A self-fluorescing near-infrared fluorophore, a preparation method and application thereof in super-resolution imaging of living cells

Abstract

The application discloses a kind of spontaneous scintillation near-infrared fluorophore, preparation method and its application in the super-resolution imaging of living cell;The molecular structure formula of the spontaneous scintillation near-infrared fluorophore is as shown in formula 1);Wherein, R1 is 1-ethyl-3,3-dimethyl-4,5-benzoindole-2-yl or 1-ethyl-quinoline-4-yl;R2 and R3 are methyl or ethyl, R4 is the absorption wavelength of the spontaneous scintillation near-infrared fluorophore molecule of the application, which can be up to 700-800nm, can significantly reduce phototoxicity in living cell imaging.In addition, such molecules have good biocompatibility, and can realize the super-resolution imaging and dynamic tracking of endoplasmic reticulum and mitochondria.

Description

Technical Field

[0001] This invention belongs to the field of organic synthesis and super-resolution imaging technology of fluorescent probes, specifically relating to a type of spontaneously scintillated near-infrared fluorophore, its preparation method, and its application in super-resolution imaging of live cells. Background Technology

[0002] Single-molecule localization-based super-resolution imaging technology breaks through the traditional optical diffraction limit, enabling the visualization of previously invisible molecular details in biological systems, attracting significant attention in the life sciences. Single-molecule localization super-resolution imaging offers advantages such as high imaging resolution and low equipment requirements, but it imposes specific requirements on fluorescent dyes. They must complete a transition from a dark to a bright state before photobleaching, and different dye molecules must be sparsely distributed in space when emitting light simultaneously to avoid signal overlap. Self-flashing fluorophores, such as rhodamine dyes, are subject to pH regulation and undergo a thermal equilibrium transition between their open-ring fluorescent state and spirocyclic dark state. One of the main limitations in developing single-molecule localization super-resolution imaging technology is the need for high-intensity illumination during the imaging process, which not only accelerates photobleaching of fluorophores but also causes phototoxicity in living cells. Current probe chemistry research mainly focuses on improving the photophysical properties of probes to adapt to high-power irradiation, neglecting the significant impact on cell health during imaging. According to literature reports, as the excitation wavelength increases, the phototoxicity of light to living cells also decreases significantly. Therefore, in probe chemistry research, it is necessary to further redshift the excitation wavelength of fluorescent probes to reduce phototoxicity in live cell imaging, while still ensuring that the fluorescent probes have excellent photophysical properties to achieve high spatiotemporal resolution. Summary of the Invention

[0003] The purpose of this invention is to provide a class of spontaneously scintillated near-infrared fluorophores, their preparation method, and their application in super-resolution imaging of live cells. These molecules have absorption wavelengths down to 700-800 nm, significantly reducing phototoxicity in live-cell imaging. Furthermore, these molecules exhibit good biocompatibility, enabling super-resolution imaging and dynamic tracking of the endoplasmic reticulum and mitochondria.

[0004] To achieve the above-mentioned objectives, the present invention adopts the following technical solution.

[0005] This invention provides a self-scinding near-infrared fluorophore, the molecular structure of which is shown in Formula 1):

[0006]

[0007] In the formula, R1 is 1-ethyl-3,3-dimethyl-4,5-benzoindole-2-ylene or 1-ethyl-quinoline-4-ylene, with the following structural formulas: R2 and R3 are independently methyl or ethyl, and R4 is... or

[0008] In this invention, R2 and R3 are ethyl groups, and R4 is a ethyl group.

