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Ratiometric Imaging of Tissue by Two-Photon Microscopy: Observation of a High Level of Formaldehyde around Mouse Intestinal Crypts Subhankar Singha, Yong Woong Jun, Juryang Bae, and Kyo Han Ahn Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b00044 • Publication Date (Web): 20 Feb 2017 Downloaded from http://pubs.acs.org on February 21, 2017
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Analytical Chemistry
Subhankar Singha,† Yong Woong Jun,† Juryang Bae, and Kyo Han Ahn* Department of Chemistry, Pohang University of Science and Technology (POSTECH), 77 Cheongam-Ro, Nam-Gu, Pohang 37673, Republic of Korea. ABSTRACT: Ratiometric imaging by two-photon microscopy can offer a viable tool for the relative quantification of biological analytes inside tissue with minimal influence from environmental factors that affect fluorescence signal. We demonstrate the ratiometric imaging of formaldehyde at the sub-organ level using a two-photon fluorescent probe, which involves pixel-to-pixel ratiometric data transformation. This study reveals for the first time a high level of formaldehyde around the crypts of mouse small intestine, implicating its possible protective role along with the released antimicrobials from the Paneth cells.
Detection of a molecule of interest in living systems with relative quantitative information is an essential but challenging task in life sciences. Among various tools, fluorescence microscopy combined with an appropriate probe has become the most popular tool in studying biological processes occurring at the cellular level, owing to its unrivalled spatiotemporal resolution.1–3 As the intensity of fluorescence signal is highly sensitive to the environment, use of intensitybased probes, however, provides less reliable information on the target analyte. For an environment-insensitive analysis, a ratiometric probe with “ratiometric imaging” capability is highly demanded.4–6 Such a ratiometric imaging probe should signal separate fluorescent bands for the probe itself and for its analyte-bound one (or its reaction product with the analyte), which is necessary to extract relative quantitative information on the analyte through pixel-to-pixel ratiometric data transformation into the corresponding ratiometric image for the entire area/volume of interest. Formaldehyde (HCHO), a reactive carbonyl species, is known as a probable human carcinogen and a sick house syndrome gas.7,8 Recent studies, however, indicate that formaldehyde is enzymatically generated in cells, presenting in the human body at high concentrations ranging from 100 μM in blood to 400 μM in cells,9–11 whereas other reactive carbonyl species such as methylglyoxal and acetaldehyde remain in the lower concentration range (a few μM 12 and 0.1–68 μM in blood,13 respectively, in case of normal people). A few studies on the biological roles of endogenous formaldehyde proposed that it is related to the storage, preservation, and retrieval of long-term memory.14 Formaldehyde and ‘masked’ formaldehyde compounds are also reported to show antibacterial/antimicrobial activities by virtue of its ability to penetrate into the interior of bacterial spores.15–17 On the contrary, abnormally high levels of endogenous formaldehyde are related to various diseases including cancer,18 neurodegenerative diseases,19,20 diabetes, chronic liver and heart disorders.21 In spite of existence and production of such a
high level of formaldehyde in human, the biological roles of formaldehyde are yet to be elucidated. To investigate the biological roles and significance of formaldehyde, a noninvasive detection system with ratiometric imaging capability is highly demanded. Recently, several reaction-based sensing schemes to fluorescently detect formaldehyde in living species have been disclosed by Chang,22 Chan23 and Lin24–26 groups, independently, which involve chemical reactions between formaldehyde and an amine. Those probes are effective for cellular imaging, albeit under one-photon excitation at short wavelengths. For tissue imaging, additional issues such as photobleaching of the probe and autofluorescence from tissue become serious concerns under one-photon excitation at the shorter wavelengths. Such issues can be significantly alleviated by two-photon imaging under excitation in the lowenergy, near-infrared wavelength region, which allows imaging of tissues at the deeper region.27 Although a few twophoton probes appeared recently, but none of those has the ratiometric imaging capability for monitoring of formaldehyde in cells and tissue.28–30 Herein, we disclose a two-photon formaldehyde probe, in particular, with the ratiometric imaging capability, which enabled us to observe the high level of formaldehyde around the Paneth cells of small intestine with implications of its antibacterial/antimicrobial role for the first time.
