Preparation method and application of cytoplasmic membrane covalent labeling fluorescent probe based on non-membrane permeability

Abstract

The invention relates to the technical field of fluorescent probes, and discloses a preparation method and application of a cytoplasmic membrane covalent labeling fluorescent probe based on non-membrane permeability, and the preparation method comprises the following steps: S1, synthesizing 4-(2, 3, 3-trimethyl-3H-indole-1-onium-1-yl) butane-1-sulfonate; s2, synthesizing a 2-halogen substituted N-((1E, 2Z)-3-9 (phenylamino) allylidene) phenylamine derivative; and S3, synthesizing a target product, namely the Sulfo-Cy5-X fluorescent probe. The novel cytoplasmic membrane fluorescent probe is prepared by adopting a membrane impermeability-based cytoplasmic membrane covalent labeling principle and introducing a sulfonic acid group with high water solubility and a covalent reaction site, so that a long-time and specific labeling function on the cytoplasmic membrane is realized; according to the method, the problems of short imaging time and low water solubility of a traditional fluorescent probe are solved, and a more ideal tool is provided for in-situ observation of dynamic change of the cytoplasmic membrane.

Description

Technical Field

[0001] This invention relates to the field of fluorescent probe technology, and in particular to a method for preparing and applying a non-membrane-permeable cell membrane covalently labeled fluorescent probe. Background Technology

[0002] In recent years, fluorescence imaging has been widely used in biomolecular detection, medical diagnosis, and other fields due to its convenient operation, high sensitivity, excellent photostability, and real-time detection capabilities. As a key structure of cells, the cell membrane not only maintains cell morphology and homeostasis as a physical barrier, but also regulates core life activities such as cell metabolism, proliferation, and differentiation through selective substance transport, signal transduction, cell recognition, and immune defense. To delve deeper into these dynamic processes, fluorescent probes have become an indispensable tool for studying the physiological mechanisms of the cell membrane.

[0003] Traditional fluorescent probes for cell membranes typically achieve specific labeling of the cell membrane through non-covalent mechanisms, such as the classic FM and Di series cell membrane probes. These probes often employ lipophilic long alkyl chains or introduce positive charges to form amphiphilic molecules, utilizing the principle of "like dissolves like" and electrostatic forces to achieve weak binding to the cell membrane, thereby displaying signals on the cell membrane surface. However, in biological applications such as washing and fixation, these probes still suffer from problems such as easy internalization by cells, short staining time, and decreased brightness after washing. Furthermore, dye aggregation caused by poor solubility can interfere with experimental results.

[0004] Based on this, a method for preparing and applying a non-membrane-permeable cell membrane covalently labeled fluorescent probe is proposed. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing and applying a non-membrane-permeable covalently labeled fluorescent probe for the cytoplasmic membrane in order to solve the above-mentioned problems.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A method for preparing a non-membrane-permeable cell membrane covalently labeled fluorescent probe includes the following steps:

[0008] S1: Synthesis of 4-(2,3,3-trimethyl-3H-indol-1-onthium-1-yl)butane-1-sulfonate;

[0009] S2: Synthesize 2-halogen-substituted N-((1E,2Z)-3-9(phenylamino)allyl)phenylammonium derivatives;

[0010] S3: Synthesize the target product, the Sulfo-Cy5-X fluorescent probe.

[0011] Preferably, the specific operation of step S1 is as follows: 2,3,3-trimethylindole and 1,4-butanesulfonate lactone are mixed in toluene solution, and then the mixture is refluxed. After the reaction is completed, a red solid appears on the wall of the round-bottom flask. After evaporation and drying, it is washed with ethyl acetate, and the final product is dried into a red powder to obtain 4-(2,3,3-trimethyl-3H-indole-1-onthium-1-yl)butane-1-sulfonate.

[0012] Preferably, in step S1, the ratio of 2,3,3-trimethylindole, 1,4-butyryl lactone, and toluene is 18.84 mmol: 58.07 mmol: 50 mL.

