CN110836879B - Cell membrane long-time multicolor fluorescence imaging reagent and preparation method and application thereof - Google Patents
Cell membrane long-time multicolor fluorescence imaging reagent and preparation method and application thereof Download PDFInfo
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Abstract
The invention provides a long-time multicolor fluorescence imaging reagent for cell membranes, and a preparation method and application thereof. The cell membrane imaging reagent is prepared by a two-step method: (1) grafting a certain amount of polyethylene glycol-cholesterol molecules on the surface of a polyamide-amine dendrimer (PAMAM); (2) and grafting different fluorescent molecules on the product obtained in the first step to obtain the final membrane imaging reagent. The imaging reagent is simple to prepare, good in water dispersibility and biocompatibility, rapid in imaging, wide in applicability and long in imaging effect maintaining time. The fluorescent molecules in the imaging reagent are various in types, so that cell membrane imaging with different colors can be realized. In addition, the reagent can perform in-situ long-time imaging on cell membranes/walls of bacteria and fungi and cell membranes of epidermal cells of live zebra fish embryos in addition to excellent membrane imaging capability on various mammalian cells cultured in vitro.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a long-time multicolor fluorescence imaging reagent for cell membranes, and a preparation method and a use method thereof.
Background
The cell membrane has important physiological functions, not only can maintain the intracellular environment of stable metabolism of cells, but also can regulate and select substances to enter and exit the cells, ensures the relative stability of the intracellular environment, and enables various biochemical reactions to be orderly operated. The cell membrane is closely related to important vital activities such as cell attachment, migration, proliferation, endocytosis, apoptosis and signal transduction in function. The change of the state of the cell membrane can reflect the progress of various biochemical reactions in the cell and the change of the activity of the cell to a certain extent, so that the research on the structure and the function of the cell membrane can obtain important information about the behavior and the state of the cell. The cell membrane fluorescence imaging technology is the simplest and most important means in cell membrane research, and can be used for not only visually observing the structure of a cell membrane, but also dynamically tracking the change of the cell membrane so as to observe a plurality of important biological activities including exocytosis, pinocytosis, cell division and apoptosis.
The imaging effect of the current commonly used cell membrane fluorescent dyes (such as DiD family, FM family, CellMask and the like) is not ideal, and the method faces a plurality of problems of easy endocytosis by cells, insufficient fluorescence stability, incompatibility with immunofluorescence staining and the like. Secondly, the imaging effect of most of the current commercial cell membrane dyes can not be maintained for a long time, and the cell membrane dyes are very easy to be absorbed by endocytosis of cells or fall off from the cells, so that the requirement of long-time cell membrane dyeing observation can not be met.
In addition, currently commercially available membrane dyes have difficulty in achieving membrane/wall imaging of bacterial and fungal cells. Finally, most of the existing cell membrane dyes can only realize the imaging of in vitro cultured cells, but cannot realize the cell membrane imaging of in vivo cells (such as zebrafish embryonic epidermal cells). In conclusion, there is an urgent market demand for developing a cell membrane fluorescence probe that can image for a long time, has stable imaging effect and color diversity, and can realize in vivo layer cell membrane imaging.
Disclosure of Invention
The purpose of the invention is as follows: in order to solve the problems, the invention provides a cell membrane long-time multicolor fluorescence imaging reagent and a preparation method and application thereof. The imaging probe has good water dispersibility and biocompatibility, and can be quickly anchored on a cell membrane for a long time to realize quick and stable cell membrane imaging. In addition, the probe can also realize long-time multicolor imaging of the cell membrane of the zebra fish embryonic epidermal cell.
The technical scheme is as follows: in order to achieve the purpose, the invention discloses a cell membrane long-time multicolor fluorescence imaging reagent which is obtained by covalently modifying polyethylene glycol-cholesterol molecules and fluorescent dye molecules on the surface of polyamidoamine dendrimer PAMAM.
Wherein, the generation numbers of the polyamide-amine dendrimer PAMAM are the third generation, the fourth generation and the fifth generation. The molecular weight of the polyethylene glycol in the polyethylene glycol-cholesterol molecule is 500-10000.