[0009] The present invention also provides a method for preparing the above-mentioned spontaneously scintillated near-infrared fluorophore, comprising the following steps:

[0010] Step 1: Under an inert atmosphere, compound A, benzotriazol-1-yl-oxytripyrrolylphosphide (PyBOP), an amine compound, and an acid-binding agent are dissolved in a solvent and reacted with stirring at room temperature to prepare compound B; wherein: the structural formula of compound A is... The amine compound is any one of ethylenediamine monohydrate, 2,2'-oxobis(ethylamine), or o-phenylenediamine;

[0011] When the amine compound is ethylenediamine monohydrate or 2,2'-oxobis(ethylamine), the resulting compound B is the target compound with a spontaneously scintillated near-infrared fluorophore of formula 1); when the amine compound is o-phenylenediamine, the resulting compound B continues the reaction steps below.

[0012] Step 2: Under an inert atmosphere and ice bath conditions, compound B is dissolved in a solvent, followed by the addition of glutaryl chloride and an acid-binding agent to trigger the reaction. After the reaction is completed, water is added to terminate the reaction.

[0013] Step 3: Extract the reaction solution obtained in Step 2 to concentrate the organic layer and obtain compound C, which is the near-infrared fluorophore target compound of Formula 1).

[0014] In this invention, the inert atmosphere in steps one and two is nitrogen or argon, the solvent is anhydrous dichloromethane, and the acid-binding agent is triethylamine.

[0015] In this invention, in step one, the molar ratio of compound A, PyBOP, amine compound, and acid-binding agent is 1:(1.2-1.5):(9-12):(9-12).

[0016] In this invention, in step two, the ratio of compound B, acid-binding agent and glutaryl chloride is 1:(9-12):(9-12).

[0017] In this invention, in step three, the reaction solution extraction system consists of water and dichloromethane; compound C is obtained by silica gel column chromatography, with petroleum ether, ethyl acetate, and triethylamine used as the mobile phase.

[0018] Furthermore, the present invention also provides the application of the above-mentioned spontaneously scintillated near-infrared fluorophores in the preparation of live-cell super-resolution imaging reagents; the live-cell super-resolution imaging reagents realize super-resolution structural reconstruction of mitochondria and endoplasmic reticulum and super-resolution time-lapse imaging of dynamic changes based on single-molecule localization microscopy.

[0019] The spontaneously scintillated near-infrared fluorophore of this invention consists of a classic rhodamine structure and a hemicyanine structure with a quinoline ring or benzoindole ring. This fluorophore retains the spirocyclic advantage of rhodamine, exhibiting a transition between "ring-open" and "ring-closed" states in solutions with different pH values. The "ring-closed" state is fluorescence-free, while the ring-open state exhibits strong fluorescence. Based on rhodamine derivatives, this series of fluorophores precisely modulates the molecular pK by linking different side chain groups R4 via acyl chloride condensation. a Numerical variations affect the switching rate. Furthermore, the absorption and emission wavelengths of the hemicyanine fluorophore are both in the near-infrared region (700-800 nm), while the absorption wavelength range of fluorophores with different R1 substituents is 730-740 nm, and the emission wavelength range is 760-770 nm. With R1 as 1-ethyl-3,3-dimethyl-4,5-benzoindole-2-ylene, the structural formula is... R4 is Taking a self-scinding near-infrared fluorophore compound as an example, the principle is shown in the following equation:

[0020]

[0021] In alkaline or neutral solutions, the compound exists in a non-fluorescent "ring-closed" state; in acidic solutions, the compound transitions to an open-ring state, exhibiting strong fluorescence, and simultaneously, the nitrogen in the benzoindole moiety is protonated to N2. + The latter exhibits a significantly increased conjugated structure compared to the former, resulting in a substantial redshift in both ultraviolet absorption and fluorescence emission. The spontaneously scintillated near-infrared fluorophores enable super-resolution imaging based on single-molecule localization, facilitating dynamic observation of physiological activities within living cells and helping us to further understand the fine structures within cells.

[0022] This invention has outstanding substantive features and significant progress compared to the prior art, specifically in that:

[0023] First, the spontaneously flashing near-infrared fluorophores of the present invention have low phototoxicity and good biocompatibility, making them easy to enter cells.

