CN109735326B

CN109735326B - Fluorescent probe, preparation method thereof and super-resolution imaging method

Info

Publication number: CN109735326B
Application number: CN201811529076.8A
Authority: CN
Inventors: 吴长锋; 刘志贺; 刘洁; 孙泽洲
Original assignee: Southern University of Science and Technology
Current assignee: Shenzhen Judian Biotechnology Co Ltd
Priority date: 2018-12-13
Filing date: 2018-12-13
Publication date: 2021-11-16
Anticipated expiration: 2038-12-13
Also published as: CN109735326A

Abstract

The invention relates to a fluorescent probe, a preparation method thereof and a super-resolution imaging method. The material of the fluorescent probe comprises semiconductor polymer quantum dots and fluorescent dye, wherein the semiconductor polymer quantum dots are donors of fluorescence resonance energy transfer, the fluorescent dye is an acceptor of the fluorescence resonance energy transfer, and the fluorescent probe can generate fluorescence scintillation. Based on the adjustment of a fluorescence resonance energy transfer mechanism, the donor semiconductor polymer quantum dots can transfer energy to the acceptor fluorescent dye, so that the fluorescence intensity of the fluorescent dye is enhanced, and the semiconductor polymer quantum dots are quenched by fluorescence, thereby realizing the random fluorescence scintillation of the fluorescent probe.

Description

Fluorescent probe, preparation method thereof and super-resolution imaging method

Technical Field

The invention relates to the field of fluorescence imaging, in particular to a fluorescent probe, a preparation method thereof and a super-resolution imaging method.

Background

Organisms are composed of complex cellular processes and persist in their natural environment. To further appreciate the biological processes in these complex environments, scientists have developed a variety of biological imaging techniques. Among these techniques, bioluminescence imaging has been widely developed due to simple imaging conditions and compatibility with biological samples. However, the conventional fluorescence imaging technology is limited by the optical diffraction limit, and cannot distinguish the spatial structure below 200nm, which causes obstacles to the research of the biological process of the subcellular structure. The super-resolution fluorescence microscope technology breaks through the limitation of the traditional optical diffraction on the imaging resolution, and can obtain complex biological processes such as cell dynamics under the nanoscale. Super-resolution imaging techniques include stimulated emission depletion microscopy (STED), light activated positioning microscopy (PALM), random optical reconstruction microscopy (STORM), structured light illumination microscopy (SIM), and super-resolution optical fluctuation imaging (SOFI). In addition to improvements and upgrades to traditional wide-field fluorescence microscope frameworks, typical super-resolution imaging microscopy techniques typically rely on the photophysical properties of the fluorescent probe material. The SOFI is an emerging super-resolution imaging technology first proposed by t.derringger in 2009, and has become the mainstream super-resolution imaging method due to its balanced temporal and spatial resolution, as well as background reduction capability and simple optical setup. The SOFI is an imaging technique that achieves improved resolution based on random fluctuations in fluorescence intensity of mutually independent fluorescent chromophores. The fluorescent material only needs to generate the fluctuation of the fluorescence intensity under the laser irradiation without having the state transition of OFF-ON. Originally, fluorescent scintillating quantum dots (Qdots) were used in SOFI imaging to improve their algorithms and imaging levels. Later, fluorescent proteins were used for the SOFI study. However, the disadvantage of low fluorescence intensity of Qdots and fluorescent proteins, the excitation light power has to be increased during imaging, which causes great damage to the biological sample.

At the beginning of the development of the SOFI nanomicroscope, inorganic quantum dots (QD525, QD625, QD705, etc.) were used as fluorescent probes. However, even through various modifications or alterations, the biological toxicity caused by heavy metal ions in the commonly used Qdots is still a concern. For live cell SOFt super-resolution microscopic imaging of fluorescent protein, Peter Dedecker in 2012 first proposed the utilization of fluorescent protein (Dronpa) and red reversible light-emitting fluorescent protein to realize two-color live cell SOFI super-resolution microscopic imaging. At present, RSFPs are single in variety, the number of fluorescence photons of commonly used green RSFPs Dronpa is low, and the optical stability needs to be improved.

