to that of H2S.19 Besides, H2Sn also exhibits high potency in regulating the activity of transcription factors and tumor suppressors.20-23 However, the fundamental chemistry and biology functions of H2Sn are still poorly understood though H2Sn might be the real signaling molecule rather than H2S in some biological processes. Given the indispensable role of H2Sn and the controversy about which is the signaling molecule on earth, developing excellent fluorescence probes with high selectivity for H2Sn over H2S is desperately needed. Among all kinds of fluorescence probes, small-molecule fluorescent probe is particularly invaluable and powerful to monitor the generating, distribution, accumulating, and dynamic fluctuation of active biological species due to their real-time, sensitive, and noninvasive characteristics and unrivaled spatiotemporal resolution ability.24-26 However, the research progress of small-molecule fluorescent probes for visualizing intracellular H2Sn are still in its infancy because there are short of proper understanding about its reactivity until Xian



Page 4 of 50

and co-workers developed two fluorescent probe for H2Sn by taking advantage of H2Sn-mediated benzodithiolone formation.27-28 Making use of the reducing property and high nucleophilicity of H2Sn29, a number of fluorescent probes have been developed for H2Sn detection in living systems.30-42 The fundamental chemistry of H2Sn has been revealed by these probes and researchers can understand more about the RSS biology than ever.43-45 It has been widely acknowledged that ratiometric two-photon fluorescent probe is always superior, as it possesses not only the excellent characteristic of two-photon fluorescent probe, such as localized excitation, lower tissue background fluorescence and self-absorption, deeper penetration depth, and little tissues damage,46-50 apart from the need of a costly high-intensity (105-108 W cm2) pulse laser source for the two-photon excitation and the additional photobleaching pathway activated by higher photon density, but also the advantages of ratiometric fluorescent probe in accurately evaluating the analyte’s concentration and eliminating systematic errors which derives from the probe’s concentration, light collection efficiency, optical distance, and sample environment, etc.51-52 Unfortunately, the ratiometric two-photon fluorescent probes for H2Sn can be counted on one's fingers 30, 43. From another perspective, research results imply that generation of H2Sn is inextricably associated with H2S redox homeostasis in mitochondria and the mitochondrial fraction of H2Sn are more than half in a cell.13,

53-54

Hence, it is

especially appreciative to develop a ratiometric two-photon fluorescent probe to visualize the distribution and fluctuation of mitochondrial H2Sn. As far as we know, such a significant approach has not been reported yet. Moreover, fluorescent probe


Page 5 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


with high photostablity is always needed on account of the expectation to resist photobleaching well during fluorescent bio-imaging. Thus, here are key challenges for imaging and sensing intracellular H2Sn: (1) How to establish powerful ratiometric two-photon approach to detect mitochondrial H2Sn; (2) How to improve the selectivity over other RSS, such as H2S and biothiols, while for the probe based on H2Sn-induced nucleophilic substitution reaction and spontaneous cyclization; (3) How to obtain a small-molecule based fluorescent probe with excellent photobleaching resistance and biocompatibility. Taking these requirements into consideration and following our previous study on intracellular H2Sn detection30, we present herein the design and synthesis of a photostable two-photon fluorescent probe, Mito-NRT-HP (Scheme 1), for ratiometric visualizing H2Sn in mitochondria. Mito-NRT-HP showed not merely high two-photon absorption cross section and excellent ratiometric fluorescence response towards H2Sn, but several promising advantages such as high aqueous solubility, super photobleaching resistance, extremely favorable cellular membrane permeability and good biocompatibility. More interestingly, this nitro-activated fluorobenzoiate55 involved fluorescent probe will not participate in irreversible reaction with H2S and biothiols even in high concentrations. Besides the above-mentioned properties, fluorescence imaging experiments revealed that this probe could be applied to specifically track mitochondrial H2Sn in both living cancer cells and normal cells and the probe was favorable for application in complex biosystems. (Scheme 1 in here)



Page 6 of 50

2. EXPERIMENTAL SECTION Materials and instruments Reagents

and

starting

4-amino-1,8-naphthalic 4-dimethylaminopyridine,

materials

including

2-fluoro-5-nitro

anhydride, triphosgene,

benzoic

acid,

N,N-diisopropylethylamine,

trimethylamine,

1,2-diaminopropane,

4-hydroxybenzaldehyde, (4-carboxybutyl) triphenylphosphonium bromide and other chemicals were obtained from commercial sources. The dried solvents of analytical grade used in this study were prepared according to standard procedures before use. LPS (Escherichia coli O111:B4) was purchased from Sigma-Aldrich Chemical Co. Ltd. Mito Tracker Red CMXRos and Lyso Tracker Red DND-99 were purchased from shanghai YEASEN Biotech Co., Ltd. and CellTiter 96® AQueous One Solution Cell Proliferation Assay (1,000 assays) was purchased from Promega (Beijing) Biotech Co., Ltd. (4-hydroxymethyl)phenyl-2-fluoro-5-nitrobenzoate was synthesized according to our previous method.30 The reactions were performed without protection except where noted and monitored by thin-layer chromatography (TLC). 1H NMR and 13C

NMR spectra were recorded on a JNM-ECS 400M spectrometer and referenced to

TMS. ESI-MS were performed on a Bruker Esquire 6000 Ion Trap System. The absolute quantum yield Ф and two-photon fluorescence was measured by using an Edinburgh Instrument FLS920 spectrophotometer. All pH measurements were made with a pH-10C digital pH meter. The absorption spectra were recorded using a Varian Cary 5000 UV-Vis-NIR spectrophotometer and fluorescence spectra were recorded by using a Hitachi F-7000 spectrophotometer. Fluorescence imaging experiments


Page 7 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


were performed with a Leica TCS SP8 confocal fluorescence microscope for one-photon imaging (λex = 405 nm) and an Olympus FV1000 MPE two-photon microscope for two-photon imaging (λex = 800 nm). The imaging signals at 425-475 nm and 550-600 nm range were both collected by internal PMTs in a 12 bit unsigned 1024×1024 pixels. Synthesis of Mito-NRT-HP Synthesis of N-(2-aminoethyl)-4-Amino-1,8-naphthalimide. To a solution of 4-Amino-1,8-naphthalic anhydride (540 mg, 2.5 mmol) in 50 ml of ethanol, added triethylamine (600 mg, 10 mmol) under stirring. The reaction mixture was stirred at 80 oC for 8 hours and then cooled to room temperature. The solvent was removed and a small amount of water was added. The crude product was obtained by suction filtration and washed with water (50 mL) and diethyl ether (25 mL) in sequence. After dried in a vacuum oven at 50 oC overnight, this product was used in next step without further purification. 1H NMR (400 MHz, d6-DMSO) δ 8.59 (t, J = 10.6 Hz, 1H), 8.43 (dd, J = 11.2, 4.9 Hz, 1H), 8.19 (d, J = 8.3 Hz, 1H), 7.65 (t, J = 7.8 Hz, 1H), 7.41 (s, 2H), 6.85 (d, J = 8.4 Hz, 1H), 4.08-3.98 (m, 2H), 2.82-2.73 (m, 2H). Synthesis of Mito-NRT. To a solution of (4-carboxybutyl)triphenylphosphonium bromide (443 mg, 1.0 mmol) in 10 mL dry DMF, added EDCI (191 mg, 1.0 mmol) and DMAP (122 mg, 1.0 mmol). And the mixture was stirred at room temperature for an hour under Ar protection. Then, the reaction was kept stirring overnight after compound 2 (255 mg, 1.0 mmol) was added. The solvent was evaporated under reduced pressure after the reaction and the residue was purified by silica gel



chromatography with CH2Cl2/CH3OH (v/v, 20:1 to 5:1) as eluent, yielding Mito-NRT as claybank solid (531 mg, 78%). 1H NMR (400 MHz, d6-DMSO) δ 8.62 (d, J = 8.1 Hz, 1H), 8.34 (dd, J = 7.3, 1.0 Hz, 1H), 8.12 (d, J = 8.4 Hz, 1H), 7.93-7.86 (m, 4H), 7.83-7.74 (m, 12H), 7.62 (dd, J = 8.3, 7.4 Hz, 1H), 7.46 (s, 2H), 4.04 (t, J = 6.1 Hz, 2H), 3.55 (t, J = 14.4 Hz, 2H), 2.04 (t, J = 7.0 Hz, 2H), 1.70-1.61 (m, 2H), 1.57-1.46 (m, 2H). 13C NMR (101 MHz, d6-DMSO) δ 171.67, 164.01, 163.09, 152.79, 134.86, 133.63, 133.53, 130.27, 130.16, 123.75, 121.83, 119.35, 118.95, 118.10, 108.01, 107.49, 36.64, 34.10, 25.80, 21.17. ESI-MS m/z [(M-Br)+]: 600.4.