[0013] Preferably, the specific operation of step S2 is as follows: at 0°C, an ethanol solution of (Z)-2,3-dihalo-4-oxobut-2-enoic acid is added dropwise to an ethanol solution of aniline. In a 250 mL round-bottom flask, the mixture is heated under reflux for 4 h. After cooling to room temperature, the mixture is evaporated to dryness to obtain a precipitate. The precipitate is washed with ethyl acetate to obtain a yellow solid, which yields a 2-halogen-substituted N-((1E,2Z)-3-(phenylamino)allyl)phenylammonium derivative.

[0014] Preferably, in step S2, the ratio of (Z)-2,3-dihalo-4-oxobut-2-enoic acid, aniline, and ethanol is 19.40 mmol: 42.68 mmol: 100 mL.

[0015] Preferably, the specific operation of step S3 is as follows: an ethanol solution of 4-(2,3,3-trimethyl-3H-indol-1-onth-1-yl)butane-1-sulfonate (0.61 g, 2.07 mmol) is added dropwise to an ethanol solution of a mixture of 2-halogen-substituted N-((1E,2Z)-3-9(phenylamino)allyl)phenylammonium derivative, sodium acetate, and acetic anhydride. The mixture is heated under nitrogen in the dark and refluxed for 16 hours. After cooling to room temperature, the solvent in the mixture is removed by vacuum evaporation. Ethanol is added to dissolve the mixture. Ethyl acetate is added and a blue solid precipitates. The mixture is filtered under vacuum, washed with ethyl acetate, and dried to obtain a blue solid product.

[0016] Preferably, in step S3, the ratio of 4-(2,3,3-trimethyl-3H-indol-1-onthium-1-yl)butane-1-sulfonate, 2-halogen-substituted N-((1E,2Z)-3-9(phenylamino)allyl)phenylammonium derivative, sodium acetate, acetic anhydride, and ethanol is 20 mmol: 10 mmol: 20 mmol: 5 mL: 100 mL.

[0017] Preferably, the fluorescent probe is a Sulfo-Cy5-X fluorescent probe, the structural formula of which is shown below:

[0018]

[0019] Preferably, the fluorescent probe prepared by the method described above, which is based on a non-membrane-permeable covalently labeled fluorescent probe for cell membranes, is used in dynamic tracking of cell membranes.

[0020] In summary, due to the adoption of the above technical solution, the beneficial effects of the present invention are:

[0021] 1. This application is the first to employ the principle of covalent labeling of the cell membrane based on its membrane impermeability. A novel fluorescent probe for the cell membrane (Sulfo-Cy5-X) is prepared by introducing highly water-soluble sulfonic acid groups and covalent reaction sites, achieving long-term, specific labeling of the cell membrane. This method overcomes the problems of short imaging time and low water solubility inherent in traditional fluorescent probes, providing a more ideal tool for in-situ observation of dynamic changes in the cell membrane. This technology has significant practical application value in life science research and clinical medicine. Attached Figure Description

[0022] Figure 1 The photophysical properties of Sulfo-Cy5-Br and its covalent reaction with BSA provided in the embodiments of the present invention are shown. (A) UV-Vis absorption spectrum of aqueous solution of Sulfo-Cy5-Br (10.0 μM);

[0023] (B) Fluorescence emission spectrum of Sulfo-Cy5-Br in aqueous solution (10.0 μM);

[0024] (C) Fluorescence spectra of Sulfo-Cy5-Br (10.0 μM) in PBS before and after the addition of BSA (50.0 mg / mL);

[0025] (D) SDS-PAGE analysis of Sulfo-Cy5-Br (20.0 μM). Dye-BSA fluorescent conjugate (line 1) and BSA (line 2) stained with Coomassie Brilliant Blue (left, (a)) and gel fluorescence scanning imaging (right, (b)). (EX = 640 nm, EM = 666 nm);

[0026] Figure 2 The image shows laser scanning confocal microscopy images of HeLa cells stained with Sulfo-Cy5-Br (20.0 μM, 20 min) at different time points according to an embodiment of the present invention. (Scale bar)

[0027] =10μm;

[0028] Figure 3 A laser scanning confocal microscopy image of HeLa cell membrane damage monitored under specific conditions using Sulfo-Cy5-Br dye (20.0 μM, 20 min) according to an embodiment of the present invention is shown. Scale bar = 10 μm;