[0024] Secondly, the near-infrared fluorophore of the present invention has high selectivity for pH, and the reversible "on-off" ring endows the fluorophore with spontaneous scintillation properties.

[0025] Third, the near-infrared fluorescent molecular probe of the present invention absorbs and emits in the near-infrared region, avoiding interference from the autofluorescence of biological tissues, and has strong biological penetration.

[0026] Fourth, in the state where the strong fluorescence of the self-sparkling near-infrared fluorophore of the present invention is turned on, the molecules are positively charged, which can effectively locate mitochondria in living cells.

[0027] Fifth, the method for preparing the spontaneously scintillated near-infrared fluorophores of the present invention has the advantages of simple synthesis process, readily available raw materials, low cost and high yield.

[0028] Sixth, the self-scinding near-infrared fluorophore of this invention can achieve super-resolution imaging of fine structures within living cells. Attached Figure Description

[0029] Figure 1 This is the 1H NMR spectrum of compound 3 in deuterated chloroform.

[0030] Figure 2 This is the high-resolution mass spectrum of compound 3.

[0031] Figure 3 This is a graph showing the fluorescence changes of compound 3 in buffer solutions at different pH values.

[0032] Figure 4 This is a co-localization imaging image of mitochondria in living cells using compound 3 and Mito-Tracker Green, a commercially available dye that targets mitochondria.

[0033] Figure 5 This is a super-resolution image of mitochondria in a living cell located by compound 3.

[0034] Figure 6 This is a super-resolution dynamic image of the mitochondria in living cells located by compound 3.

[0035] Figure 7 This is a co-localization imaging of compound 8 and the commercially available dye ER-Tracker Green, which targets the endoplasmic reticulum, in transfected / untransfected live cells.

[0036] Figure 8 These are super-resolution images and diffraction restriction maps of the endoplasmic reticulum in living cells where compound 8 is located.

[0037] Figure 9 This is a super-resolution dynamic image of the endoplasmic reticulum in living cells located by compound 8. Detailed Implementation

[0038] The technical solution of the present invention will be further described in detail below through specific embodiments.

[0039] Unless otherwise specified, all reagents and consumables used in the experiments of this invention can be purchased commercially. Unless otherwise specified, the experimental methods are conventional. All reagents used in the examples are commercially available analytical grade or chemically pure.

[0040] Benzotriazole-1-yl-oxytripyrrolidinephosphide hexafluorophosphate (PyBOP) and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU) were purchased from TCI.

[0041] Triethylamine and o-phenylenediamine were purchased from Aladdin.

[0042] Glutaryl chloride was purchased from J&K Scientific.

[0043] Analytical instruments:

[0044] Ultraviolet spectrophotometer (UV-2550, Shimadzu)

[0045] Fluorescence spectrophotometry (FS5, Edinburgh)

[0046] Mass spectrometer (LCQ - Advantages)

[0047] Nuclear magnetic resonance spectrometer (Bruker 300MHz)

[0048] Microscope (ECLIPSE TE2000-U, Nikon).

[0049] The synthetic methods for compounds 1 and 6 can be found in published literature, such as:

[0050] (1) Yuan, L.; Lin, W.; Yang, Y.; Chen, HA unique class of near-infrared functional fluorescent dyes with carboxylic-acid-modulated fluorescence ON / OFF switching: rational design, synthesis, optical properties, theoretical calculations, and applications for fluorescence imaging in living animals. J. Am. Chem. Soc. 2012, 134, 1200-1211.

[0051] (2) Vegesna, GK; Janjanam, J.; Bi, J.; Luo, FT; Zhang, J.; Olds, C.; Tiwari, A.; Liu, H. pH-activatable near-infrared fluorescent probes for detection of lysosomal pH inside living cells. J.Mater.Chem.B 2014, 2, 4500-4508.