Synthesis of Mito-NRT-HP. To an airtight reaction flask contains Mito-NRT（340 mg，0.5 mmol）and DMAP (25 mg, 0.2 mmol) injected 20 mL of dry CH2Cl2 with a syringe, and 250 μL of trimethylamine was added gradually. The CH2Cl2 solution of triphosgene (150 mg, 0.5 mmol) was injected to the reaction half day later. Then added compound 2 (174 mg, 0.6 mmol) under Ar protection. After stirred overnight, the solvent was evaporated under reduced pressure and the residue was purified by silica gel chromatography with CH2Cl2/CH3OH (v/v, 50:1 to 20:1) as eluent, yielding Mito-NRT-HP as faint yellow solid (629 mg, 63%). 1H NMR (400 MHz, d6-DMSO) δ 10.43 (d, J = 1.6 Hz, 1H), 8.83 (dd, J = 6.2, 3.0 Hz, 1H), 8.73 (d, J = 8.6 Hz, 1H), 8.67-8.59 (m, 1H), 8.43 (dd, J = 9.0, 7.8 Hz, 2H), 8.21 (d, J = 8.3 Hz, 1H), 7.98-7.86 (m, 4H), 7.85-7.72 (m, 15H), 7.65 (d, J = 8.4 Hz, 2H), 7.42 (d, J = 8.5 Hz, 2H), 5.33 (s, 2H), 4.13-4.04 (m, 3H), 3.55 (t, J = 12.9 Hz, 2H), 3.17 (d, J = 5.3 Hz, 1H), 2.02 (t, J = 6.6 Hz, 2H), 1.63 (dt, J = 14.4, 7.2 Hz, 2H), 1.55-1.46 (m, 2H), 0.85 (ddd, J = 7.5, 2.5, 1.8 Hz, 2H). 13C NMR (101 MHz, d6-DMSO) δ 171.68, 165.83, 163.66, 163.11,


Page 8 of 50

Page 9 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


160.36, 153.92, 149.88, 143.75, 140.56, 134.83, 134.53, 133.61, 133.51, 130.81, 130.21, 130.09, 129.75, 129.64, 128.42, 127.85, 127.68, 126.27, 123.83, 122.36, 122.04, 121.78, 119.35, 118.93, 118.63, 118.52, 118.08, 117.26, 65.97, 36.37, 34.10, 26.31, 21.21, 13.91. ESI-MS m/z [(M-Br)+]: 917.1. Spectroscopy studies The stock solution of Mito-NRT-HP was prepared at 10-3 M in DMSO. The aqueous solutions of various biological ions were prepared from corresponding perchlorate or sodium salt and the stock solutions of reactive small biological molecule were prepared or diluted with their analytical reagent. In selectivity and interference measurements, a final concentration of 1.0 mM was used for Na+, K+ and GSH, 0.1 mM for Ca2+, Mg2+, Cl-, Br-, I-, HSO32-, NO3-, S2O32-, H2PO4-, NO2-, OAc-, CO32-, Cystine, GSSG, AA, ADP and ATP, 50 μM for Cys, Hcy, Na2S, H2O2 and NaClO, and 10 μM for Zn2+, Fe3+, Fe2+ and Mn2+. But beyond that, the concentrations of H2S2 and H2Sn were referred to their sodium salt in this study. For a typical test, 20 μL of stock solution Mito-NRT-HP and an appropriate aliquot of each analyte stock solution into were added into 2.0 mL of PBS buffer solution (10 mM，pH 7.40) in a quartz cuvette with a cell volume of 3.0 mL. The resulting solution was kept at room temperature for 15 min before its fluorescence spectra were recorded. The two-photon absorption (TPA) cross section values (δ) were measured according to the reported process by using fluorescein as reference compound.30, 56 Cell culture and fluorescence imaging



Cells (HeLa cells or BHK cells) were cultured in DMEM (Dulbecco's Modified Eagle's Medium, High Glucose) culture medium (containing 10% fetal bovine serum, FBS) at an atmosphere of 5% CO2 and 95% with a humidified incubator set at 37 oC for 48 h. After removing DMEM culture medium, the adherent cells were treated with trypsin and incubated with cell culture medium (about 5×108 cells per liter) for 24 h. Then, the cells were seeded in a confocal culture dish before subjected to imaging. For cytotoxicity assay, HeLa cells were initially propagated in a 25 cm2 tissue culture flask in DMEM supplemented with 10% (v/v) FBS, penicillin (100 μg/mL), and streptomycin (100 μg/mL) in a CO2 incubator, and then the cells were inoculated in 96-well plate at a density of 104 cells per well. After incubated with various concentrations (0, 5, 10, 30, 50 and 100 µM) of Mito-NRT or Mito-NRT-HP for 24 h, MTS reagent (10 μL/well, 0.5 mg/mL) was added to each well and incubated for additional 4 h. The optical densities were measured by using a microplate reader (BIO-RAD Model 680, USA) at 490 nm, which were directly proportional to the number of viable cells. Each case of concentration was done in quintuplicate and the average value was taken ultimately. Imaging of samples of acute organ injury model The LPS induced acute organ injury model of mice was established by following literature reports43, 57-60. Male C57BL/6 mice of normal body weight were purchased and housed in standard conditions to adjust to the environment before they were randomly divided into two groups. For first group, 10 mg/kg body weight of LPS in 100 μL 0.9% saline was administered with an intraperitoneal injection. For another


Page 10 of 50

Page 11 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


group, 100 μL 0.9% saline was administered as control. Then, mice were injected with 10 μM Mito-NRT-HP in 50 μL mixture solution of DMSO/saline (1:99, v/v). Mice were euthanized, and their intestine, lung, liver, kidney and spleen tissue samples were harvested at 1.0 h after injection of Mito-NRT-HP. 3. RESULTS AND DISCUSSION The aforementioned concerns encouraged us to develop a mitochondrial targeted two-photon fluorescent for ratiometric sensing H2Sn. As shown in Scheme 1, 1,8-naphthalimide derivates are common fluorophores with excellent photophysical properties including tunable intramolecular charge transfer (ICT) fluorescence, admirable

two-photon

properties,

favourable

photostability

and

reduced

pH-interference. Moreover, this kind of molecular structure can be easily functionalized through delicate modification at appropriate positions, which should be beneficial for multipurpose molecular design. Thus, fluorescent probe Mito-NRT-HP was rationally designed based on the following considerations: (1) Bis-electrophilic 2-fluoro-5-nitrobenzoate moiety was introduced as receptor unit to capture H2Sn, in which fluoro group is expected to be replaced by H2Sn, forming a –SSH adducts and then releasing the extricated fluorophore following a spontaneous and fast intramolecular cyclization. In addition, the electron-withdrawing property of fluorobenzoates group was expected to change the ICT fluorescence, resulting distinct blue shifts in both absorption and emission bands. Notably, the ratiometric fluorescent probe can provide a built-in correction to eliminate the environmental effects, thus allowing the accurate measurements for biological samples. (2) The lipophilic



Page 12 of 50

triphenylphosphonium cation was integrated into the molecular structure of Mito-NRT-HP as the targeting group for visualizing H2Sn in mitochondria. We also anticipated such cationic molecular could exhibit intensive water solubility. (3) Though mercapto group (–SH) in RSS hold various degrees of electrophilicity, as the representative GSH can often interfere the detection of H2Sn. However, H2Sn can react with Mito-NRT-HP to form an intermediate sulfide (R-SSH) adducts which then undergoes a cyclization to release fluorophore, while the intermediate product of GSH is restricted to undergo the intramolecular cyclization to change the fluorescence. Moreover, the intermediate product of GSH is able to further react with H2Sn to give the

R-SSH

adducts.