[0029] Figure 4 The images shown are laser scanning confocal microscopy images of HeLa cells at different time points after co-incubation with Sulfo-Cy5-Br (20.0 μM, 20 min) and lipopolysaccharide (400 μg) according to embodiments of the present invention. Scale bar = 10 μm. Detailed Implementation

[0030] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0031] Please see Figures 1-4 The present invention provides a technical solution:

[0032] A cell membrane covalently labeled fluorescent probe based on membrane impermeability, its preparation method, and its application. The fluorescent probe is a Sulfo-Cy5-X fluorescent probe, and its structural formula is shown below:

[0033]

[0034] The Sulfo-Cy5-X fluorescent probe is based on the molecular framework of Cy5 near-infrared anthocyanin fluorescent dye. The introduction of two sulfonate groups significantly enhances the probe's water solubility and imparts a negative charge. This negative charge effectively prevents the probe molecules from penetrating the cell membrane via osmosis. Simultaneously, the introduction of halogen atoms provides sites for covalent reactions with the cell membrane surface proteome, enabling rapid binding to the cell membrane surface. The heavy atom effect of the halogen atoms further reduces the fluorescence background, ensuring high-fidelity imaging of the cell membrane. Furthermore, based on the above design strategy, this series of probe molecules can maintain a long-term and stable presence on the cell membrane.

[0035] Based on the above design principles, this invention designs a cell membrane covalently labeled fluorescent probe based on membrane impermeability.

[0036] This invention also provides a method for preparing a covalently labeled fluorescent probe for cell membranes based on membrane impermeability, comprising the following steps:

[0037] S1: Synthesis of 4-(2,3,3-trimethyl-3H-indol-1-onthium-1-yl)butane-1-sulfonate;

[0038] S2: Synthesize 2-halogen-substituted N-((1E,2Z)-3-9(phenylamino)allyl)phenylammonium derivatives;

[0039] S3: Synthesize the target product, the Sulfo-Cy5-X fluorescent probe.

[0040] The invention will be further described in detail below through specific embodiments.

[0041] Example 1:

[0042] This invention provides a method for preparing a cell membrane covalently labeled fluorescent probe based on membrane impermeability. The structural formula of the fluorescent probe Sulfo-Cy5-Br is shown below:

[0043]

[0044] The synthetic route is shown below:

[0045]

[0046] The preparation method of the Sulfo-Cy5-Br fluorescent probe specifically includes the following steps:

[0047] S1: Synthesis of 4-(2,3,3-trimethyl-3H-indol-1-onthiol-1-yl)butane-1-sulfonate: 2,3,3-trimethylindole (3.00 g, 18.84 mmol) and 1,4-butyrylolactone (7.90 g, 58.07 mmol) were mixed in 50 mL of toluene solution, and the mixture was then refluxed. After the reaction was complete, a red solid appeared on the wall of the round-bottom flask. After rotary evaporation to dryness, the solid was washed several times with ethyl acetate, and the final product was dried as a red powder. Yield: 83%.

[0048] Product NMR data: 1 H NMR(400MHz, Methanol-d4)δ7.94(d,J=5.2Hz,1H),7.75(d,J=3.3Hz,1H),7.68-7.61(m,2H),4.56(t ,J=7.8Hz,2H),2.90(t,J=7.1Hz,2H),2.15(p,J=8.2,7.5Hz,2H),1.95(p,J=7.3Hz,2H),1.61(s,6H). 13CNMR(101MHz,Methanol-d4)δ196.74,142.00,141.21,130.36,129.16,123.17,115.29,55.01,49.76,47.47,26.02,21.85,21.36.

[0049] S2: Synthesis of N-((1E,2Z)-2-bromo-3-(phenylamino)allyl)benzamide: At 0 °C, an ethanol solution of (Z)-2,3-dibromo-4-oxobut-2-ene (5.00 g, 19.40 mmol) was slowly added dropwise to an ethanol solution of aniline (3.97 g, 42.66 mmol). The mixture was heated under reflux for 4 h in a 250 mL round-bottom flask, cooled to room temperature, and evaporated to dryness to obtain a precipitate. The precipitate was washed repeatedly with ethyl acetate to give a yellow solid. Yield: 86%.