[0052] (3)Tan, B.; Zhao, C.; Wang, J.; Tiemuer, A.; Zhang, Y.; Yu, H.; Liu, Y. Rational design of pH-activated upconversion luminescent nanoprobes for bioimaging of tumor acidic microenvironment and the enhancement of photothermal therapy. Acta Biomater. 2023, 155, 554-563.

[0053] (4) Yu, H.; Sun, W.; Tiemuer, A.; Zhang, Y.; Wang, HY; Liu, Y. Mitochondria targeted near-infrared chemodosimeter for upconversion luminescence bioimaging of hypoxia. Chem. Commun. 2021, 57, 5207-5210.

[0054] Example 1: Preparation of near-infrared fluorescent probe compound 3

[0055]

[0056] In the formula, R2 and R3 are both ethyl groups.

[0057] Compound 1 (50.0 mg, 0.08 mmol) and PyBOP (50.1 mg, 0.096 mmol) were dissolved in an appropriate amount of anhydrous dichloromethane, and the mixture was stirred for 30 minutes under nitrogen protection. Then, triethylamine (75.6 mg, 0.8 mmol) and o-phenylenediamine (80.8 mg, 0.8 mmol) were added to the reaction solution, and the mixture was reacted at room temperature for 6 hours to obtain compound 2.

[0058] Under nitrogen protection and an ice bath, compound 2 (100 mg, 0.12 mmol) was dissolved in an appropriate amount of anhydrous dichloromethane. Glutaryl chloride (405.6 mg, 2.4 mmol) and triethylamine (121.4 mg, 1.2 mmol) were added to the reaction solution. After stirring for 30 minutes, water was added to terminate the reaction. Excess glutaryl chloride was removed with excess water, and the organic phase was extracted by dichloromethane / water extraction. The crude product was dried under reduced pressure. The crude product was purified by silica gel column chromatography. Under a mobile phase of ethyl acetate / petroleum ether / triethylamine (v / v / v = 1:5:0.05), the final yellow product, compound 3 (50.8 mg, yield 51.2%), was obtained.

[0059] The mass spectrum of compound 3 prepared in Example 1 was confirmed by a mass spectrometer (model: LCQ-Advantages) and a nuclear magnetic resonance spectrometer (model: Bruker 300MHz), and the results are as follows. Figure 1 and Figure 2 As shown, the relevant parameters are 1 H NMR(300MHz,Chloroform-d)δ8.03(d,J=8.6Hz,1H),7.97(dd,J=6.5,1.7Hz,1H),7.78(d,J=8.2Hz,1H),7.72(d,J=8.5Hz,1H),7.5 1(td,J=7.4,1.4Hz,2H),7.48–7.38(m,2H),7.25–7.16(m,2H),6.99(dd,J=8.5,4.5Hz,2H),6.68(d,J=8.0Hz,1H),6.60(d,J=8.6H z,2H),6.32(d,J=8.9Hz,2H),5.42(d,J=12.6Hz,1H),3.80–3.72(m,2H),3.40–3.32(m,4H),2.60(s,1H),2.38(s,1H),2.08(dd,J= 12.6,6.1Hz,1H),1.99(s,6H),1.72–1.61(m,6H),1.40(d,J=16.2Hz,2H),1.31–1.24(m,6H),1.24–1.08(m,6H).HRMS(m / z):[M+H] + calcd.for C 53 H 55 N4O5 + :827.41725; found:827.41604.

[0060] Example 2: Preparation experiment of near-infrared fluorescent probe compound 4.

[0061]

[0062] In the formula, R2 and R3 are both ethyl groups.