These

design

concepts

are

responsible

for

the

mitochondrial-targeted ratiometric H2Sn probe, Mito-NRT-HP. The synthetic details were elaborated in the experimental section and the characterizing data were presented in the Supporting Information. After reacted with H2Sn, the 19F NMR peak of Mito-NRT-HP at -96.9 ppm was disappeared as the result of mercapto group induced nucleophilic aromatic substitution (Fig. S1). Meanwhile, the mass peak at m/z = 600.2 [M-Br]+ was characteristic of fluorophore Mito-NRT. The sensing performances of Mito-NRT-HP were investigated either. It's interesting to note that surfactant CTAB or Tween 80 was needed for most of H2Sn probes to stabilize H2Sn in the buffer solution or that to enhance their active reaction,55, 61-62 but Mito-NRT-HP could capture and response H2Sn well in PBS buffer solution (10 mM, pH 7.40) without introducing additional surfactant. That maybe because of the introduction of cationic triphenylphosphonium group in molecular structure.


Page 13 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Moreover, Mito-NRT-HP showed an improved water solubility compared with NRT-HP, reported in our previous work.30 Then, Na2S2 (the donor of H2Sn) was used to study the spectral response capability of the well-designed probe in PBS buffer solution because H2S2 is the major component of H2Sn and there is a rapid dynamic equilibrium between H2S2 and H2Sn.28,

41, 46

As shown in Fig. 1, Mito-NRT-HP

showed an absorption peak at 378 nm. Upon titrating Na2S2 with different concentrations, this peak gradually disappeared and a new absorption band around 435 nm formed with a distinct isosbestic point at 405 nm. As a result, the solution color changed from achromatic to yellow. Under excitation at 405 nm, the initial fluorescence emission peak at 478 nm decreased while a new emission peak at 546 nm continuously increased with a significant bathochromic shift approximately 68 nm, implying a ratiometric fluorescent response towards H2Sn accompanied by the fluorescent color changed from blue to green. It's worth noting that the emerging absorption band and emission peak are characteristic of Mito-NRT (Fig. S2), suggesting the generation of Mito-NRT upon reaction of Mito-NRT-HP with H2Sn. Thus, the H2Sn-mediated spectral changes are likely due to the enhanced ICT effect in Mito-NRT. As indicated in Fig. S3, the shapes and positions of excitation spectrum are similar to that of absorption spectrum, regardless of Mito-NRT-HP or Mito-NRT, implying that the fluorescent emissions of the two molecules are originated from S0→S1 transition under photoexcitation. (Fig. 1 in here)



Page 14 of 50

For further study, long-term stability of Mito-NRT-HP was evaluated. As shown in Fig. S4, the fluorescence spectrum of Mito-NRT-HP maintained despite Mito-NRT-HP was stocked in dimethyl sulfoxide solution for five months. This observation means that Mito-NRT-HP is stable under normal circumstance. Moreover, the fluorescence intensity ratio I546

nm/I478 nm

of Mito-NRT-HP could

remain stable and was not obviously affected by the alteration of pH values in the range from 6.0 to 8.0 in PBS buffer solution (Fig. S5, see the Supporting Information). The photostability of Mito-NRT-HP was then investigated by using a 150 W xenon lamp as an excitation source and the emission spectra were recorded every 10 min during 4.0 h. To our satisfaction, the fluorescence spectra and their intensity ratios at 546 nm and 478 nm were comparatively stable within the scanning period (Fig. S6). Small molecular organic dyes also suffer from the issues of photo-oxidation, leading to the structural damage and degradation. A comparative study was also conducted to assess the oxidation resistance of Mito-NRT-HP by using 1,3-diphenylisobenzofuran (DPBF) which can react with singlet oxygen in a quantitative batching to produce non-absorbing compound as control reagent. As shown in Fig. 1c, the absorption band at 410 nm reduced gradually as the DPBF solution was illuminated using xenon lamp with the wavelength of 405 nm, indicating the production of singlet oxygen. Interestingly, no distinct change was observed for the absorption spectra of Mito-NRT-HP in the same circumstances (Fig. S7). From these results, we can draw a conclusion that Mito-NRT-HP is stable to media, light


Page 15 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


and air, which is favorable for its application in H2S2 sensing and intracellular imaging. Compared with most aforementioned fluorescent probe for H2Sn, Mito-NRT-HP exhibits relatively short response time, which is beneficial to the real-time detection of H2Sn. Notably, the absorption spectra in Fig. S8 showed a two-stage changing process in which a shoulder peak appeared at around 468 nm in less than 1 min upon the addition of Na2S2 solution and then the absorption bands at 384 nm and 468 nm gradually decreased while the new absorption peak at 435 nm increasing accompanied by the formation of two well-defined isoemissive point at 405 nm and 465 nm, respectively, which was the typical absorption spectrogram of the conversion process from one chromophore to another. This result confirmed, to a certain extent, the proposed sensing mechanism. The unstable intermediate adduct (R-SSH) was firstly formed via a fast nucleophilic aromatic substitution in the presence of H2S2, and then the intermediate adduct further undergone a spontaneous intramolecular cyclization to release free fluorophore. As indicated in Fig. S9, the time-dependent emission spectra were no longer changed at 10 min in the presence of Na2S2. And that the dynamics curves showed in Fig. S10 mean that the total concentration of Na2S2 existed in solution hardly affected the time it takes for Mito-NRT-HP to reach equilibrium in reaction. The analysis of fluorescence titration implied that the linear relationship between the fluorescence intensity ratio and the concentration of Na2S2 much depend on the ratios of different fluorescent emission wavelengths (Fig. 1e). The fluorescence intensity ratio at 470 nm and 532 nm (I470 nm/I532 nm) was linearly proportional the total



Page 16 of 50

concentration of Na2S2 from 0 to 8 μM while the fluorescence intensity ratio at 546 nm and 478 nm (I546 nm/I478 nm) showed a linear relationship with the concentration of Na2S2 from 12 up to 25 μM. The regression equations were I470 nm/I532 nm = -0.2102 × [Na2S2] + 2.1853 with R2 = 0.9947 and I546 nm/I478 nm = 1.0933 × [Na2S2] -10.5909 with R2 = 0.9887, respectively. Moreover, the limit of detection (LOD) measured to be 10 nM according to the 3σ/k criterion under the testing experimental conditions, indicating that Mito-NRT-HP is sensitive in the quantitative determination of H2Sn. Moreover, Mito-NRT-HP showed an improved water solubility compared with NRT-HP. As shown in Fig. S11, a clear and transparent solution was achieved when 50 µM of Mito-NRT-HP was added into water. However, the solution of NRT-HP became turbid in the same condition. This result indicates that Mito-NRT-HP possessed a superior water solubility, which has been confirmed by DLS experiments. Considering all above, Mito-NRT-HP could be used to detect H2S2 quantitatively under physiological conditions. Then, the selectivity of Mito-NRT-HP for H2Sn over various interfering bio-species, including physiologically important metal ions (Na+, K+, Ca2+, Mg2+, Zn2+, Fe3+, Fe2+ and Mn2+ ), coexisting anion (Cl-, Br-, I-, HSO32-, NO3-, S2O32-, H2PO4-, NO2-, OAcand CO32-), and bio-related reactive species (Cystine, Cys, Hcy, GSH, GSSG, H2S, HClO, H2O2, AA, ADP and ATP) was evaluated. As shown in Fig. 2, no obvious variations in the fluorescence intensity ratio I470

nm/I532 nm

were observed in the

presence of aforementioned interference biologically relevant species except GSH and H2O2 at higher concentrations (1.0 mM for GSH and 0.1 mM for H2O2) displayed a