General Information. The chemical reagents were purchased from Sigma-Aldrich or Alfa-Aesar and used as received. All solvents were purified and dried by standard methods prior to use. Deionized water was used to prepare all aqueous solutions. 1H and 13C NMR spectra were recorded on a Bruker 300 MHz or 500 MHz spectrometer using tetramethylsilane as the internal reference. All chemical shifts are reported in the standard notation of parts per million (ppm) using residual solvent protons as the internal standard. Mass
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spectroscopic data were obtained from the Korea Basic Science Institute (Daegu) with a JEOL JMS 700 high resolution mass spectrometer. Synthesis. Only the synthetic procedures are described here. The synthetic scheme is shown in the Supporting Information (Scheme S1). Synthesis of probe 1. A mixture of 6-(pyrrolidin-1-yl)-2naphthaldehyde (aldehyde 2 prepared in the following; 225 mg, 1.0 mmol) and 2-methoxyethylamine (261 µL, 3 mmol) was refluxed in ethanol (5 mL) for 10 h. After being cooled to room temperature, the solvent was evaporated and it was kept under high vacuum for 12 h to afford “dry” imine product. The crude imine was then dissolved in THF (5 mL), cooled down to –78 °C, and treated slowly with 1.0 M allyl magnesium bromide in THF (1.2 mL, 1.2 mmol). The reaction mixture was allowed to attain room temperature within 30 min, quenched with MeOH (0.5 mL), and then filtered through a pad of Celite. The filtrate was concentrated and directly subjected to column chromatography (eluent: MeOH/CH2Cl2 = 5/95) to afford pure probe 1 (188 mg, 58%) as a white solid: 1 H NMR (500 MHz, CDCl3) 𝛿 7.66 (d, J = 9.0 Hz, 1 H), 7.61 (d, J = 8.5 Hz, 1 H), 7.58 (s, 1 H), 7.35 (dd, J = 8.5, 1.5 Hz, 1 H), 6.98 (dd, J = 9.0, 2.0 Hz, 1 H), 6.75 (d, J = 2.0 Hz, 1 H), 5.78–5.72 (m, 1 H), 5.13–5.03 (m, 2 H), 3.74 (t, J = 7.0 Hz, 1 H), 3.46–3.50 (m, 1 H), 3.44–3.38 (m, 5 H), 3.33 (s, 3 H), 2.68–2.63 (m, 2 H), 2.50 (t, J = 7.0 Hz, 2 H), 2.06–2.03 (m, 4 H); 13C NMR (125 MHz, CDCl3) 𝛿 146.0, 136.3, 135.8, 134.8, 128.8, 126.2, 126.1, 125.8, 117.4, 115.9, 104.8, 72.2, 63.0, 58.4, 48.0, 47.2, 43.0, 25.6; HRMS: m/z calcd for C21H28N2O [M+] 324.2202; found 324.2200 [M+]. Synthesis of 6-(pyrrolidin-1-yl)-2-naphthaldehyde (2). A mixture of pyrrolidine (3.8 mL, 45.0 mmol), 6-bromo-2naphthol (2.0 g, 9.0 mmol), Na2S2O5 (3.6 g, 18.0 mmol), and DI water (20 mL) in a sealed tube was stirred at 145 °C for 48 h. After being cooled to room temperature, the reaction mixture was diluted with 20 mL of water and the product was extracted with CH2Cl2 (30 mL × 2). The combined organic solution was concentrated and then purified by flash column chromatography (eluent: ethyl acetate/hexane = 5/95) to afford 1-(6-bromonaphthalen-2-yl)pyrrolidine (1.93 g, 78%) as a white solid. 1-(6-Bromonaphthalen-2-yl)pyrrolidine (1.66 g, 6.0 mmol) prepared in the above was dissolved in anhydrous THF (15 mL), cooled to –78 °C under argon, and then treated with 1.6 M n-BuLi in hexane (0.8 mL, 7.2 mmol). The reaction mixture was stirred at -30 °C for 1 h and then treated with anhydrous DMF (0.7 mL, 9.0 mmol), and it was allowed to attain 0 °C. After being stirred at 0 °C for 1 h, the reaction mixture was quenched with saturated ammonium chloride (5 mL). The product was extracted with ethyl acetate (30 mL × 2), and then purified by silica gel column chromatography (eluent: ethyl acetate/hexane = 5/95) to yield aldehyde 2 (0.95 g, 70%): 1H NMR (300 MHz, CDCl3): δ 9.99 (s, 1H), 8.13 (s, 1H), 7.90 (d, J = 9.6 Hz, 1H), 7.61 (d, J = 8.4 Hz, 1H), 7.00 (dd, J = 9.0, 2.4 Hz, 1H), 6.73 (d, J = 2.4 Hz, 1H), 3.43 (t, J = 6.6 Hz, 4H), 2.11–2.06 (m, 4H); 13C NMR (75 MHz, CDCl3) 𝛿 192.0, 148.4, 139.1, 135.3, 131.1, 130.3, 126.6, 124.9, 123.7, 116.5, 104.8, 47.9, 25.7; HRMS: m/z calcd for C15H15NO [M+] 225.1154; found 225.1150 [M+]. Fluorescence assays with probe 1. All of the solvents used were of analytical grade. A stock solution of formaldehyde (40 mM) was prepared by addition of 6.2 mg of paraformaldehyde