[0063] Compound 1 (50.0 mg, 0.08 mmol) and PyBOP (50.1 mg, 0.096 mmol) were dissolved in an appropriate amount of anhydrous dichloromethane and stirred under nitrogen protection for 30 min. Then, triethylamine (75.6 mg, 0.8 mmol) and ethylenediamine monohydrate (62.5 mg, 0.8 mmol) were added to the reaction solution, and the reaction was continued for 4 h. After the reaction was completed, the reaction solvent was dried by rotary evaporation, and the crude product was purified with petroleum ether / ethyl acetate / triethylamine mobile phase (v / v / v = 4 / 1 / 0.005) to give compound 4 (39.0 mg, 73.3% yield) as a yellow solid. 1 H NMR(300MHz,Chloroform-d)δ8.05(d,J=8.6Hz,1H),7.85(d,J=8.7Hz 1H),7.78(d,J=8.6Hz,1H),7.73(d,J=8.6Hz,1H),7.58(d,J=12.7Hz,1H),7.44(td,J=6.8,1.5Hz,3H),7.26(d,J=8.7Hz ,1H),7.18(dd,J=6.4,1.7Hz,1H),7.01(d,J=8.7Hz,1H),6.37–6.32(m,2H),6.28(dd,J=6.8,2.1Hz,1H),5.45(d,J=12. 7Hz,1H),3.77(q,J=7.1Hz,2H),3.37(m,6H),3.16(qd,J=6.4,4.1Hz,2H),2.60(td,J=6.5,4.3Hz,2H),2.05(s,6H),1.9 6(d,J=2.7Hz,1H),1.85–1.76(m,1H),1.71–1.65(m,2H),1.31(t,J=6.1Hz,3H),1.17(t,J=12.2Hz,6H).HRMS(m / z)[M+H] + calcd.for C 44 H 49 N4O2 + :665.3811; found:665.38420.

[0064] Example 3: Preparation experiment of near-infrared fluorescent probe compound 5.

[0065]

[0066] In the formula, R2 and R3 are both ethyl groups.

[0067] Compound 1 (50.0 mg, 0.08 mmol) and PyBOP (50.1 mg, 0.096 mmol) were dissolved in an appropriate amount of anhydrous dichloromethane and stirred under nitrogen protection for 30 min. Then, triethylamine (75.6 mg, 0.8 mmol) and 2,2'-oxobis(ethylamine) (83.3 mg, 0.8 mmol) were added to the reaction solution and the reaction was continued for 6 h. After the reaction was completed, the reaction solvent was evaporated to dryness, and the crude product was purified by silica gel column chromatography. Compound 5 (38.0 mg, 67.1% yield) was obtained as a yellow solid in a mobile phase of petroleum ether / ethyl acetate / triethylamine (v / v / v = 4 / 1 / 0.005). 1 H NMR(300MHz,Chloroform-d)δ8.05(d,J=8.6Hz,1H),7.83(d,J=8.6Hz,1H),7.81(d,J=6.6Hz 1H),7.75(d,J=8.6Hz,1H),7.47-7.43(d,J=12.7Hz,1H),7.45(td,J=6.8,1.5Hz,3H),7.23(m,1H),7.18(dd,J=6.4,1 .7Hz,1H),7.02(d,J=8.7Hz,1H),6.36–6.32(m,2H),6.28(dd,J=6.8,2.1Hz,1H),5.45(d,J=12.7Hz,1H),3.77(q,J=7 .1Hz,2H),3.37(h,J=7.1Hz,6H),3.16(qd,J=6.4,4.1Hz,2H),2.60(td,J=6.5,4.3Hz,2H),2.05(s,6H),1.96(d,J=2. 7Hz,1H),1.85–1.76(m,1H),1.72–1.68(m,2H),1.32–1.28(t,J=11.2Hz,3H),1.14(t,J=6.7Hz,6H).HRMS(m / z)[M+H] + calcd.for C 46 H 53 N4O3 + :709.4037; found:709.40932.

[0068] Example 4: Preparation experiment of near-infrared fluorescent probe compound 7.

[0069]

[0070] In the formula, R2 and R3 are both ethyl groups.