Page 17 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


slight response, suggesting Mito-NRT-HP showed a specific response to H2Sn over these potentially competing species in physiological condition. Meanwhile, as shown in Fig. S12, the fluorescence intensity ratio I546 nm/I478 nm of Mito-NRT-HP solution could remain unchanged in the presence of various bio-species except Na2Sn. In addition, experiments for interference test revealed that the coexistence of these interfering bio-species had no obvious interference with H2Sn-triggered fluorescence intensities ratio change in PBS buffer solution. However, the presence of excess competitive biothiol species in the test system could prolong the response time of Na2S2. Even as the concentration of coexisting GSH was up to 1.0 mM, the fluorescence intensity ratio I470 nm/I532 nm was little changed. The reason might be that the intermediate adduct generated from the reaction between Mito-NRT-HP and GSH can still react with H2Sn to form Mito-NRT. Therefore, Mito-NRT-HP may not get over the problem of the depletion by sulfhydryl compounds, such as GSH. Since it was reported that H2Sn can be generated from the reaction of H2S and HClO in aqueous solution, we also have tested the fluorescence response of Mito-NRT-HP for the reaction of H2S with HClO. Fig S13 showed that the co-existence of H2S and HClO was essential for the inducing of the fluorescence response of Mito-NRT-HP, as neither HClO nor H2S alone efficiently changed the fluorescence intensity ratio I470 nm/I532 nm.

This confirms the in-situ generation of H2Sn in the presence of HClO as

ROS in the aqueous solution of H2S. These results indicated that the Mito-NRT-HP could be employed for the selective detection of H2Sn under physiological conditions. (Fig. 2 in here)



Encouraged by the excellent sensing performance of Mito-NRT-HP, the characters of two-photon induced fluorescence were then investigated. Since it is usually only a small fraction of photons can be absorbed by two-photon fluorophore to produce the fluorescence, two-photon excitation is an alternative approach to determining two-photon absorption cross section, provided that the dye is fluorescent and that its fluorescent quantum efficiency is known. The two-photon absorption cross section values (δ) for Mito-NRT-HP and Mito-NRT were determined in PBS buffer solution with fluorescein as the reference molecule. As shown in Fig. 3a, Mito-NRT-HP could be sensitized efficiently with two-photon excitation wavelength during the range from 750 nm to 825 nm with the maximum two-photon absorption value detected to be 290 GM at 810 nm, which is also larger than those previously reported H2Sn probes. After complexion with Na2S2, to our delight, the available excitation wavelength range has largely stayed the same as initial. As expected, both Mito-NRT-HP and Mito-NRT exhibited high fluorescence quantum yield (φ) in PBS buffer solution, the absolute quantum yield was measured to be 36.8% and 18.5%, respectively. Fig. 3b showed two-photon fluorescence spectra of Mito-NRT-HP upon continuous addition of Na2S2 under the two-photon excitation at 808 nm in PBS buffer solution (10 mM, pH 7.40). With increasing total concentrations of Na2S2, the new appeared emission peak at 546 nm gradually increased while the emission peak at 478 nm went down, leading to the ratiometric determination of H2Sn. This trends and patterns of change were similar to that under one-photon excitation, indicating that Mito-NRT-HP is capable in detecting H2Sn under both one-photon and two-photon excitation.


Page 18 of 50

Page 19 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(Fig. 3 in here) Given that Mito-NRT-HP exhibits comparative stability, highly selectivity, fast-response, and impressive sensitivity, we set out to verify whether these merits are effectual in mapping the intracellular H2Sn in living cells. Firstly, the cytotoxicity of Mito-NRT-HP and Mito-NRT were examined in HeLa cells by the standard MTS assay. As shown in Fig. S14, the cellular viability of cells was estimated to be >70% after incubation with 0-100 μM of Mito-NRT-HP for 24 h, meanwhile, more than 80% of the cells remained viable in the same case as cells were incubated with Mito-NRT, indicating that both probe and its reaction product have low cytotoxicity. As shown in Fig. 4, an obvious fluorescence was observed in HeLa cells soon after feeding the cells with Mito-NRT-HP, which revealed the amazing cell membrane penetrability of Mito-NRT-HP. Then, a long-term scanning imaging study was carried out to investigate the light stability of Mito-NRT-HP. The Mito-NRT-HP loaded HeLa cells were scanned continuously under the irradiating of laser integrated in the confocal microscopy. As depicted in Fig. S15, the fluorescence intensities both in blue channel and green channel were hardly weakened after 30 min irradiation, indicating

the

good

photobleaching

resistance

of

Mito-NRT-HP.

Since

non-fluorescent dichloro-dihydro-fluorescein diacetate (DCFH-DA) could be oxidized into fluorescent product by intracellular reactive oxygen, it was often used to evaluate the level of reactive oxygen in living cells. Thus, co-staining experiment was performed with Mito-NRT-HP and DCFH-DA to check the oxidation resistance of Mito-NRT-HP in biological samples. Results in Fig. S16 showed that bright and



increasing fluorescence belonging to oxidation product of DCFH-DA could be observed, revealing the presence or generation of reactive oxygen species. But fluorescence assigned to the co-incubated Mito-NRT-HP was not suppressed. This observation has implied that reactive oxygen species had no depletion effect on the fluorescence of Mito-NRT-HP. In addition, we also conducted an experiment to assess whether Mito-NRT-HP is effectual in measuring H2Sn levels in different cell lines. (Fig. 4 in here) As shown in Fig. 5, HeLa cells and BHK cells were cultured in the same confocal culture dish because the two cell lines can be easily identified by their morphology, HeLa cells are wedge like while BHK cells are spindle shaped. As expected, bright fluorescence signals were obtained in both HeLa cells and BHK cells after loaded with Mito-NRT-HP. The fluorescence intensity profiles for region of interesting (ROI) and overlap Z-scan image in Fig. S17 demonstrated that the fluorescence was evident in the cytoplasm other than nucleus and the membrane, which was observed for both HeLa cells and BHK cells. The slight difference in the fluorescence intensity ratios of green channel to blue channel reflected the different endogenous levels of H2Sn. (Fig. 5 in here) Mitochondria plays a key role in maintaining the redox environment during energy metabolism and is related to cell apoptosis. The majority of intracellular H2Sn is held in mitochondria. Our probe Mito-NRT-HP might promise a significant efficacy in


Page 20 of 50

Page 21 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


targeting mitochondria because of the lipophilic triphenylphosphonium cation. To confirm this prediction, mitochondria tracker (Mito Tracker Red CMXRos) and lysosome tracker (Lyso Tracker Red DND-99) were used as co-staining dyes. As illustrated in Fig. 6, a conspicuous overlap between the blue fluorescence of Mito-NRT-HP and the red fluorescence of Mito Tracker Red CMXRos has been achieved. Meanwhile, the variations in the fluorescence intensity profiles of the linear regions of interest (ROI) crossing HeLa cell kept synchronization consistently.

Furthermore,

the

fluorescence

intensity

scatter

plot

of

Mito-NRT-HP (blue channel) and Mito Tracker Red CMXRos (red channel) is highly correlative with the overlap coefficient of 0.94. In contrast, the fluorescence intensity scatter plot of Mito-NRT-HP was not merged with that of Lyso Tracker Red DND-99 and the overlap coefficient is only 0.42. The above-mentioned results confirmed Mito-NRT-HP predominantly accumulated in the mitochondria rather than other parts and made Mito-NRT-HP very attractive for measuring the distribution and contribution of H2Sn in biological systems. (Fig. 6 in here) As inspired by the favorable two-photon properties and fluorescent imaging performance, we have examined the capability of Mito-NRT-HP in two-photon imaging and bio-sensing. As shown in Fig. 7a, the fluorescence intensity ratio of green channel to blue channel was relatively low when cells incubated with



Page 22 of 50

Mito-NRT-HP only. However, when the cells were pretreated with Na2S2, an increase in the fluorescence intensity ratio was detected (Fig. 7b). In the control group, cells were pretreated with N-methylmaleimide (NMM), an RSS mercapto group blocking agent, to remove endogenous H2Sn before incubated with Mito-NRT-HP. Compared with Fig. 7a, the fluorescence intensity ratio of this group is obviously decreased, which demonstrates that Mito-NRT-HP is sensitive enough to illuminate the basic level of H2Sn in living cells. Literature reported that the levels of H2Sn could be significantly elevated by stimulating the cells with lipopolysaccharides (LPS) to induce the over-expression of cystathionine γ-lyase (CSE).63