Preferably, the modification density of the polyethylene glycol-cholesterol molecules is 10% -80% of the number of terminal amino groups of the PAMAM.
The fluorescent dye molecule is Tetramethyl Rhodamine Isothiocyanate (TRITC) or indocyanine dye. Wherein the indocyanine dye is selected from sulfo-Cy2, sulfo-Cy3, sulfo-Cy5, sulfo-Cy5.5, sulfo-Cy7, Cy2, Cy3, Cy5, Cy5.5 or Cy 7.
The invention also discloses a preparation method of the cell membrane long-time multicolor fluorescence imaging reagent, which comprises the following steps:
(1) weighing N-hydroxysuccinimide ester-polyethylene glycol-cholesterol and polyamide-amine dendrimer (PAMAM), respectively dissolving in dimethyl sulfoxide, mixing after dissolving, reacting at room temperature, purifying after the reaction is finished, and freeze-drying to obtain an imaging reagent precursor;
(2) dissolving the imaging reagent precursor prepared in the step (1) in a phosphate buffer solution, rapidly mixing with N-hydroxysuccinimide ester-indocyanine dye molecules (sulfo-Cy2 NHS ester, sulfo-Cy3NHS ester, sulfo-Cy 5NHS ester, sulfo-Cy5.5 NHS ester, sulfo-Cy7 NHS ester, Cy2 NHS ester, Cy3NHS ester, Cy5NHS ester, Cy5.5 NHS ester or Cy7 NHS ester), reacting at room temperature, purifying after the reaction is finished, and freeze-drying to obtain the indocyanine dye-based cell membrane long-time multicolor fluorescence imaging reagent (Cy is abbreviated as 2-PAMAM-PEG-Chol, Cy 3-PAMAM-PEG-ol, Cy5-PAMAM-PEG-Chol, Cy5.5-PAM-PEG-Chol or PAMAM 7-PAM-Chol);
or dissolving the imaging reagent precursor prepared in the step (1) in a sodium carbonate/sodium bicarbonate buffer solution, mixing with TRITC molecules, reacting at room temperature, purifying after the reaction is finished, and freeze-drying to obtain the TRITC-based long-time multicolor fluorescence imaging reagent for the cell membrane (abbreviated as TRITC-PAMAM-PEG-Chol).
Preferably, the molecular weight of the polyethylene glycol in the N-hydroxysuccinimide ester-polyethylene glycol-cholesterol molecule is 500-10000. Further preferably, the molecular weight of the polyethylene glycol is 1000-. Still more preferably, the polyethylene glycol has a molecular weight of 2000.
Preferably, the generation numbers of the PAMAM are the third generation, the fourth generation and the fifth generation. More preferably, the number of generations of the PAMAM is the fourth generation.
Preferably, the modification density of the N-hydroxysuccinimide ester-polyethylene glycol-cholesterol molecule is 10% -80% of the number of terminal amino groups of the PAMAM, and more preferably 50%.
In the step (1), the quantity ratio of the N-hydroxysuccinimide ester-polyethylene glycol-cholesterol to the PAMAM is 6.4-51.2: 1.
In the step (1), the reaction time is more than 4h, and the purification method comprises the following steps: and (3) dialyzing and purifying the reacted solution in dimethyl sulfoxide dialysate for more than 24h by using a dialysis bag with the molecular weight cutoff of 1000-10000, and then replacing the dialysate with ultrapure water to continue dialyzing and purifying for more than 24 h.
Preferably, in the step (2), the imaging reagent precursor is mixed with the N-hydroxysuccinimide ester-indocyanine dye molecules (sulfo-Cy2 NHS ester, sulfo-Cy3NHS ester, sulfo-Cy 5NHS ester, sulfo-Cy5.5 NHS ester, sulfo-Cy7 NHS ester, Cy2 NHS ester, Cy3NHS ester, Cy5NHS ester, Cy5.5 NHS ester or Cy7 NHS ester) in a ratio of 1:1 to 1:5, more preferably 1: 1.