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(95% purity) to 5 mL of PBS buffer (pH 7.4), and the resulting mixture was heated at 90 °C for 1 h in a closed vial to make a clear solution. The solution was allowed to cool down to room temperature before spectrometric measurements. Solutions of other biologically relevant analytes such as methylglyoxal, acetaldehyde, glucose, pyruvate, H2O2, Cys, and GSH were prepared by dissolving each of the corresponding reagents in distilled water. A stock solution of H2S was prepared by dissolving Na2S.9H2O in distilled water. A stock solution of probe 1 was prepared in dimethyl sulfoxide (DMSO) at a concentration of 1 mM. For spectroscopic measurement, the probe stock solution was diluted to 10 μM in 10 mM PBS buffer solutions of different pH values as needed, which was treated with an analyte solution and then kept at 37 °C in an incubator. After specified time, a required amount of the mixed solution was transferred to a cuvette (1 mL) for spectroscopic measurement. Fluorescence spectra were recorded on a Photon Technical International Fluorescence system using a 1.0 mL quartz cuvette. Absorption spectra were measured using a HP Agilent 8453 spectrophotometer. All pH measurements were made with a Thermo scientific, Orion 2 star pH benchtop. Preparation of cell samples and their confocal imaging. MCF7 human breast cancer cells were obtained from Korean Cell Line Bank. MCF7 cells were incubated in DMEM supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) penicillin-streptomycin (PS) at 37 °C in a humidified atmosphere of 5% of CO2 in the air. Cells were passaged when they reached approximately 80% confluence. Cells were seeded onto a cell culture dish at a density of 1.0 × 105 cells, which was incubated at 37 °C overnight under 5% CO2 in the air. For imaging experiments, cells were incubated in three conditions: For the detection of endogenous formaldehyde, cells were incubated in DMEM containing the probe (10 μM) for 30 min and washed with PBS (phosphate buffered saline) three times to remove the remaining probe, and then incubated further for 2.5 h. In the negative control experiment with sodium bisulfite, cells were incubated in DMEM containing sodium bisulfite (200 μM) for 30 min and washed with PBS three times to remove the remaining sodium sulfite. Then the cells were incubated in DMEM containing the probe (10 μM) for 30 min, washed with PBS three times to remove the remaining probe, and then incubated for additional 2.5 h. In the positive control experiment with an exogenous formaldehyde source, the cells were incubated in DMEM containing the probe (10 μM) for 30 min and washed with PBS (phosphate buffered saline) three times to remove the remaining probe and then incubated further with formaldehyde (1 mM) for 2.5 h. Fluorescent cellular images were recorded by confocal microscopy, using Leica TCS SP5 II Advanced System equipped with multiple visible laser lines (405, 458, 476, 488, 496, 514, 561, 594, and 633 nm) and a 40× objective lens (obj. HCX PL APO 40×/ 1.10 W CORR CS, Leica, Germany). For this one-photon imaging experiment, cells were excited by 405 nm laser line and fluorescent emission was collected in two emission channels; λem, blue = 410–440 nm and λem, green = 500–600 nm. Acquired images were processed using LAS AF Lite (Leica, Germany), and all the images were converted into the corresponding pixel-to-pixel ratiometric images. Preparation of mouse organ tissue samples and their TPM imaging. The experimental procedures regarding mouse tissues herein were performed in accordance with protocols
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Vision II, Coherent) at 140 fs pulse width and 80 MHz pulse repetition rate (TCS SP5 II, Leica, Germany) and a 20× objective lens (obj. HCX PL APO 20×/ 1.10 W CORR CS, Leica, Germany). The two-photon excitation wavelength was tuned to 760 nm for the probe. Each emission light was spectrally resolved into multi-channels (λem, blue = 400–440 nm, λem, green = 470–550 nm). The tissue samples prepared as above were mounted on a tight-fitting holder. The excitation laser power was approximately 9.3 mW. The images were consisted of 1024 × 1024 pixels, and the scanning speed was maintained as 100 MHz during the entire imaging.