[0050] Product NMR data: 1 H NMR (400MHz, Methanol-d4) δ9.16 (s, 2H), 7.61 (d, J = 7.7Hz, 4H), 7.52 (t, J = 7.9Hz, 4H), 7.38 (t, J = 7.3Hz, 2H). 13 C NMR (101MHz, Methanol-d4) δ157.05,138.32,129.70,127.29,119.34,89.12.

[0051] S3: Synthesis of 4-(2-((1E,3Z)-3-bromo-5-((E)-3,3-dimethyl-1-(4-sulfonylbutyl)indololin-2-ylidene)pent-1,3-dien-1-yl)-3,3-dimethyl-3H-indol-1-onthium-1-yl)butane-1-sulfonic acid: An ethanol solution of 4-(2,3,3-trimethyl-3H-indol-1-onthium-1-yl)butane-1-sulfonate (0.98 g, 3.32 mmol) was slowly added dropwise to an ethanol solution of a mixture of N-((1E,2Z)-2-bromo-3-(phenylamino)allyl)benzamide (0.50 g, 1.31 mmol), sodium acetate (0.41 g, 5.00 mmol), and acetic anhydride (5 mL, catalytic amount), and heated under nitrogen at reflux for 16 hours in the dark. After cooling to room temperature, the solvent in the mixture was removed by vacuum evaporation. A small amount of ethanol was added to dissolve the mixture, and an appropriate amount of ethyl acetate was added, precipitating a blue solid. The mixture was then filtered under vacuum, washed with ethyl acetate, and dried to give the blue solid product. Yield: 63%.

[0052] Product NMR data: 1H NMR (400MHz, Methanol-d4) δ8.43(d,J=13.3Hz,2H),7.55(d,J=7.5Hz,2H),7.46(d,J=4.2Hz,4H),7.33-7.31( m,2H),6.51(d,J=13.2Hz,2H),4.25(t,J=7.2Hz,4H),2.93(t,J=7.3Hz,4H),2.07-1.97(m,8H),1.77(s,12H). 13 C NMR (101MHz, Methanol-d4) δ174.90,149.83,141.97,141.56,128.55,125.63,122.15,115.96,111.42,104.95,102.43.

[0053] Performance Characterization

[0054] The Sulfo-Cy5-Br fluorescent probe prepared in the first example was subjected to UV-Vis absorption and fluorescence emission spectra measurements. The UV-Vis absorption spectrum of its aqueous solution is shown below. Figure 1 As shown in Figure a: Under sunlight, the Sulfo-Cy5-Br fluorescent probe solution appears blue, with a maximum absorption wavelength of 640 nm. Its fluorescence emission spectrum test results are as follows... Figure 1 As shown in b: its fluorescence emission spectrum was measured, and the maximum emission wavelength of the Sulfo-Cy5-Br fluorescent probe aqueous solution was 666 nm.

[0055] To evaluate the fluorescence response of Sulfo-Cy5-Br dye to cell membrane surface proteomes, we investigated its interaction with bovine serum albumin (BSA) in phosphate-buffered saline (PBS). The results showed that Sulfo-Cy5-Br exhibited weak fluorescence under UV irradiation, but its fluorescence intensity significantly increased upon the addition of BSA (e.g., ...). Figure 1 (As shown). Specifically, the fluorescence of Sulfo-Cy5-Br increased by approximately 3.1-fold after the addition of BSA. This indicates that the probe can exhibit a stronger fluorescence signal through a covalent reaction with the protein. Figure 1 As shown in Figure d, we have demonstrated that this covalent interaction between the Sulfo-Cy5-Br dye and proteins is reliable and effective. This discovery provides an important experimental basis for studying protein dynamics on the cell membrane surface. To verify the performance of the membrane-impermeable covalently labeled fluorescent probe Sulfo-Cy5-X in long-term stable imaging of the cytoplasmic membrane, we used Sulfo-Cy5-Br to perform membrane imaging experiments on HeLa cells at different time points, as shown in Figure d. Figure 2As shown in the figure. Real-time monitoring revealed that this dye can stably label and continuously observe the cell membrane, providing an important experimental basis for studying the dynamic changes of the cell membrane.