In the step (2), the ratio of the imaging reagent precursor to the TRITC molecule is 1:1 to 1:5, and more preferably 1: 1.
In the step (2), the phosphate buffer solution is pH 6.8-8.0, the reaction time is more than 4h, and the purification method comprises the following steps: the solution after the reaction was purified in ultrapure water using a dialysis bag having a molecular weight cut-off of 1000-.
In the step (2), the sodium carbonate/sodium bicarbonate buffer solution is a sodium carbonate/sodium bicarbonate buffer solution with the pH value of 8.5-10.5, the reaction time is more than 4h, and the purification method comprises the following steps: the solution after the reaction was purified in ultrapure water using a dialysis bag having a molecular weight cut-off of 1000-.
Furthermore, the invention also provides application of the cell membrane long-time multicolor fluorescence imaging reagent in cell membrane imaging, and the reagent can realize rapid and long-time multicolor imaging of cell membranes of in vitro cultured cells and living epidermal cells. Wherein the cell membrane imaging of the living epidermal cells mainly refers to the cell membrane imaging of the zebra fish embryonic epidermal cells.
Has the advantages that: compared with the prior art, the invention has the following advantages:
(1) and (3) stable imaging for a long time: at present, most of commercialized cell membrane imaging fluorescent dyes can not realize long-time cell membrane imaging, and the imaging reagent prepared by the invention can realize a stable cell membrane imaging effect at least as long as 8 hours.
(2) The safety is good: the dye has low cytotoxicity, and can not affect normal physiological state of cell in cell membrane imaging process.
(3) Membrane/wall imaging of bacterial/fungal cells: the dyes enable excellent membrane/wall imaging of bacterial/fungal cells, whereas most commercial membrane dyes do not enable membrane/wall imaging of bacterial and fungal cells.
(4) In vivo imaging applications: compared with the commercialized cell membrane fluorescence imaging dye, the fluorescence probe provided by the invention can be used for imaging the cell membrane of the in vitro cultured cell and in situ imaging the cell membrane of the epidermal cell of the living zebra fish embryo, and has a wide in vivo imaging application prospect.
Drawings
FIG. 1 is a schematic representation of a synthetic cell membrane long-term multicolor fluorescence imaging reagent of the present invention;
FIG. 2 is a graph showing the staining effect of the reagent of the present invention on human normal lung cells (a: TRITC-PAMAM-PEG-Chol, b: Cy3-PAMAM-PEG-Chol, c: Cy 5-PAMAM-PEG-Chol);
FIG. 3 is a confocal fluorescence image of the reagent of the present invention (Cy5-PAMAM-PEG-Chol) with respect to normal human lung cells at different times after staining and washing;
FIG. 4 is a graph showing the toxicity evaluation of the reagents of the present invention (TRITC-PAMAM-PEG-Chol, Cy3-PAMAM-PEG-Chol, and Cy5-PAMAM-PEG-Chol) against human normal lung cells;
FIG. 5 is a graph showing the staining effect of the reagent of the present invention (TRITC-PAMAM-PEG-Chol) on E.coli cells;
FIG. 6 is a graph showing the effect of the reagent of the present invention (TRITC-PAMAM-PEG-Chol) on staining Trichoderma reesei cells;
FIG. 7 is a graph showing the effect of the reagent of the present invention (Cy5-PAMAM-PEG-Chol) on the staining of the cell membrane of the embryonic epidermis of zebra fish;
FIG. 8 is a diagram showing the effect of the reagent of the present invention (Cy5-PAMAM-PEG-Chol) on the confocal imaging taken 6h after the cell membrane of the embryonic epidermis of zebra fish is stained and washed.
Detailed Description
The invention is further elucidated with reference to the drawings and the specific examples.
In the following examples, N-hydroxysuccinimide ester-polyethylene glycol 2000-cholesterol, fourth-generation polyamidoamine dendrimer (PAMAM), tetramethylrhodamine isothiocyanate (TRITC), and N-hydroxysuccinimide ester-indocyanine dye molecules (sulfo-Cy 3NHS ester and sulfo-Cy 5NHS ester) were all commercially available products.