[0071] Compound 6 (100.0 mg, 0.18 mmol) and PyBOP (111.8 mg, 0.22 mmol) were dissolved in an appropriate amount of anhydrous dichloromethane. The reaction was stirred for 5 minutes under nitrogen protection. Then, triethylamine (179.8 mg, 1.78 mmol) and o-phenylenediamine (193.4 mg, 1.78 mmol) were added to the reaction solution, and the reaction was continued for 20 minutes. Then, glutaryl chloride (304.2 mg, 1.8 mmol) and triethylamine (182.16 mg, 1.8 mmol) were added to the reaction solution. The reaction was stirred for 20 minutes under nitrogen protection. After the reaction was completed, the excess glutaryl chloride was removed with excess water. The crude product was purified by silica gel column chromatography. Compound 7 (10.03 mg, yield 14.3%) was given as a yellow solid under a mobile phase of dichloromethane / methanol / triethylamine (v / v / v = 40:1:0.04). 1 H NMR(300MHz,Chloroform-d)δ8.13(d,J=9.2Hz,2H),7.96(dd,J=12.8Hz,2H),7.58(d,J=8.7Hz,1H),7.54(d,J =8.8Hz,1H),7.48(m,2H),7.40–7.35(t,J=14.8Hz,2H),7.17–7.10(m,3H),7.07–7.00(m,2H),6.41–6.28(m,4 H),3.82–3.80(t,J=6.8Hz,2H),3.28(m,2H),3.25(m,2H)3.13–3.06(m,2H),2.42–2.40(t,J=6.4Hz,2H),2.37 –2.35(m,2H),2.22–2.20(t,J=4.6Hz,2H),1.97(m,4H),1.74(m,2H),1.38(t,J=14.4Hz,9H).HRMS(m / z)[M+H] + calcd.for C 48 H 49 N4O5 + :761.3703; found:761.3285.

[0072] Example 5: Measurement experiment of fluorescence emission of spontaneously scintillated near-infrared fluorophore compounds in buffer solutions of different pH values.

[0073] Weigh 8.27 mg of compound 3 and dissolve it in 10 mL of ethanol to prepare a 1 mM probe stock solution. Prepare 13 5 mL centrifuge tubes. Add 15 μL of the probe stock solution to each tube, then dilute to 3 mL with citrate-disodium hydrogen phosphate buffer at pH values ​​of 3.0, 3.5, 4.0, 4.5, 5.0, 5.4, 5.8, 6.2, 6.6, 7.0, 7.4, 7.8, and 8.0, respectively. At this point, the probe concentration in each centrifuge tube is 5 μM. After thorough mixing, add the solutions sequentially to cuvettes for fluorescence emission detection (excitation wavelength 720 nm). The obtained data are processed using Origin software. Figure 3 ,like Figure 3 As shown, the fluorescence signal at 770 nm gradually increases as the pH of the solution gradually decreases, indicating that the near-infrared probe has a good pH response mechanism in solution.

[0074] The spectral properties and pKa values ​​of compounds 3, 4, 5, and 7 are shown in Table 1. These compounds have pKa values ​​around 6, which is in the dark state. Imaging of live cells is performed under physiological pH conditions (7.2-7.4), so these compounds exhibit good scintillation behavior. In addition, the absorption and emission of these compounds are both in the near-infrared region. Near-infrared excited probes have low phototoxicity, avoiding interference from the autofluorescence of biological tissues. They also have strong bio-penetration and biocompatibility, making it easy to enter cells. Therefore, these compounds all have the ability to achieve super-resolution imaging. The binding modes are diverse, such as connecting to the targeting group of specific organelles (triphenylphosphine targeting mitochondria), or achieving specific targeting through the technology of transfecting SNAP-Tag labeled proteins.

[0075] Table 1 Spectral properties of the compounds

[0076]

[0077]

[0078] Example 6: Near-infrared fluorescent probe with targeting function

[0079]

[0080] When Y1 = -OH, the compound is used to label mitochondria;

[0081] At that time, the compound was used to target the SNAP-Tag protein.