Accordingly, we incubated

living cells with LPS (1 μg/ml) for 16 hours in DMEM culture medium and subsequently stained with Mito-NRT-HP, the fluorescence intensity ratio is moderately enhanced (Fig. 7d), and this confirmed the elevation of H2Sn. When HeLa cells were pre-treated with L-Cys (another H2Sn enhancer) which can enhance H2Sn levels by involving in the enzyme-mediated H2S formation pathway64, but only a slender increase in fluorescence intensity ratio was observed (Fig. 7e). These results indicate that Mito-NRT-HP is competent in monitoring basal and the fluctuations of endogenous H2Sn in the mitochondria of living cells, which is of significance to unravel the physiological and pathological functions of H2Sn. (Fig. 7 in here)


Page 23 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Finally, fluorescence imaging experiments were carried to demonstrate the potential application of Mito-NRT-HP in biological systems. Because H2Sn could be formed from CSE- mediated cysteine metabolism as a result of the overexpression of LPS, LPS induced acute organ injury model was established by injecting 10 mg/kg LPS into male C57BL/6 mice with 100 μL of 0.9% saline as control. After 16 h, mice were euthanized and their intestine, lung, liver, kidney and spleen organs were harvested at 1.0 h after the injection of 10 μM Mito-NRT-HP. The tissue slices were achieved from these organs and the fluorescence imaging experiments were performed by utilizing two-photon microscopy. As shown in Fig. S18, we also confirmed that two-photon imaging could contribute to the longer imaging depths. By contrast, two-photon fluorescence imaging showed higher resolution than one-photon fluorescence imaging in both blue channel and green channel (Fig. S19). Moreover, the fluorescence in both blue channel and green channel were totally different in different tissue slices. As shown in Fig. 8, the tissue slices of liver, kidney and spleen exhibited a relatively strong fluorescence. This might because Mito-NRT-HP expressed varying degrees of enrichment in different organs. Compared to the control samples, the samples of LPS induced organ injury exhibited an enhanced fluorescence in the 575-625 nm channel and a weak signal in the 425-475 nm channel, implying that the samples of LPS-induced organ injury could be identified from normal samples. These results indicate that Mito-NRT-HP was potentially competent in diagnosing LPS-induced organ injury. (Fig. 8 in here)



4. CONCLUSION In summary, we have developed a mitochondria-accessing one- and two-photon fluorescent probe Mito-NRT-HP for intracellular H2Sn detection. Mito-NRT-HP exhibits preeminent selectivity toward H2Sn over excess amount of other RSS and biologically important substances. Moreover, experimental results revealed that the incorporation of triphenylphosphine group into molecular structure not only endowed the crafted probe targeting ability, but also improved probe’s aqueous solubility, photobleaching resistance, membrane permeability and biocompatibility. Besides, the H2Sn-induced shift of emission peaks was up to approximately 68 nm, allowed the proposed probe to quantitatively response H2Sn with high resolution. To the best of our knowledge, this is the first mitochondria-targeted ratiometric probe for H2Sn. And with this well-designed probe, the distribution of endogenous H2Sn could be mapped. The study implied that Mito-NRT-HP is probably suitable for stimulated emission depletion (STED) nanoscopy application65-66 and it offers us a powerful assay for unravelling the diverse biological functions of H2Sn biological systems. ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: XXXXX


Page 24 of 50

Page 25 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Synthesis route for Mito-NRT-HP, supplementary figures, and characterization data (PDF). AUTHOR INFORMATION Corresponding Author *E/mail: [email protected] (W.S. Liu). *E/mail: [email protected] (X.C. Wang). *E/mail: [email protected] (J.X. Ru). ORCID Qingxin Han: 0000-0001-6417-4585 Li Wang: 0000-0002-4677-5851 Huie Jiang: 0000-0003-3699-4126 Weisheng Liu: 0000-0001-5448-6315 Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS We are grateful for the financial support from the NSFC (Grant 21431002, 21804084), China Postdoctoral Science Foundation Funded Project (Grant



2018M643557) and Doctoral Start-up Fund of Shanxi University of Science and Technology (Grant 2018BJ-19). REFERENCES

1.

Giles, G. I.; Tasker, K. M.; Jacob, C., Hypothesis: The Role of Reactive Sulfur Species in Oxidative Stress. Free Radical Biol. Med. 2001, 31 (10), 1279-1283.

2.

Gruhlke, M. C. H.; Slusarenko, A. J., The Biology of Reactive Sulfur Species (RSS). Plant Physiol. Biochem. 2012, 59, 98-107.

3.

Lin, V. S.; Chen, W.; Xian, M.; Chang, C. J., Chemical Probes for Molecular Imaging and Detection of Hydrogen Sulfide and Reactive Sulfur Species in Biological Systems. Chem. Soc. Rev. 2015, 44 (14), 4596-4618.

4.

Chen, W.; Xu, S.; Day, J. J.; Wang, D. F.; Xian, M., A General Strategy for Development of Near-Infrared Fluorescent Probes for Bioimaging. Angew. Chem. Int. Edit. 2017, 56 (52), 16611-16615.

5.

Szabo, C., Hydrogen Sulphide and Its Therapeutic Potential. Nat. Rev. Drug. Discov. 2007, 6 (11), 917-935.

6.

Yang, G.; Wu, L.; Jiang, B.; Yang, W.; Qi, J.; Cao, K.; Meng, Q.; Mustafa, A. K.; Mu, W.; Zhang, S.; Snyder, S. H.; Wang, R., H2S as a Physiologic Vasorelaxant: Hypertension in Mice with Deletion of Cystathionine Γ-Lyase. Science 2008, 322 (5901), 587.

7.

Li, L.; Rose, P.; Moore, P. K., Hydrogen Sulfide and Cell Signaling. Annu. Rev. Pharmacol. Toxicol. 2011, 51, 169-87.


Page 26 of 50

Page 27 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


8.

Vandiver, M. S.; Snyder, S. H., Hydrogen Sulfide: A Gasotransmitter of Clinical Relevance. J. Mol. Med. 2012, 90 (3), 255-263.

9.

Fukuto, J. M.; Carrington, S. J.; Tantillo, D. J.; Harrison, J. G.; Ignarro, L. J.; Freeman, B. A.; Chen, A.; Wink, D. A., Small Molecule Signaling Agents: The Integrated Chemistry and Biochemistry of Nitrogen Oxides, Oxides of Carbon, Dioxygen, Hydrogen Sulfide, and Their Derived Species. Chem. Res. Toxicol. 2012, 25 (4), 769-793.

10. Vitvitsky, V.; Kabil, O.; Banerjee, R., High Turnover Rates for Hydrogen Sulfide Allow for Rapid Regulation of Its Tissue Concentrations. Antioxid. Redox Signaling 2012, 17 (1), 22-31. 11. Greiner, R.; Pálinkás, Z.; Bäsell, K.; Becher, D.; Antelmann, H.; Nagy, P.; Dick, T. P., Polysulfides Link H2S to Protein Thiol Oxidation. Antioxid. Redox Signaling 2013, 19 (15), 1749-1765. 12. Toohey, J. I., Sulfur Signaling: Is the Agent Sulfide or Sulfane? Anal. Biochem. 2011, 413 (1), 1-7. 13. Shibuya, N.; Tanaka, M.; Yoshida, M.; Ogasawara, Y.; Togawa, T.; Ishii, K.; Kimura, H., 3-Mercaptopyruvate Sulfurtransferase Produces Hydrogen Sulfide and Bound Sulfane Sulfur in the Brain. Antioxid. Redox Signaling 2008, 11 (4), 703-714. 14. Nagy, P.; Winterbourn, C. C., Rapid Reaction of Hydrogen Sulfide with the Neutrophil Oxidant Hypochlorous Acid to Generate Polysulfides. Chem. Res. Toxicol. 2010, 23 (10), 1541-1543. 15. Ida, T.; Sawa, T.; Ihara, H.; Tsuchiya, Y.; Watanabe, Y.; Kumagai, Y.; Suematsu, M.; Motohashi, H.; Fujii, S.; Matsunaga, T.; Yamamoto, M.; Ono, K.; Devarie-Baez, N. O.; Xian, M.; Fukuto, J. M.; Akaike, T., Reactive Cysteine Persulfides and S-Polythiolation Regulate Oxidative Stress and Redox Signaling. Proc. Natl. Acad. Sci. 2014, 111 (21), 7606.