[0056] This invention further demonstrates the application of the cell membrane covalently labeled fluorescent probe Sulfo-Cy5-X, based on membrane impermeability, in cell membrane damage recognition and programmed cell death processes.

[0057] The following will provide a detailed explanation through specific application examples.

[0058] Application Example 1

[0059] Sulfo-Cy5-Br fluorescent probe monitors HeLa cell membrane damage.

[0060] Specific implementation methods:

[0061] The human cervical cancer cell line HeLa was routinely cultured at 37°C and 5% CO2 in Dulbecco's Modified Eagle Medium (DMEM; GENOM, Zhejiang, China), which was supplemented with 10% fetal bovine serum (FBS; ExCellBio, Shanghai, China) and 1% penicillin / streptomycin (Gibco, Gaithersburg, Maryland, USA).

[0062] We treated cells with 2,2'-azobis(2-methylpropanediamine) dihydrochloride (AAPH) and paraformaldehyde (PFA), respectively. AAPH, as a free radical inducer, can induce oxidative stress in cells, ultimately leading to apoptosis; while PFA is a commonly used biological fixative that can fix protein structures and maintain the original morphology and structure of tissues.

[0063] After chemically inducing varying degrees of cell membrane damage, the cells were incubated with 20.0 μM Sulfo-Cy5-Br at 22°C for 20 min. Fluorescence imaging was then performed using a Leica TCS SP5 laser confocal scanning microscope (Germany) with an excitation wavelength of 633 nm. Figure 3As shown, after treating HeLa cells with AAPH (100mM) for 90 min, less fluorescence signal was observed inside the cells, indicating a relatively small degree of cell membrane damage. Subsequently, after treating HeLa cells with AAPH (100mM) for 90 min and then fixing them with PFA for 30 min, it was observed that the intensity of the intracytoplasmic fluorescence signal tended to increase when cell permeability changed. Specifically, when cells approached death, the permeability of the nuclear membrane changed, causing the nucleus to light up. In conclusion, because the Sulfo-Cy5-Br dye itself is non-membrane permeable, it is sensitive to chemically induced cell damage or changes in cell membrane permeability. Therefore, the degree of cell membrane damage can be visually distinguished using a microscope.

[0064] Application Example 2

[0065] The Sulfo-Cy5-Br fluorescent probe monitors the process of cell necrosis and apoptosis.

[0066] The specific implementation method and instrument requirements are the same as in Application Example 1.

[0067] Here, we induced programmed cell death in HeLa cells using LPS, a process that typically takes several hours. Based on the long-term stability of the Sulfo-Cy5-Br fluorescent probe, we were able to utilize its real-time in-situ labeling on the cell membrane to achieve dynamic monitoring of this process throughout. In the experiment, LPS (400 μg / mL) was mixed with Sulfo-Cy5-Br (20.0 μM) and co-incubated with HeLa cells. Fluorescence imaging was performed under a Leica TCS SP5 laser confocal scanning microscope at an excitation wavelength of 633 nm. The results are as follows: Figure 4 As shown, during co-incubation for 2 hours, the cells began to swell overall, with their membrane morphology approaching a spherical shape. After 5 hours, the cell membrane permeability gradually increased, followed by a weak fluorescent signal in the cell nucleus. Notably, this process lasted for 3 hours, but no cell swelling, blistering, or rupture was observed, and the cell membrane remained intact, indicating that this was a necrotizing apoptotic process. Application examples 1-2 further validated the advantages of the Sulfo-Cy5-X series of fluorescent probes in long-term real-time monitoring of cell membrane contour function, providing reliable technical support for cell research in the field of biology.

[0068] This fluorescent probe achieves strong membrane anchoring through a covalent reaction with cell membrane surface proteins. It remains stably bound to the cell membrane before and after washing, with its fluorescence intensity remaining essentially unchanged, thus enabling long-term cell membrane imaging. The sulfonic acid group in this fluorescent probe not only endows it with excellent membrane impermeability but also provides advantages such as high water solubility and low cytotoxicity, which are beneficial for cellularization and in vivo imaging applications. This fluorescent probe plays an important tool role in the life sciences field, providing strong support for related research.