[0082] Example 7 Preparation of Compound 8

[0083] In a dry round-bottom flask, compound 3 (3.3 mg, 4 μmol) and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU, 1.1 mg, 4 μmol) were dissolved in anhydrous dimethylformamide (DMF, 1 mL). 4-((4-(aminomethyl)benzyl)oxy)-6-chloropyrimidin-2-amine (CLP, 1.1 mg, 4 μmol) and N,N-diisopropylethylamine (DIPEA, 3.8 mg, 0.03 mmol) were slowly added dropwise to DMF and stirred at room temperature for 10 hours. The reaction mixture was diluted with CH₂Cl₂, washed with saturated NaCl, dried over anhydrous Na₂SO₄, filtered, and concentrated under vacuum. The crude product was purified by silica gel column chromatography (CH₂Cl₂ / MeOH = 100:0 to 95:5) to give compound 8 as a green solid (2.8 mg, 65% yield).

[0084] Example 8: Colocalization Imaging Experiment of Mitochondria in Live Cells

[0085] Live HeLa cells were co-incubated with 2 μM compound 3 and 0.2 μM Mito-Tracker Green (a commercially available dye targeting mitochondria) for 30 minutes. After washing three times with PBS, fluorescence images were acquired using a total internal reflection fluorescent microscope (TIRF). The excitation wavelengths were 488 nm (Mito-Tracker Green) and 721 nm (compound 3). The fluorescence image of Mito-Tracker Green was a single frame, while the fluorescence image of compound 3 was a superimposed image of 500 frames due to its scintillation properties. The Pearson correlation coefficient was calculated to be 0.90 using ImageJ, demonstrating that compound 3 can achieve targeted imaging of mitochondria in live cells. Figure 4 ).

[0086] Example 9: Super-resolution imaging of mitochondria in living cells.

[0087] Live HeLa cells were stained with 2 μM compound 3 for 30 minutes, washed with PBS (Phosphate Buffered Saline), and then imaged in phenol red-free DMEM (Dulbecco's modified Eagle medium). Super-resolution imaging of live cells was also performed on a TIRF microscope. (2 kW / cm²) 2 Under wide-field illumination, a 721nm laser is used for excitation, the pixel size is fixed at 106nm, and imaging video is acquired at a rate of 20 frames per second. Figure 5 and Figure 6 This indicates that the super-resolution reconstruction results not only clearly present the microstructure of mitochondria, but also allow observation of their dynamic changes in fusion and division.

[0088] Example 10: Colocalization imaging experiment of endoplasmic reticulum in living cells.

[0089] For untransfected cells, incubation was performed for 30 minutes with 5 μM Compound 8 and 1 μM ER-Tracker Green (a commercial dye targeting the endoplasmic reticulum). After washing with PBS, images were imaged in phenol red-free DMEM. For transfected cells, the transfection protocol was as described in Example 11. Transfected HeLa cells were incubated for 2 hours with 5 μM Compound 8. Cells were washed three times with PBS, and the DMEM medium was changed twice at 30-minute intervals. During the second change, 1 μM ER-Tracker Green was added and incubated for 30 minutes. After washing with PBS, images were imaged in phenol red-free DMEM. Figure 7 Imaging results showed that compound 8 can achieve targeted imaging of the endoplasmic reticulum in living cells.

[0090] Example 11: Super-resolution imaging of the endoplasmic reticulum in living cells.

[0091] HeLa cells were pre-seeded in culture dishes to approximately 80% confluence, and then transiently transfected with the ER-SNAP plasmid using Lipofectamine 3000 and P3000 (Invitrogen) according to standard protocol. The ER-SNAP plasmid was designed based on the ER signal peptide KDEL sequence. Transfected HeLa cells were incubated with 5 μM compound 8 for 2 hours, washed three times with PBS, and then the DMEM medium was changed twice at 30-minute intervals. Finally, imaging was performed in phenol red-free DMEM. Figure 8 and Figure 9 The imaging results show that, compared to diffraction images, super-resolution reconstructed images can observe endoplasmic reticulum structures at higher resolutions.