16. Yadav, P. K.; Martinov, M.; Vitvitsky, V.; Seravalli, J.; Wedmann, R.; Filipovic, M. R.; Banerjee, R., Biosynthesis and Reactivity of Cysteine Persulfides in Signaling. J. Am. Chem. Soc. 2016, 138 (1), 289-299. 17. Paulsen, C. E.; Carroll, K. S., Cysteine-Mediated Redox Signaling: Chemistry, Biology, and Tools for Discovery. Chem. Rev. 2013, 113 (7), 4633-4679. 18. Zhang, D.; Macinkovic, I.; Devarie-Baez, N. O.; Pan, J.; Park, C.-M.; Carroll, K. S.; Filipovic, M. R.; Xian, M., Detection of Protein S-Sulfhydration by a Tag-Switch Technique. Angew. Chem. Int. Ed. 2014, 53 (2), 575-581. 19. Kimura, Y.; Mikami, Y.; Osumi, K.; Tsugane, M.; Oka, J.-i.; Kimura, H., Polysulfides Are Possible H2S-Derived Signaling Molecules in Rat Brain. FASEB J. 2013, 27 (6), 2451-2457. 20. Ono, K.; Akaike, T.; Sawa, T.; Kumagai, Y.; Wink, D. A.; Tantillo, D. J.; Hobbs, A. J.; Nagy, P.; Xian, M.; Lin, J.; Fukuto, J. M., Redox Chemistry and Chemical Biology of H2S, Hydropersulfides, and Derived Species: Implications of Their Possible Biological Activity and Utility. Free Radical Biol. Med. 2014, 77, 82-94. 21. C. Estevam, E.; Faulstich, L.; Griffin, S.; Burkholz, T.; Jacob, C., Polysulfides in Biology: From Intricate Chemistry to an Astonishing yet Hidden Biological Activity. Curr. Org. Chem. 2016, 20 (2), 211-217. 22. Kimura, H., Signaling Molecules: Hydrogen Sulfide and Polysulfide. Antioxid. Redox Signaling 2014, 22 (5), 362-376. 23. Mishanina, T. V.; Libiad, M.; Banerjee, R., Biogenesis of Reactive Sulfur Species for Signaling by Hydrogen Sulfide Oxidation Pathways. Nat. Chem. Biol. 2015, 11, 457.


Page 28 of 50

Page 29 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


24. Chan, J.; Dodani, S. C.; Chang, C. J., Reaction-Based Small-Molecule Fluorescent Probes for Chemoselective Bioimaging. Nat. Chem. 2012, 4, 973. 25. Li, X.; Gao, X.; Shi, W.; Ma, H., Design Strategies for Water-Soluble Small Molecular Chromogenic and Fluorogenic Probes. Chem. Rev. 2014, 114 (1), 590-659. 26. Yuan, L.; Lin, W.; Zheng, K.; He, L.; Huang, W., Far-Red to near Infrared Analyte-Responsive Fluorescent Probes Based on Organic Fluorophore Platforms for Fluorescence Imaging. Chem. Soc. Rev. 2013, 42 (2), 622-661. 27. Chen, W.; Liu, C.; Peng, B.; Zhao, Y.; Pacheco, A.; Xian, M., New Fluorescent Probes for Sulfane Sulfurs and the Application in Bioimaging. Chem. Sci. 2013, 4 (7), 2892-2896. 28. Liu, C.; Chen, W.; Shi, W.; Peng, B.; Zhao, Y.; Ma, H.; Xian, M., Rational Design and Bioimaging Applications of Highly Selective Fluorescence Probes for Hydrogen Polysulfides. J. Am. Chem. Soc. 2014, 136 (20), 7257. 29. Hartle, M. D.; Pluth, M. D., A Practical Guide to Working with H2S at the Interface of Chemistry and Biology. Chem. Soc. Rev. 2016, 45 (22), 6108-6117. 30. Han, Q.; Mou, Z.; Wang, H.; Tang, X.; Dong, Z.; Wang, L.; Dong, X.; Liu, W., Highly Selective and Sensitive One- and Two-Photon Ratiometric Fluorescent Probe for Intracellular Hydrogen Polysulfide Sensing. Anal. Chem. 2016, 88 (14), 7206-7212. 31. Kawagoe, R.; Takashima, I.; Uchinomiya, S.; Ojida, A., Reversible Ratiometric Detection of Highly Reactive Hydropersulfides Using a FRET-Based Dual Emission Fluorescent Probe. Chem. Sci. 2017, 8 (2), 1134-1140.



32. Chen, W.; Yue, X.; Sheng, J.; Li, W.; Zhang, L.; Su, W.; Huang, C.; Song, X., Rational Development of a Highly Selective Ratiometric Fluorescent Probe for Hydrogen Polysulfides. Sens. Actuators, B 2018, 258, 125-132. 33. Gao, M.; Yu, F.; Chen, H.; Chen, L., Near-Infrared Fluorescent Probe for Imaging Mitochondrial Hydrogen Polysulfides in Living Cells and in Vivo. Anal. Chem. 2015, 87 (7), 3631-3638. 34. Yu, F.; Gao, M.; Li, M.; Chen, L., A Dual Response Near-Infrared Fluorescent Probe for Hydrogen Polysulfides and Superoxide Anion Detection in Cells and In vivo. Biomaterials 2015, 63, 93-101. 35. Chen, W.; Rosser, E. W.; Zhang, D.; Shi, W.; Li, Y.; Dong, W.-J.; Ma, H.; Hu, D.; Xian, M., A Specific Nucleophilic Ring-Opening Reaction of Aziridines as a Unique Platform for the Construction of Hydrogen Polysulfides Sensors. Org. Lett. 2015. 17 (11), 2776-2779. 36. Zeng, L.; Chen, S.; Xia, T.; Hu, W.; Li, C.; Liu, Z., Two-Photon Fluorescent Probe for Detection of Exogenous and Endogenous Hydrogen Persulfide and Polysulfide in Living Organisms. Anal. Chem. 2015, 87 (5), 3004-3010. 37. Shang, H.; Chen, H.; Tang, Y.; Guo, R.; Lin, W., Construction of a Two-Photon Fluorescent Turn-on Probe for Hydrogen Persulfide and Polysulfide and Its Bioimaging Application in Living Mice. Sens. Actuators, B 2016, 230, 773-778. 38. Li, K.-B.; Chen, F.-Z.; Yin, Q.-H.; Zhang, S.; Shi, W.; Han, D.-M., A Colorimetric and near-Infrared Fluorescent Probe for Hydrogen Polysulfides and Its Application in Living Cells. Sens. Actuators, B 2018, 254, 222-226.