Organic dyes are the most widely used probes in fluorescence imaging. Single dye molecules exhibit scintillation due to intersystem crossing to the dark triplet state, but the use of dyes in SOFI encounters several challenges: (1) photobleaching of dyes under continuous irradiation limits the acquisition time, but the SOFI algorithm requires recording a section of fluorescent signal that fluctuates with time; (2) the low signal-to-noise ratio of the dye limits the image quality of the SOFI; (3) the typical intersystem crossing rate of organic dyes results in their "on/off" blinking on the microsecond timescale, whereas most acquisitions of SOFIs are limited to the millisecond timescale. The dye-loaded latex or the dye-loaded silica nanoparticles exhibit better brightness and photostability than single dye molecules. However, the fluorescence of the dye-loaded nanoparticles is derived from dye molecule clusters, and the fluctuation of different dye molecules can compensate each other to make the dye molecules have no fluorescence flicker characteristics. Therefore, the application of fluorescent dyes in high-order SOFI nano-imaging remains very challenging.

The polymer quantum dots (Pdots) are widely applied to a fluorescence imaging tracing analysis technology as a new generation of excellent biological fluorescent probes. Pdots with a particle size greater than 15nm exhibit stable fluorescence kinetics without significant light flicker, whereas small Pdots often observe light flicker when the size is reduced below 10nm, but the fluorescence brightness still does not allow for higher contrast fluorescence images.

Disclosure of Invention

Accordingly, it is necessary to provide a fluorescent probe with adjustable fluorescence blinking characteristics and high fluorescence brightness.

In addition, a preparation method of the fluorescent probe and a super-resolution imaging method based on fluorescence resonance energy transfer regulation are also provided.

A fluorescent probe is made of materials including semiconductor polymer quantum dots and fluorescent dye, wherein the semiconductor polymer quantum dots are donors of fluorescence resonance energy transfer, the fluorescent dye is acceptors of the fluorescence resonance energy transfer, and the fluorescent probe can generate fluorescence scintillation.

In one embodiment, the semiconducting polymer quantum dot comprises at least one of poly [9, 9-dioctylfluorenyl-2, 7-diyl) ] and poly [ 2-methoxy-5- (2-ethylhexyloxy) -1,4- (1-cyanoethylene-1, 4-phenyl) ] and the fluorescent dye comprises at least one of coumarin6 and 2,9,16, 23-tetra-tert-butyl-29H, 31H phthalocyanine.

In one embodiment, the weight ratio of the semiconductor polymer quantum dots to the fluorescent dye is 100: 0.5-100: 1.

In one embodiment, the material of the fluorescent probe further comprises styrene maleic anhydride copolymer.

In one embodiment, the weight ratio of the styrene maleic anhydride copolymer to the semiconductor polymer quantum dots is 1:2 to 1: 20.

A preparation method of a fluorescent probe comprises the following steps:

mixing semiconductor polymer quantum dots, a fluorescent dye and an organic solvent to obtain a precursor solution, wherein the semiconductor polymer quantum dots are donors of fluorescence resonance energy transfer, and the fluorescent dye is an acceptor of the fluorescence resonance energy transfer;

injecting the precursor solution into water with the volume ratio of the precursor solution to the precursor solution of 5: 1-20: 1 under the ultrasonic oscillation condition to obtain an ultrasonic solution;

removing the organic solvent from the sonicated solution to obtain a residue;

and filtering the residue to obtain the fluorescent probe.

In one embodiment, the concentration of the semiconductor polymer quantum dots in the precursor solution is 100mg/L, and the concentration of the fluorescent dye is 0.5 mg/L-1 mg/L.

A super-resolution imaging method based on fluorescence resonance energy transfer regulation comprises the following steps:

carrying out subcellular structure labeling on a fluorescent probe to obtain a label, wherein the fluorescent probe comprises semiconductor polymer quantum dots and fluorescent dye, the semiconductor polymer quantum dots are donors of fluorescence resonance energy transfer, the fluorescent dye is an acceptor of the fluorescence resonance energy transfer, and the fluorescent probe can generate fluorescence scintillation;

capturing a fluorescence image of the marker;

and performing cumulant analysis on the fluorescence image.

In one embodiment, the step of performing cumulative analysis on the fluorescence image comprises: and sequentially carrying out second-order cumulant analysis, third-order cumulant analysis and fourth-order cumulant analysis on the fluorescence image.

In one embodiment, the number of frames for capturing the fluorescent image of the marker is 200 to 1000 frames.

The material of the fluorescent probe comprises semiconductor polymer quantum dots and fluorescent dye, wherein the semiconductor polymer quantum dots and the fluorescent dye are respectively a donor and an acceptor of fluorescence resonance energy transfer, so that under the regulation and control of a fluorescence resonance energy transfer mechanism, the donor semiconductor polymer quantum dots can transfer energy to the acceptor fluorescent dye, the fluorescence intensity of the fluorescent dye is enhanced, the semiconductor polymer quantum dots are quenched by fluorescence, and the random fluorescence scintillation of the fluorescent probe is realized.