Example 1
The synthesis process of the cell membrane long-time multicolor fluorescence imaging reagent is as follows:
The reagent obtained by the above method is named as TRITC-PAMAM-polyethylene glycol-cholesterol (abbreviated as TRITC-PAMAM-PEG-Chol), and the schematic diagram of the above reaction is shown in FIG. 1.
Example 2
Similar to example 1, except that 27mg of N-hydroxysuccinimide ester-polyethylene glycol 2000-cholesterol was replaced with 43.2mg of N-hydroxysuccinimide ester-polyethylene glycol 2000-cholesterol in step 1, i.e., the mass ratio of N-hydroxysuccinimide ester-polyethylene glycol 2000-cholesterol to PAMAM was 51.2: 1. In step 2, 464 mug of TRITC molecules are replaced by 92.8 mug, namely the ratio of the imaging reagent precursor to the TRITC molecules is 1: 5.
Example 3
Similar to example 1, except that 27mg of N-hydroxysuccinimide ester-polyethylene glycol 2000-cholesterol was replaced with 5.4mg of N-hydroxysuccinimide ester-polyethylene glycol 2000-cholesterol in step 1, i.e., the mass ratio of N-hydroxysuccinimide ester-polyethylene glycol 2000-cholesterol to PAMAM was 6.4: 1. In step 2, 278.4 μ g of TRITC molecules was substituted for 92.8 μ g of TRITC molecules, i.e., the ratio of the imaging reagent precursor to the TRITC molecules was 1: 3.
Example 4
and 2, weighing 20mg of the imaging reagent precursor prepared in the step 1, dissolving the imaging reagent precursor in a phosphate buffer solution with the pH value of 7.4, quickly mixing the imaging reagent precursor with 161.5 mu g of sulfo-Cy3NHS ester (namely the ratio of the imaging reagent precursor to sulfo-Cy3NHS ester molecules is 1:1), reacting for more than 4 hours at room temperature, dialyzing and purifying for 24 hours in ultrapure water by using a dialysis bag with the molecular weight cutoff of 3500 after the reaction is finished, and freeze-drying to obtain the long-time cell membrane orange fluorescence imaging reagent.
The reagent obtained by the method is marked as Cy 3-PAMAM-polyethylene glycol-cholesterol (abbreviated as Cy 3-PAMAM-PEG-Chol).
Example 5
Similar to example 4, except that 27mg of N-hydroxysuccinimide ester-polyethylene glycol 2000-cholesterol was replaced with 43.2mg of N-hydroxysuccinimide ester-polyethylene glycol 2000-cholesterol in step 1, i.e., the mass ratio of N-hydroxysuccinimide ester-polyethylene glycol 2000-cholesterol to PAMAM was 51.2: 1. In step 2, 161.5 mu g of sulfo-Cy3NHS ester is replaced by 807.5 mu g of sulfo-Cy3NHS ester, namely the ratio of the imaging reagent precursor to the sulfo-Cy3NHS ester molecules is 1: 5.
Example 6
Similar to example 4, except that 27mg of N-hydroxysuccinimide ester-polyethylene glycol 2000-cholesterol was replaced with 5.4mg of N-hydroxysuccinimide ester-polyethylene glycol 2000-cholesterol in step 1, i.e., the mass ratio of N-hydroxysuccinimide ester-polyethylene glycol 2000-cholesterol to PAMAM was 6.4: 1. In step 2, 161.5 mu g of sulfo-Cy3NHS ester is replaced by 484.5 mu g of sulfo-Cy3NHS ester, namely the ratio of the imaging reagent precursor to the sulfo-Cy3NHS ester molecules is 1: 3.
Example 7
Similar to example 4, except that 161.5. mu.g of sulfo-Cy3NHS ester was replaced with 167.4. mu.g of sulfo-Cy 5NHS ester in step 2, i.e.the ratio of the imaging reagent precursor to the sulfo-Cy 5NHS ester molecules when mixed was 1:1 mass ratio.