Design of two-photon ratiometric probe for formaldehyde. To realize ratiometric fluorescence response, we have implemented a pro-aza-Cope rearrangement moiety in a two-photon absorbing dye in such a way that the rearrangement can induce significant intramolecular charge transfer (ICT). Probe 1 thus designed has a sechomoallylamine moiety attached to 1-(naphthalen-2yl)pyrrolidine (Figure 1a). The probe, upon treatment with formaldehyde, will form the iminium ion that undergoes cationic 2-aza-Cope rearrangement and subsequent hydrolysis to afford 6-(pyrrolidin-1-yl)-2-naphthaldehyde, a donoracceptor type two-photon absorbing dye (Figure 1a and S1 in the Supporting Information). We chosed the pyrrolidine group as an electron donor part for the dye due to its ability to cause bright fluorescence in aqueous medium compared to that of the conventional N,N-dimethylamino donor.31 Probe 1 was readily synthesized from aldehyde 2 through imine formation with 2-methoxyethylamine, followed by nucleophilic addition with allylmagnesium bromide (details in the Experimental Sections). Probe 1 itself showed strong blue emission at 438 nm with a high quantum yield (Φ = 0.92) under one-photon excitation at 400 nm. Upon the gradual addition of formaldehyde in the physiological concentration range (0–800 µM), the emission
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approved by the Pohang University of Science and Technology Committee on Animal Research and followed the guidelines for the use of experimental animals established by The Korean Academy of Medical Science. We made every effort to minimize animal suffering and reduce the number of animals used to prepare samples for imaging. Balb/C type mice (6 weeks) were used for this experiment. Basically experiment was done under light protected conditions (in a dark-room and using aluminum foil). The mouse was dissected after dislocation of the cervical vertebra. Blood perfusion with phosphate buffered saline (PBS, 1X solution) was performed for elimination of blood. The five organs (brain, lung, liver, kidney and colon) were dissected and washed with PBS buffer, and then sliced with a vibrating blade microtome (VT1000S, Leica, Germany) as 50 μm thickness. For imaging experiments, tissues were incubated in three conditions: For the detection and quantification of endogenous formaldehyde, tissues were incubated in DMEM containing 10 μM of the probe for 30 min and washed with PBS for three times to remove the remaining probe and incubated further for 2.5 h. In negative control experiment with sodium bisulfite, tissues were incubated in DMEM containing sodium bisulfite (200 μM) for 30 min and washed with PBS for three times to remove the remaining sodium sulfite, followed by incubation in DMEM containing 10 μM of the probe for 30 min and washed with PBS for three times to remove the remaining probe and further incubation for 2.5 h. In positive control experiment with exogenous formaldehyde, tissues were incubated in DMEM containing 10 μM of the probe for 30 min and washed with PBS (phosphate buffered saline) for three times to remove the remaining probe and incubated further for 2.5 h with formaldehyde (1 mM). All incubations were carried out at 37 °C under 5% CO2 in the air. The stained samples were placed on a slide glass for imaging, after washing with PBS three times to remove remaining probe. Fluorescence images of the tissue samples were recorded by two-photon microscopy (TPM). TPM imaging was performed using a Ti-Sapphire laser (Chameleon Vision II, Coherent) at 140 fs pulse width and 80 MHz pulse repetition rate (TCS SP5 II, Leica, Germany) through a 20× objective lens (obj. HCX PL APO 20×/ 1.10 W CORR CS, Leica, Germany). The two-photon excitation wavelength for the probe was tuned to 760 nm. Each emission light was spectrally resolved into two channels (λem, blue = 400–440 nm, λem, green = 470–550 nm). The tissue samples prepared as above were mounted on a tight-fitting holder. The excitation laser power