[0092] In summary, the spontaneously scintillated near-infrared fluorophore of this invention possesses a pH-responsive mechanism and exhibits a thermodynamic equilibrium between its fluorescent (open-ring) and dark (spiro-ring) states. By altering the R1 and R4 groups located on this fluorophore, the near-infrared absorption / emission wavelength range and its pK of the molecule can be controlled. aBecause these probes possess optical switching properties, they can be applied to super-resolution imaging studies based on single-molecule localization. Compared to commonly used visible-light-excited dyes, the fluorescent probes in this invention have absorption wavelengths in the near-infrared region (700-800 nm), which significantly reduces the impact of phototoxicity during imaging and makes them suitable for imaging studies of live cells. The fluorescent probes in this invention possess excellent properties such as a large molar extinction coefficient, high fluorescence quantum yield, and good biocompatibility, enabling super-resolution imaging and dynamic tracking of mitochondria and endoplasmic reticulum in live cells. Furthermore, the fluorescent probes in this invention have advantages such as simple synthesis process, low cost, and high yield.

[0093] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them; although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications can still be made to the specific implementation of the present invention or equivalent substitutions can be made to some technical features without departing from the spirit of the technical solutions of the present invention, and all such modifications and substitutions should be covered within the scope of the technical solutions claimed in the present invention.

Claims

1. A self-scinding near-infrared fluorophore, characterized in that, Its molecular structure is shown in Formula 1): ； Formula 1) In the formula, R1 is ; R2 and R3 are independently methyl or ethyl, and R4 is... .

2. The spontaneously scintillated near-infrared fluorophore according to claim 1, characterized in that, R2 and R3 are ethyl groups, and R4 is an ethyl group. .

3. A method for preparing a spontaneously scintillated near-infrared fluorophore according to claim 1, characterized in that, Includes the following steps: Step 1: Under an inert atmosphere, compound A, benzotriazol-1-yl-oxytripyrrolylphosphide (PyBOP), an amine compound, and an acid-binding agent are dissolved in a solvent and reacted with stirring at room temperature to prepare compound B; wherein: the structural formula of compound A is... The structural formula of compound B is In the formula, R2 and R3 are independently methyl or ethyl; the amine compound is o-phenylenediamine; Step 2: Under an inert atmosphere and ice bath conditions, compound B is dissolved in a solvent, followed by the addition of glutaryl chloride and an acid-binding agent to trigger the reaction. After the reaction is completed, water is added to terminate the reaction. Step 3: Extract the reaction solution obtained in Step 2 to concentrate the organic layer and obtain compound C, which is the target compound of Formula 1).

4. The preparation method according to claim 3, characterized in that, In steps one and two, the inert atmosphere is nitrogen or argon, the solvent is anhydrous dichloromethane, and the acid-binding agent is triethylamine.

5. The preparation method according to claim 3, characterized in that, In step one, the molar ratio of compound A, PyBOP, amine compound, and acid-binding agent is 1:(1.2-1.5):(9-12):(9-12).

6. The preparation method according to claim 3, characterized in that, In step three, the extractants for the reaction solution are water and dichloromethane; compound C is obtained by silica gel column chromatography, with petroleum ether, ethyl acetate and triethylamine as the mobile phase.

7. The application of the spontaneously scintillated near-infrared fluorophore according to claim 1 in the preparation of a live-cell super-resolution imaging reagent.

8. The application according to claim 7, characterized in that, The live-cell super-resolution imaging reagent is based on single-molecule localization microscopy to achieve super-resolution structural reconstruction of mitochondria and endoplasmic reticulum, as well as time-lapse imaging of super-resolution dynamic changes.

Images