The reagent obtained by the method is marked as Cy 5-PAMAM-polyethylene glycol-cholesterol (abbreviated as Cy 5-PAMAM-PEG-Chol).
Example 8
Similar to example 4, except that 27mg of N-hydroxysuccinimide ester-polyethylene glycol 2000-cholesterol was replaced with 43.2mg of N-hydroxysuccinimide ester-polyethylene glycol 2000-cholesterol in step 1, i.e., the mass ratio of N-hydroxysuccinimide ester-polyethylene glycol 2000-cholesterol to PAMAM was 51.2: 1. In step 2, 161.5. mu.g of sulfo-Cy3NHS ester is replaced by 837. mu.g of sulfo-Cy 5NHS ester, i.e., the ratio of the imaging reagent precursor to the sulfo-Cy 5NHS ester molecules is 1: 5.
Example 9
Similar to example 4, except that 27mg of N-hydroxysuccinimide ester-polyethylene glycol 2000-cholesterol was replaced with 5.4mg of N-hydroxysuccinimide ester-polyethylene glycol 2000-cholesterol in step 1, i.e., the mass ratio of N-hydroxysuccinimide ester-polyethylene glycol 2000-cholesterol to PAMAM was 6.4: 1. In step 2, 161.5 mu g of sulfo-Cy3NHS ester is replaced by 502.2 mu g of sulfo-Cy 5NHS ester, namely the ratio of the imaging reagent precursor to the sulfo-Cy 5NHS ester molecule is 1: 3.
Example 10
Observing the staining of the cell membrane of the human normal lung cell (HP) by TRITC-PAMAM-PEG-Chol, Cy3-PAMAM-PEG-Chol or Cy5-PAMAM-PEG-Chol prepared in examples 1, 4 and 7 is as follows:
after culturing HP cells in a 96-well plate for 24 hours, TRITC-PAMAM-PEG-Chol, Cy3-PAMAM-PEG-Chol or Cy5-PAMAM-PEG-Chol were dispersed in a cell culture medium at a concentration of 30. mu.g/mL and added to a 96-well plate containing HP cells (100. mu.L per well) at 37 ℃ with 5% CO2After incubation for 5min in the environment, the cells were washed with phosphate buffer and observed.
And (3) confocal fluorescence microscope imaging observation: TRITC-PAMAM-PEG-Chol, Cy3-PAMAM-PEG-Chol and Cy5-PAMAM-PEG-Chol fluoresce orange, orange and red respectively under 552nm, 552nm and 638nm laser excitation (the results correspond to a, b and c in FIG. 2, respectively). As can be seen, the cell membrane of each cell in the visual field was uniformly stained with the three reagents.
Example 11
The effect of imaging the cell membrane for a long time was observed using Cy5-PAMAM-PEG-Chol prepared in example 7 as follows:
after the HP cells were cultured in a 96-well plate for 24 hours, Cy5-PAMAM-PEG-Chol fluorescence imaging reagent was dispersed in a cell culture medium at a concentration of 30. mu.g/mL and added to the HP cells-containing 96-well plate (100. mu.L per well) at 37 ℃ with 5% CO2After incubation in the environment for 5min, washing with phosphate buffer, the imaging effect was observed at 0.5, 1, 2, 3, 5, 8 and 10h after washing.
The results are shown in FIG. 3. As can be seen, Cy5-PAMAM-PEG-Chol can still achieve stable imaging on cell membranes for at least 8 hours even after washing, and part of the imaging agent is not endocytosed or shed from the cells until 10 hours after washing.