Drawings

FIG. 1 is a flow chart of a method for preparing a fluorescent probe according to an embodiment;

FIG. 2 is a flow chart of a super-resolution imaging method according to an embodiment;

FIG. 3-a is the particle size distribution diagram of PFO-Coumarin6Pdots in example 1, and FIG. 3-b is the particle size distribution diagram of CNPPV-NIR695Pdots in example 2;

FIG. 4-a shows absorption and emission spectra of PFO and Coumarin6, and FIG. 4-b shows absorption and emission spectra of CNPPV and NIR 695;

FIG. 5-a is the single particle emission spectrum of PFO-Coumarin6Pdots in example 1, and FIG. 5-b is the single particle emission spectrum of CNPPV-NIR695Pdots in example 2;

FIG. 6-a is the absorption spectrum and the emission spectrum of PFO-Coumarin6Pdots in example 1, and FIG. 6-b is the absorption spectrum and the emission spectrum of CNPPV-NIR695Pdots in example 2;

FIG. 7-a shows the fluorescence traces of pure PFO and PS-Coumarin6, and FIG. 7-b shows the fluorescence traces of pure CNPPV and PS-NIR 695;

FIG. 8-a is the fluorescence trace of PFO-Coumarin6Pdots in example 1, and FIG. 8-b is the fluorescence trace of CNPPV-NIR695Pdots in example 2;

FIG. 9 is a fluorescence resonance mechanism diagram of a fluorescent probe according to an embodiment;

FIG. 10-a, FIG. 10-b, FIG. 10-c and FIG. 10-d are respectively wide-field fluorescence imaging, 2-order SOFI imaging, 3-order SOFI imaging and 4-order SOFI imaging of PFO-Coumarin6Pdots in example 1;

FIG. 11-a is the intensity curves of the wide-field image and the different-order images of PFO-Coumarin6Pdots in example 1, and FIG. 11-b is the full width at half maximum statistic of the wide-field image and the different-order images of PFO-Coumarin6Pdots in example 1;

FIGS. 12-a and 12-c are wide field fluorescence images of microtubules labeled with PFO-Coumarin6 from example 1 in COS-7 cells; FIGS. 12-b and 12-d are four-stage SOFI nanoimaging of the fluorescence images of FIGS. 12-a and 12-c, respectively; FIGS. 12-e and 12-f are enlarged views of the area images indicated by the white boxes labeled "I" in FIGS. 12-a and 12-b, respectively; FIGS. 12-g and 12-h are enlarged views of the area images indicated by the white boxes labeled "II" in FIGS. 12-c and 12-d, respectively;

FIG. 13-a is a graph showing a fluorescence intensity distribution of a microtube indicated by a white arrow in FIGS. 12-e and 12-f, and FIG. 13-b is a graph showing a fluorescence intensity distribution of a microtube indicated by a white arrow in FIGS. 12-g and 12-h.

Detailed Description

In order that the invention may be more fully understood, reference will now be made to the following description taken in conjunction with the accompanying drawings. The detailed description sets forth the preferred embodiments of the invention. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

The fluorescent probe of an embodiment is made of a material including a semiconductor polymer quantum dot and a fluorescent dye, wherein the semiconductor polymer quantum dot is a donor of fluorescence resonance energy transfer, the fluorescent dye is an acceptor of fluorescence resonance energy transfer, and the fluorescent probe is capable of realizing fluorescence scintillation. Wherein, the bright state of the fluorescence scintillation in the fluorescent probe comes from a fluorescent dye, and the dark state of the fluorescence scintillation comes from a semiconductor polymer quantum dot.

The fluorescent probe can be based on

The principle of resonance energy transfer (FRET) is to activate cooperative light scintillation from clusters of dye molecules. The fluorescence emitted from the fluorescent dye acceptor cluster doped in the semiconductor polymer quantum dot can be effectively modulated by the FRET process to produce a discrete, single photon emission-like optical scintillation characteristic. The fluorescence of the fluorescent dye clusters is randomly "on" and "off" indicating that the energy transfer is regulated by a random process. Single particle spectra show that fluorescence of dye-doped Pdots at the single particle level is dominated by the emission peak of the dye acceptor. The results of single particle fluorescence scintillation characteristics evident from single particle luminescence kinetics studies indicate that the fluorescent "on" state is entirely attributable to the dye acceptor. The "off" state during fluorescence blinking of the dye molecule is primarily derived from the semiconducting polymer. The blinking of dye-containing Pdots is activated by a cluster of semiconducting polymer donors and fluorescent dye molecules, a mechanism that is quite unique compared to previously reported optical blinking systems. Both dye-doped Pdots show a Power-law (Power-law) scintillation process, similar to photon statistics for single photon emission systems. This feature highlights the possibility of using FRET pathways to convert multiple dye clusters into quantitative single photon emission applications.