[0069] The above description of the embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for preparing a non-membrane-permeable cell membrane covalently labeled fluorescent probe, characterized in that, Includes the following steps: S1: Synthesis of 4-(2,3,3-trimethyl-3H-indol-1-onthium-1-yl)butane-1-sulfonate; S2: Synthesize 2-halogen-substituted N-((1E,2Z)-3-9(phenylamino)allyl)phenylammonium derivatives; S3: Synthesize the target product, the Sulfo-Cy5-X fluorescent probe.

2. The method for preparing a non-membrane-permeable cell membrane covalently labeled fluorescent probe according to claim 1, characterized in that, The specific operation of step S1 is as follows: 2,3,3-trimethylindole and 1,4-butanesulfonate lactone are mixed in toluene solution, and then the mixture is refluxed. After the reaction is completed, a red solid appears on the wall of the round-bottom flask. After evaporation and drying, it is washed with ethyl acetate. The final product is dried into a red powder to obtain 4-(2,3,3-trimethyl-3H-indole-1-onthium-1-yl)butane-1-sulfonate.

3. The method for preparing a non-membrane-permeable covalently labeled fluorescent probe for cell membranes according to claim 2, characterized in that, In step S1, the ratio of 2,3,3-trimethylindole, 1,4-butyryl lactone, and toluene is 18.84 mmol: 58.07 mmol: 50 mL.

4. The method for preparing a non-membrane-permeable cell membrane covalently labeled fluorescent probe according to claim 1, characterized in that, The specific operation of step S2 is as follows: at 0°C, the ethanol solution of (Z)-2,3-dihalo-4-oxobut-2-enoic acid is added dropwise to the ethanol solution of aniline. In a 250 mL round-bottom flask, the mixture is heated under reflux for 4 h. After cooling to room temperature, it is dried by rotary evaporation to obtain a precipitate. The precipitate is washed with ethyl acetate to obtain a yellow solid, which yields a 2-halogen-substituted N-((1E,2Z)-3-(phenylamino)allyl)phenylammonium derivative.

5. The method for preparing a non-membrane-permeable cell membrane covalently labeled fluorescent probe according to claim 4, characterized in that, In step S2, the ratio of (Z)-2,3-dihalo-4-oxobut-2-enoic acid, aniline, and ethanol is 19.40 mmol: 42.68 mmol: 100 mL.

6. The method for preparing a non-membrane-permeable cell membrane covalently labeled fluorescent probe according to claim 1, characterized in that, The specific operation of step S3 is as follows: an ethanol solution of 4-(2,3,3-trimethyl-3H-indol-1-onth-1-yl)butane-1-sulfonate (0.61 g, 2.07 mmol) is added dropwise to an ethanol solution of a mixture of 2-halogen-substituted N-((1E,2Z)-3-9(phenylamino)allyl)phenylammonium derivative, sodium acetate, and acetic anhydride. The mixture is heated under nitrogen in the dark and refluxed for 16 hours. After cooling to room temperature, the solvent in the mixture is removed by vacuum evaporation. Ethanol is added to dissolve the mixture. Ethyl acetate is added and a blue solid precipitates. The mixture is filtered under vacuum, washed with ethyl acetate, and dried to obtain a blue solid product.

7. The method for preparing a non-membrane-permeable covalently labeled fluorescent probe for cell membranes according to claim 6, characterized in that, In step S3, the ratio of 4-(2,3,3-trimethyl-3H-indol-1-onth-1-yl)butane-1-sulfonate, 2-halogen-substituted N-((1E,2Z)-3-9(phenylamino)allyl)phenylammonium derivative, sodium acetate, acetic anhydride, and ethanol is 20 mmol: 10 mmol: 20 mmol: 5 mL: 100 mL.

8. The method for preparing a non-membrane-permeable cell membrane covalently labeled fluorescent probe according to claim 1, characterized in that, The fluorescent probe is the Sulfo-Cy5-X fluorescent probe, and its structural formula is shown below:

9. The application of the fluorescent probe prepared by the method for preparing a non-membrane-permeable cell membrane covalently labeled fluorescent probe according to claim 1 in cell membrane dynamic tracking.

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