Example 12
Evaluation examples 1,4 and 7, cytotoxicity of TRITC-PAMAM-PEG-Chol, Cy3-PAMAM-PEG-Chol, and Cy5-PAMAM-PEG-Chol, as follows: HP cells were arranged at 5X 104The fluorescence probes are inoculated into a 96-well plate at the density of one/mL, cultured for 24h, incubated with TRITC-PAMAM-PEG-Chol, Cy3-PAMAM-PEG-Chol and Cy5-PAMAM-PEG-Chol for 24h in a dark place, and finally evaluated for cytotoxicity by using an MTT detection method, and the results are shown in FIG. 4. The results of the experiment show no toxicity at the imaging concentration and only slight toxicity at higher concentrations (e.g., 100. mu.g/mL).
Example 13
Using the TRITC-PAMAM-PEG-Chol prepared in example 1 as an example, the staining of the cell membrane/wall of E.coli in gram-negative bacteria was observed as follows:
suspension culture of Escherichia coli in 37 deg.C shaking table to logarithmic growth phase (OD)6000.5), 1mL of the bacterial solution is centrifuged by a centrifuge at 12000rpm for 5min, the supernatant is discarded, and 100. mu.L of TRITC-PAMAM-PEG-Chol (30. mu.g/mL in physiological saline) is used for resuspension and precipitation to stain the bacteria. After staining for 5min, the stained bacterial liquid was centrifuged at 12000rpm for 5min by a centrifuge, the supernatant was discarded and then the pellet was resuspended in physiological saline, and then centrifuged again at 12000rpm for 5min by a centrifuge and then the supernatant was discarded, after which the pellet was resuspended in 100. mu.L of physiological saline. Finally, 10. mu.L of the resuspended bacterial solution is dropped on a glass slide and placed on a confocal microscope for observation by a 100-fold lens.
The results of confocal fluorescence microscope imaging observation are shown in fig. 5: TRITC-PAMAM-PEG-Chol emits orange fluorescence under the excitation of 552nm laser. As can be seen, the E.coli cell membranes/walls were stained uniformly in the visual field.
Example 14
Using the TRITC-PAMAM-PEG-Chol prepared in example 1 as an example, the staining of the cell membrane/wall of Trichoderma reesei in fungi was observed as follows:
the same procedure as in example 13.
The results of confocal fluorescence microscope imaging observation are shown in fig. 6: TRITC-PAMAM-PEG-Chol emits orange fluorescence under the excitation of 552nm laser. As can be seen, the cell membrane/wall of Trichoderma reesei was stained uniformly in the visual field.
Example 15
Using Cy5-PAMAM-PEG-Chol prepared in example 7 as an example, the staining of the epidermal cells of zebrafish embryos was observed as follows: selecting normally-developed zebra fish eggs to carry out a staining experiment, and when the zebra fish embryos develop to 60hpf, taking 1 embryo to place in the zebra fish culture solution containing 30 microgram/mLCy 5-PAMAM-PEG-Chol for staining for 10 min. Subsequently, the zebra fish embryos are washed by phosphate buffer, then the washed embryos are sucked into a 96-well plate, a small amount of MS222 anesthetic is added to anaesthetize the zebra fish, and finally, the 96-well plate filled with the zebra fish embryos is placed under a fluorescence confocal microscope for observation. Three-dimensional scanning fluorescence imaging is carried out on the zebra fish embryo by using a confocal microscope, and a three-dimensional zebra fish epidermal cell membrane fluorescence imaging picture is obtained by 3D reconstruction of the picture through software.
Confocal results show (figure 7) that Cy5-PAMAM-PEG-Chol can realize high-quality fluorescence imaging of cell membranes of embryonic epidermis of zebra fish.
Example 16
The long-term imaging effect of Cy5-PAMAM-PEG-Chol prepared in example 7 on zebra fish embryo staining after washing was observed, and the steps were as follows: selecting normally-developed zebra fish eggs to carry out a staining experiment, and when the zebra fish embryos develop to 60hpf, taking 1 embryo to place in the zebra fish culture solution containing 30 microgram/mLCy 5-PAMAM-PEG-Chol for staining for 10 min. And then washing the zebra fish embryo with a phosphate buffer, sucking the embryo which is washed and continuously cultured for 6 hours into a 96-well plate, adding a small amount of MS222 anesthetic to anaesthetize the zebra fish, and finally placing the 96-well plate filled with the zebra fish embryo under a fluorescence confocal microscope for observation. Three-dimensional scanning fluorescence imaging is carried out on the zebra fish embryo by using a confocal microscope, and a three-dimensional zebra fish epidermal cell membrane fluorescence imaging picture is obtained by 3D reconstruction of the picture through software.