was approximately 9.3 mW. The images were consisted of 1024 × 1024 pixels, and the scanning speed was maintained as 100 MHz during the entire imaging. Preparation of mouse intestinal tissue samples and their TPM imaging. Balb/C type mice (6 weeks) were used for this experiment. Basically experiment was done under light protected conditions (in a dark-room and using aluminum foil). The mouse was dissected after dislocation of the cervical vertebra. Intestine is removed and sectioned. Sections of jejunum 0.5 cm long (small intestine) and colon 0.5 cm long were immersed into the probe 1 solution (10 μM in HEPES buffer, pH 7.4) after a longitudinal incision. Incubation were carried out for 3 h at 37 °C and then washed with PBS buffer for three times. The stained samples were placed on a slide glass flatways. Fluorescence images of the tissue samples were recorded by two-photon microscopy (TPM). TPM imaging was performed using a Ti-Sapphire laser (Chameleon
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Analytical Chemistry
Fluorescence intensity (au)
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Figure 1. Sensing scheme, fluorescence titration, and selectivity data. (a) Sensing scheme of formaldehyde with probe 1. (b) Fluorescence titration of probe 1 (10 μM) with formaldehyde (0–800 μM). Inset: plot of fluorescent intensity ratio (I533 nm/I438 nm) depending on [HCHO]. (c) Response of probe 1 toward biologically relevant species (methylglyoxal, acetaldehyde, glucose, pyruvate, H2O2, H2S, Cys, GSH and formaldehyde; all are evaluated at 500 μM, except H2S at 100 μM). All data were obtained after 3 h incubation at 37 °C in PBS buffer (pH = 7.4), under excitation at 400 nm.
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Analytical Chemistry from probe at 438 nm gradually decreased whereas a new peak arose at 533 nm (Φ = 0.17) (Figure 1b and S2 in the Supporting Information). The fluorescence intensity ratios between the two peaks at 438 nm and 533 nm showed a good linear relationship with the formaldehyde concentration, which provided a basis for the quantitative imaging of formaldehyde in tissue. The limit of detection was determined to be around 10 µM (300 ppb) (Figure S3 in the Supporting Information), allowing its applicability for the detection of the intracellular formaldehyde (200–400 µM).9–11 Probe 1 also showed excellent selectivity toward formaldehyde over the potentially competing reactive carbonyl species and carbonyl containing molecules (methylglyoxal, acetaldehyde, glucose, and pyruvate), as well as over reactive oxygen species such as H2O2 and reactive sulfur species such as cysteine, glutathione and hydrogen sulfide (Figure 1c and S4 in the Supporting Information). Even in the presence of those biomolecules at their physiologically relevant concentrations, probe 1 shows excellent sensing behavior towards formaldehyde (Figure S5 i) Probe + NaHSO3
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Figure 2. Detection of endogenous formaldehyde in cells and tissues. (a) Confocal pixel-to-pixel ratiometric images (IGreen/IBlue) of MCF7 cells in different conditions: cells i) incubated with NaHSO3 (200 μM, 30 min) followed by probe 1 (10 μM, 3 h), ii) treated only with probe 1 (10 μM, 3 h), and iii) incubated with probe 1 (10 μM, 3 h) followed by exogenous formaldehyde (1 mM, 2.5 h). (b) Two-photon pixel-to-pixel ratiometric images (IGreen/IBlue) of mouse kidney tissue in different conditions: tissues i) incubated with NaHSO3 (200 μM, 30 min) followed by probe 1 (10 μM, 3 h), ii) incubated only with probe 1 (10 μM, 3 h), and iii) incubated with probe 1 (10 μM, 3 h) followed by exogenous formaldehyde (1 mM, 2.5 h). (c, d) Bar graphs representing the average ratio values in each conditions of Figure 2a and Figure 2b. Conditions for OPM and TPM: λex, OPM = 405 nm, λem, blue = 410–440 nm, and λem, green = 500–600 nm (for Figure 2a); λex, TPM = 760 nm, λem, blue = 400–440 nm, and λem, green = 470–550 nm (for Figure 2b). *: p