Specifically, the semiconductor polymer quantum dot includes at least one of poly [9, 9-dioctylfluorenyl-2, 7-diyl) ] (PFO) and poly [ 2-methoxy-5- (2-ethylhexyloxy) -1,4- (1-cyanoethylene-1, 4-phenyl) ] (CNPPV). The fluorescent dye includes at least one of Coumarin 6(Coumarin6) and 2,9,16, 23-T-tert-butyl-29H, 31H phthalocyanine (NIR 695).

Wherein PFO is an organic conjugated polymer emitting blue light, the weight-average molecular weight Mw is 147000, the dispersity coefficient is 3.0, and the structural formula is shown as follows:

CNPPV is an organic conjugated polymer emitting orange-yellow fluorescence, MW is 15000, dispersity coefficient is 5.9, and structural formula is as follows:

coumarin6 is a green fluorescent emitting dye having the formula:

NIR695 is a near infrared fluorescent emitting dye, the structural formula is shown below:

specifically, PFO and Coumarin6, CNPPV and NIR695 are donor-acceptor pairs for fluorescence resonance energy transfer, respectively.

The fluorescent dyes described above are not limited to Coumarin6 and NIR 695. Experiments prove that most of fluorescent dyes can show independent scintillation characteristics when being doped into semiconductor polymer quantum dots.

Further, the weight ratio of the semiconductor polymer quantum dots to the fluorescent dye is 100: 0.5-100: 1.

further, the fluorescent probe may further include a functional substance to enable the fluorescent probe to be used for cell labeling. Specifically, the functional substance is styrene maleic anhydride copolymer (PSMA). Further, PSMA has a weight average molecular weight of 1700, and a structural formula as shown below:

the weight ratio of the styrene maleic anhydride copolymer to the semiconductor polymer quantum dots is 1: 2-1: 20. Further, the weight ratio of the styrene maleic anhydride copolymer to the semiconductor polymer quantum dots is 1: 5.

The semiconductor polymer fluorescent quantum dots have outstanding optical properties such as high brightness and photobleaching resistance.

Referring to fig. 1, a method for preparing a fluorescent probe according to an embodiment includes the following steps:

s110: and mixing the semiconductor polymer quantum dots, the fluorescent dye and the organic solvent to obtain a precursor solution.

The semiconductor polymer quantum dots are donors of fluorescence resonance energy transfer, and the fluorescent dye is an acceptor of fluorescence resonance energy transfer. The organic solvent is tetrahydrofuran, dimethyl sulfoxide or dimethylformamide. Further, the organic solvent is tetrahydrofuran.

Specifically, the process of mixing the semiconductor polymer quantum dots and the fluorescent dye with the organic solvent also comprises the process of mixing with the styrene maleic anhydride copolymer. The mixing process specifically comprises:

providing a semiconductor polymer quantum dot solution, a fluorescent dye solution and a styrene maleic anhydride copolymer solution. Wherein, the concentration of the semiconductor polymer quantum dot solution is 1000ppm, the concentration of the fluorescent dye solution is 1000ppm, and the concentration of the styrene maleic anhydride copolymer solution is 1000 ppm. The solvents in the above solutions are all the above organic solvents. Then, mixing the semiconductor polymer quantum dot solution, the fluorescent dye solution and the styrene maleic anhydride copolymer solution according to the volume ratio of 100: (0.5-1): (5-50) mixing. Therefore, in the precursor solution, the mass ratio of the semiconductor polymer quantum dots, the fluorescent dye and the styrene maleic anhydride copolymer is 100: (0.5-1.0): (5-50).

Specifically, in the precursor solution, the concentration of the semiconductor polymer quantum dots is 100mg/L, the concentration of the fluorescent dye is 0.5 mg/L-1 mg/L, and the concentration of the styrene maleic anhydride copolymer is 5 mg/L-50 mg/L.

The organic solvent is adopted to dissolve the semiconductor polymer quantum dots and the fluorescent dye, because the organic solvent can be mutually soluble with water, the semiconductor polymer quantum dots and the fluorescent dye have better solubility in the organic solvent, and the molecular chains of the semiconductor polymer quantum dots and the fluorescent dye are in a freely extending state. Among them, tetrahydrofuran has a low boiling point, and is advantageously removed by heating in the subsequent step.