The confocal result shows (figure 8) that after Cy5-PAMAM-PEG-Chol staining, washing with phosphate buffer solution and continuing culturing for 6 hours, the cell membrane imaging effect of the zebra fish epidermal cells is still good, which indicates that the imaging reagent has stable imaging capability of the living zebra fish epidermal cells.
Claims (10)
1. A long-time multicolor fluorescence imaging reagent for cell membranes is characterized in that the imaging reagent is obtained by covalently modifying polyethylene glycol-cholesterol molecules and fluorescent dye molecules on the surface of polyamidoamine dendrimer PAMAM.
2. The reagent for long-time multicolor fluorescence imaging of cell membranes according to claim 1, wherein the generation numbers of the polyamidoamine dendrimer PAMAM are the third generation, the fourth generation and the fifth generation.
3. The reagent for long-time multicolor fluorescence imaging of cell membranes according to claim 1, wherein the molecular weight of polyethylene glycol in the polyethylene glycol-cholesterol molecule is 500-10000.
4. The reagent for long-time multicolor fluorescence imaging of cell membranes according to claim 1, wherein the modification density of the polyethylene glycol-cholesterol molecules is 10% -80% of the number of terminal amino groups of PAMAM.
5. The reagent for long-time multicolor fluorescence imaging of cell membranes according to claim 1, wherein the fluorescent dye molecule is tetramethylrhodamine isothiocyanate TRITC or an indocyanine dye.
6. The method for preparing a reagent for long-time multicolor fluorescence imaging of cell membranes according to claim 1, comprising the steps of:
(1) weighing N-hydroxysuccinimide ester-polyethylene glycol-cholesterol and polyamide-amine dendrimer PAMAM, respectively dissolving in dimethyl sulfoxide, mixing after dissolving, reacting at room temperature, purifying after the reaction is finished, and freeze-drying to obtain an imaging reagent precursor;
(2) dissolving the imaging reagent precursor prepared in the step (1) in a phosphate buffer solution, quickly mixing with N-hydroxysuccinimide ester-indocyanine dye molecules, reacting at room temperature, purifying after the reaction is finished, and freeze-drying to obtain the cell membrane long-time multicolor fluorescence imaging reagent based on the indocyanine dye;
or dissolving the imaging reagent precursor prepared in the step (1) in a sodium carbonate/sodium bicarbonate buffer solution, mixing with TRITC, reacting at room temperature, purifying after the reaction is finished, and freeze-drying to obtain the TRITC-based cell membrane long-time multicolor fluorescence imaging reagent.
7. The process according to claim 6, wherein in the step (1), the reaction time is 4 hours or more, and the purification process comprises: and (3) dialyzing and purifying the reacted solution in dimethyl sulfoxide dialysate for more than 24h by using a dialysis bag with the molecular weight cutoff of 1000-10000, and then replacing the dialysate with ultrapure water to continue dialyzing and purifying for more than 24 h.
8. The process according to claim 6, wherein the phosphate buffer solution in step (2) has a pH of 6.8 to 8.0, the reaction time is 4 hours or more, and the purification process comprises: the solution after the reaction was purified in ultrapure water using a dialysis bag having a molecular weight cut-off of 1000-.
9. The preparation method according to claim 6, wherein in the step (2), the sodium carbonate/sodium bicarbonate buffer solution has a pH of 8.5 to 10.5, the reaction time is 4 hours or more, and the purification method comprises: the solution after the reaction was purified in ultrapure water using a dialysis bag having a molecular weight cut-off of 1000-.
10. Use of the cellular membrane prolonged polychromatic fluorescence imaging agent according to claim 1 for imaging cellular membranes.
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