Page 30 of 50

Page 31 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


39. Fang, Y.; Chen, W.; Shi, W.; Li, H.; Xian, M.; Ma, H., A near-Infrared Fluorescence Off-On Probe for Sensitive Imaging of Hydrogen Polysulfides in Living Cells and Mice in Vivo. Chem. Commun. 2017, 53 (62), 8759-8762. 40. Chen, W.; Pacheco, A.; Takano, Y.; Day Jacob, J.; Hanaoka, K.; Xian, M., A Single Fluorescent Probe to Visualize Hydrogen Sulfide and Hydrogen Polysulfides with Different Fluorescence Signals. Angew. Chem. Int. Ed. 2016, 55 (34), 9993-9996. 41. A, A. H.; Sreedharan, S.; Ali, F.; Smythe, C. G.; Thomas, J. A.; Das, A., Polysulfide-Triggered Fluorescent Indicator Suitable for Super-Resolution Microscopy and Application in Imaging. Chem. Commun. 2018, 54 (30), 3735-3738. 42. Han, X.; Yu, F.; Song, X.; Chen, L., Quantification of Cysteine Hydropersulfide with a Ratiometric near-Infrared Fluorescent Probe Based on Selenium-Sulfur Exchange Reaction. Chem. Sci. 2016, 7 (8), 5098-5107. 43. Zhang, J.; Zhu, X.-Y.; Hu, X.-X.; Liu, H.-W.; Li, J.; Feng, L. L.; Yin, X.; Zhang, X.-B.; Tan, W., Ratiometric Two-Photon Fluorescent Probe for in Vivo Hydrogen Polysulfides Detection and Imaging During Lipopolysaccharide-Induced Acute Organs Injury. Anal. Chem. 2016, 88 (23), 11892-11899. 44. Gao, M.; Wang, R.; Yu, F.; You, J.; Chen, L., A near-Infrared Fluorescent Probe for the Detection of Hydrogen Polysulfides Biosynthetic Pathways in Living Cells and in Vivo. Analyst 2015, 140 (11), 3766-3772. 45. Huang, Y.; Yu, F.; Wang, J.; Chen, L., Near-Infrared Fluorescence Probe for in Situ Detection of Superoxide Anion and Hydrogen Polysulfides in Mitochondrial Oxidative Stress. Anal. Chem. 2016, 88 (7), 4122-4129.



46. Kim, H. M.; Cho, B. R., Two-Photon Probes for Intracellular Free Metal Ions, Acidic Vesicles, and Lipid Rafts in Live Tissues. Acc. Chem. Res. 2009, 42 (7), 863-872. 47. Hu, X.; Wei, T.; Wang, J.; Liu, Z.-E.; Li, X.; Zhang, B.; Li, Z.; Li, L.; Yuan, Q., Near-Infrared-Light Mediated Ratiometric Luminescent Sensor for Multimode Visualized Assays of Explosives. Anal. Chem. 2014, 86 (20), 10484-10491. 48. Kim, H. M.; Cho, B. R., Small-Molecule Two-Photon Probes for Bioimaging Applications. Chem. Rev. 2015, 115 (11), 5014-5055. 49. Qian, L.; Li, L.; Yao, S. Q., Two-Photon Small Molecule Enzymatic Probes. Acc. Chem. Res. 2016, 49 (4), 626-634. 50. Chen, Y.; Guan, R.; Zhang, C.; Huang, J.; Ji, L.; Chao, H., Two-Photon Luminescent Metal Complexes for Bioimaging and Cancer Phototherapy. Coord. Chem. Rev. 2016, 310, 16-40. 51. Zhou, X.; Yu, B.; Guo, Y.; Tang, X.; Zhang, H.; Liu, W., Both Visual and Fluorescent Sensor for Zn2+ Based on Quinoline Platform. Inorg. Chem. 2010, 49 (9), 4002-4007. 52. Zhao, Q.; Li, F.; Huang, C., Phosphorescent Chemosensors Based on Heavy-Metal Complexes. Chem. Soc. Rev. 2010, 39 (8), 3007-3030. 53. Paul, B. D.; Snyder, S. H., H2S Signalling through Protein Sulfhydration and Beyond. Nat. Rev. Mol. Cell Biol. 2012, 13, 499. 54. Jackson, M. R.; Melideo, S. L.; Jorns, M. S., Human Sulfide:Quinone Oxidoreductase Catalyzes the First Step in Hydrogen Sulfide Metabolism and Produces a Sulfane Sulfur Metabolite. Biochemistry 2012, 51 (34), 6804-6815. 55. Gupta, N.; Reja, S. I.; Bhalla, V.; Kumar, M., Fluorescent Probes for Hydrogen Polysulfides (H2Sn, N > 1): From Design Rationale to Applications. Org. Biomol. Chem. 2017, 15 (32), 6692-6701.


Page 32 of 50

Page 33 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


56. Lee, S. K.; Yang, W. J.; Choi, J. J.; Kim, C. H.; Jeon, S.-J.; Cho, B. R., 2,6-Bis[4-(P-Dihexylaminostyryl)Styryl]Anthracene Derivatives with Large Two-Photon Cross Sections. Org. Lett. 2005, 7 (2), 323-326. 57. Chen, Y.; Du, Y.; Li, Y.; Wang, X.; Gao, P.; Yang, G.; Fang, Y.; Meng, Y.; Zhao, X., Panaxadiol Saponin and Dexamethasone Improve Renal Function in Lipopolysaccharide-Induced Mouse Model of Acute Kidney Injury. PLOS ONE 2015, 10 (7), e0134653. 58. Chen, L.; Yang, S.; Zumbrun, E. E.; Guan, H.; Nagarkatti, P. S.; Nagarkatti, M., Resveratrol Attenuates Lipopolysaccharide-Induced Acute Kidney Injury by Suppressing Inflammation Driven by Macrophages. Mol. Nutr. Food Res. 2015, 59 (5), 853-864. 59. Yao, H.; Hu, C.; Yin, L.; Tao, X.; Xu, L.; Qi, Y.; Han, X.; Xu, Y.; Zhao, Y.; Wang, C.; Peng, J., Dioscin Reduces Lipopolysaccharide-Induced Inflammatory Liver Injury Via Regulating TLR4/MYD88 Signal Pathway. Int. Immunopharmacol. 2016, 36, 132-141. 60. Jiang, Q.; Yi, M.; Guo, Q.; Wang, C.; Wang, H.; Meng, S.; Liu, C.; Fu, Y.; Ji, H.; Chen, T., Protective Effects of Polydatin on Lipopolysaccharide-Induced Acute Lung Injury through TLR4-MYD88-NF-ΚB Pathway. Int. Immunopharmacol. 2015, 29 (2), 370-376. 61. Jiao, X.; Li, Y.; Niu, J.; Xie, X.; Wang, X.; Tang, B., Small-Molecule Fluorescent Probes for Imaging and Detection of Reactive Oxygen, Nitrogen, and Sulfur Species in Biological Systems. Anal. Chem. 2018, 90 (1), 533-555. 62. Yu, F.; Han, X.; Chen, L., Fluorescent Probes for Hydrogen Sulfide Detection and Bioimaging. Chem. Commun. 2014, 50 (82), 12234-12249.



63. Ida, T.; Sawa, T.; Ihara, H.; Tsuchiya, Y.; Watanabe, Y.; Kumagai, Y.; Suematsu, M.; Motohashi, H.; Fujii, S.; Matsunaga, T., From the Cover: Reactive Cysteine Persulfides and S-Polythiolation Regulate Oxidative Stress and Redox Signaling. Proc. Natl. Acad. Sci. 2014, 111 (21), 7606-7611. 64. Chen, W.; Rosser, E. W.; Matsunaga, T.; Pacheco, A.; Akaike, T.; Xian, M., The Development of Fluorescent Probes for Visualizing Intracellular Hydrogen Polysulfides. Angew. Chem. 2015, 127 (47), 14167-14171. 65. Sreedharan, S.; Gill, M. R.; Garcia, E.; Saeed, H. K.; Robinson, D.; Byrne, A.; Cadby, A.; Keyes, T. E.; Smythe, C.; Pellett, P.; Bernardino de la Serna, J.; Thomas, J. A., Multimodal Super-Resolution Optical Microscopy Using a Transition-Metal-Based Probe Provides Unprecedented Capabilities for Imaging Both Nuclear Chromatin and Mitochondria. J. Am. Chem. Soc. 2017, 139 (44), 15907-15913. 66. Li, D.; Qin, W.; Xu, B.; Qian, J.; Tang Ben, Z., AIE Nanoparticles with High Stimulated Emission Depletion Efficiency and Photobleaching Resistance for Long-Term Super-Resolution Bioimaging. Adv. Mater. 2017, 29 (43), 1703643.


Page 34 of 50

Page 35 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


SCHEMES:

Scheme 1. Design principle of Mito-NRT-HP and its reaction with H2Sn. (a) The designing of probe molecular structure. (b) Proposed mechanism for sensing H2Sn.



FIGURES:

Fig. 1 (a) Absorption spectra of Mito-NRT-HP and Mito-NRT (10 μM) in phosphate buffer solution (10 mM, pH 7.40). Insert: the color of Mito-NRT-HP (left) and the product of the reaction with Na2S2 (right). (b) UV-vis spectral changes of Mito-NRT-HP (10 μM) upon addition of increasing Na2S2 in PBS buffer solution (10 mM, pH 7.40) at room temperature. Insert: absorbance (A432 nm) changes with the concentration of Na2S2 in the range of 0-75 μM. (c) Absorption spectra of DPBF solution (red line), Mito-NRT-HP solution (pink line), and mixed solution of Mito-NRT-HP and DPBF before (black line) and after (blue line) illuminated with 150 W xenon lamp with the wavelength at 405 nm. The gray dotted line was obtained by subtracting black line with blue line. (d) Fluorescence spectra of Mito-NRT-HP upon addition of Na2S2 (0-75 μM) in PBS buffer solution (10 mM, pH 7.40), λex = 405 nm. Inset: fluorescence intensity ratios I470 nm/I532 nm (black) and I546 nm/I478 nm (red) changes with Na2S2 concentration. (e) The changes of fluorescence intensity ratio (I470 nm/I532 nm and I546 nm/I478 nm) as functions of Na2S2 concentration. (f) Time-dependent fluorescence intensity ratio I470 nm/I532 nm with time chart in the presence of Na2S2 (25 μM). Insert: visual fluorescent color of Mito-NRT-HP (left) and the product of the reaction with Na2S2 (right), the photographs were taken under a 365 nm UV lamp.


Page 36 of 50

Page 37 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Fig 2. The fluorescence intensity ratio (I470 nm/I532 nm) of 10 μM Mito-NRT-HP towards various bio-species (1.0 mM for Na+, K+ and GSH, 0.1 mM for Ca2+, Mg2+, Cl-, Br-, I-, HSO32-, NO3-, S2O32-, H2PO4-, NO2-, OAc-, CO32-, Cystine, GSSG, AA, ADP and ATP, 50 μM for Cys, Hcy, Na2S, H2O2, NaClO, Na2S2 and Na2S4, and 10 μM for Zn2+, Fe3+, Fe2+ and Mn2+) in phosphate buffer solution (10 mM, pH 7.40) at room temperature, λex = 405 nm. Black bars represent the addition of the appropriate bio-species. Red bars represent the addition of the appropriate bio-species followed by adding 50 μM of Na2S2. Error bars: S.D., n=3.

Fig 3. (a) Two-photon action spectra of 10 μM Mito-NRT-HP in the absence (black dots) and in the presence (red dots) of Na2S2 in phosphate buffer solution (10 mM, pH 7.40) at room temperature. (b) Two-photon fluorescence spectra of Mito-NRT-HP (10 μM) upon addition of Na2S2 (0-75 μM) in phosphate buffer solution, λex = 808 nm. Inset: The relationship between the intensity ratio of two-photon fluorescence (I470 nm/I532 nm) and the final concentration of Na2S2 in phosphate buffer solution.

Fig 4. Confocal fluorescence images of HeLa cells incubated with Mito-NRT-HP (10 μM) for different time (0-30 min). (a) Blue channel collected in optical windows between 425 nm and 475 nm. (b) Green channel collected in optical windows between 550 nm and 600 nm. (c) Merge channel of (a) and (b). (d) Fluorescence ratios (green/blue) of the corresponding fluorescence images. Images were acquired with the excitation at 405 nm. Scale bar: 25 μM.



Fig 5. Confocal fluorescence images of the co-incubated HeLa cells and BHK cells with Mito-NRT-HP (10 μM). The blue wires indicate the HeLa cells and the red wires indicate the BHK cells. Scale bar, 25 μm. (a) Bright-field. (b) Blue channel collected in optical windows between 425 and 475 nm. (c) Green channel collected in optical windows between 550 and 600 nm. (d) Fluorescence ratios between green channel and blue channel. (e) Overlap Z-scan image of the merged blue channel and green channel. (f) Blue fluorescence intensity profiles and fluorescence images (across the brown line in e) of HeLa and BHK cells. (g) Green fluorescence intensity profiles and fluorescence images (across the brown line in e) of HeLa and BHK cells.

Fig 6. Co-localization images of Mito-NRT-HP with Mito Tracker Red CMXRos (a-g) and Lyso Tracker Red DND-99 (h-m). (a) Bright-field image of cells co-incubated with Mito-NRT-HP (10 μM) and Mito Tracker Red CMXRos (0.2 μM). (b) Blue channel for Mito-NRT-HP. Images were collected at 425-475 nm with the excitation at 405 nm. (c) Red


Page 38 of 50

Page 39 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


channel for Mito Tracker Red CMXRos. Images were collected at 575-625 nm with the excitation at 561 nm. (d) Overlay image of (b) and (c). (e) Colocalizated points of (b) and (c). (f) Intensity scatter plot of red channel and blue channel for cells co-incubated with Mito-NRT-HP and Mito Tracker Red CMXRos. (g) Normalized intensity profile of ROI (green line in panel d) across HeLa cells. (h) Bright-field image of cells co-incubated with Mito-NRT-HP (10 μM) and Lyso Tracker Red DND-99 (1.0 μM). (i) Blue channel for Mito-NRT-HP. Images were collected at 425-475 nm with the excitation at 405 nm. (j) Red channel for Lyso Tracker Red DND-99. Images were collected at 595-645 nm with the excitation at 561 nm. (k) Overlay image of (i) and (j). (l) Colocalizated points of (i) and (j). (m) Intensity scatter plot of red channel and blue channel for cells co-incubated with Mito-NRT-HP and Lyso Tracker Red DND-99. (n) Normalized intensity profile of ROI (green line in panel k) across HeLa cells.

Fig 7. Ratiometric two-photon fluorescence images in HeLa cells. (a) Cells were incubated with Mito-NRT-HP (10 μM) for 15 min. (b) Cells were pretreated with Na2S4 (25 μM) for 30 min before incubated with Mito-NRT-HP. (c) Cells were pretreated with NEM (2 mM) for 30 min, and then incubated with Mito-NRT-HP for 15 min. (d) Cells were treated with LPS (2 μg/mL) for 16 h, followed by incubated with Mito-NRT-HP for 15 min. (e) Cells were incubated with L-Cys (0.2 mM) for 1.5 h and then loaded with Mito-NRT-HP (10 μM) for 15 min. The images were collected at 425-475 nm for blue channel and 550-600 nm for green channel with the two-photon excitation at 800 nm.



Fig 8. Ratiometric two-photon fluorescence imaging of control samples and LPS-induced organ injury samples. (a) Control tissue slice of intestine. (b) Control tissue slice of lung. (c) Control tissue slice of liver. (d) Control tissue slice of kidney. (e) Control tissue slice of spleen. (f) Tissue slice of LPS-induced intestine samples. (g) Tissue slice of LPS-induced lung samples. (h) Tissue slice of LPS-induced liver samples. (i) Tissue slice of LPS-induced kidney samples. (j) Tissue slice of LPS-induced spleen samples. The images were collected at 425-475 nm for blue channel and 550-600 nm for green channel with the two-photon excitation at 800 nm. The overlay images of blue channel and green channel were also showed.


Page 40 of 50

Page 41 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table of Contents (TOC) graphic



149x75mm (300 x 300 DPI)


Page 42 of 50

Page 43 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


296x209mm (300 x 300 DPI)



144x53mm (300 x 300 DPI)


Page 44 of 50

Page 45 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


257x179mm (144 x 144 DPI)



219x110mm (144 x 144 DPI)


Page 46 of 50

Page 47 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


105x110mm (144 x 144 DPI)



215x183mm (144 x 144 DPI)


Page 48 of 50

Page 49 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


172x117mm (150 x 150 DPI)



187x79mm (150 x 150 DPI)


Page 50 of 50

Photostable Ratiometric Two-photon Fluorescent ... - ACS Publications

Recommend Documents