S120: and injecting the precursor solution into water with the volume ratio of the precursor solution to the precursor solution of 5: 1-20: 1 under the ultrasonic oscillation condition to obtain the ultrasonic solution.

Further, the volume ratio of the precursor solution to water is 10: 1.

In the process, because water is a poor solvent of the semiconductor polymer quantum dots and the fluorescent dye molecules, in the change process of the good solvent and the poor solvent, hydrophobic interaction occurs in molecular chains and among chains of the semiconductor polymer quantum dots and the fluorescent dye molecules to generate entanglement, so that the semiconductor polymer quantum dots doped with the fluorescent dye are formed by agglomeration.

S130: the sonicated solution was stripped of the organic solvent to give a residue.

Specifically, when the organic solvent is tetrahydrofuran, the step of removing the organic solvent from the ultrasonic solution comprises: the sonicated solution was placed on a heating table and a stream of nitrogen was passed to remove the organic solvent and some of the water until the volume of the residue was 50% of the volume of the sonicated solution. Wherein the temperature of the heating table is 90 ℃. The ultrasonic solution contains a small amount of organic solvent, and has certain dissolving capacity on the semiconductor polymer quantum dots, so the organic solvent is removed by heating under the protection of nitrogen.

When the organic solvent is dimethyl sulfoxide or dimethyl formamide, the dimethyl sulfoxide and dimethyl formamide are removed by a dialysis bag dialysis method because the boiling points of the dimethyl sulfoxide and dimethyl formamide are higher than that of water.

S140: the residue was filtered to obtain a fluorescent probe.

Specifically, the step of filtering the residue to obtain the fluorescent probe comprises: and filtering the residue by using a 0.22 mu m water-based syringe filter to remove large-size aggregates in the preparation process, thereby obtaining the fluorescent probe with uniform size distribution.

The preparation method of the fluorescent probe is simple to operate, and various fluorescent dyes are coated in the semiconductor polymer quantum dots by a nano reprecipitation method, so that the fluorescent probe with uniform size, high fluorescence brightness, good light stability and fluorescence scintillation is obtained.

Referring to fig. 2, one embodiment of a super-resolution imaging method based on fluorescence resonance energy transfer modulation includes the following steps:

s210: and (3) carrying out subcellular structure labeling by using a fluorescent probe to obtain a label.

Specifically, the fluorescent probe is made of a semiconductor polymer quantum dot and a fluorescent dye, the semiconductor polymer quantum dot is a donor of fluorescence resonance energy transfer, the fluorescent dye is an acceptor of the fluorescence resonance energy transfer, and the fluorescent probe can realize fluorescence scintillation.

In order to realize the specific labeling of the fluorescent probe to the subcellular structure, the coupling of the biological streptavidin is needed to be carried out on the fluorescent probe. The bioconjugation of the fluorescent probe is based on the catalytic reaction between carboxyl on a functional substance on the surface of the fluorescent probe and amino on streptavidin under the catalysis of 1-ethyl-3- (3-dimethylaminopropyl) carboxylated diimine (EDC, Sigma). And then carrying out subcellular structure labeling on the bioconjugated fluorescent probe.

S220: a fluorescence image of the marker is taken.

Specifically, the number of frames for capturing the fluorescence image of the marker is 200-1000 frames, so as to facilitate the subsequent high-order cumulant analysis of the fluorescence image. Further, the number of frames for taking the fluorescent image of the marker was 1000 frames.

S230: and (4) carrying out cumulant analysis on the fluorescence image.

Specifically, the step of performing cumulant analysis on the fluorescence image comprises the step of sequentially performing second-order cumulant analysis, third-order cumulant analysis and fourth-order cumulant analysis on the fluorescence image. And performing high-order cumulant analysis of the fluorescence intensity fluctuation along with time in different fluorescence images to find out a unique intensity fluctuation curve of each color point. When detecting the fluctuation curve of the characteristic, individual fluorescent molecules can be distinguished from one another. The SOFI is applied to the high-order cumulants to reconstruct the super-resolution image.

The super-resolution imaging method is based on fluorescence resonance energy transfer, so that the fluorescence probe with the fluorescence scintillation characteristic is subjected to specific labeling, and the obtained fluorescence image sequence can be subjected to high-order cumulant analysis, so that the resolution of the imaging method is improved.

The following are specific examples:

it should be noted that tetrahydrofuran used in the examples was anhydrous tetrahydrofuran with a purity of 99.9% and purchased from Sigma-Aldrich, and the ultrasonic oscillation conditions used in the examples were BRASON 2810.

Example 1

(1) 10mg of poly [9, 9-dioctylfluorenyl-2, 7-diyl) ] was dissolved in 10mL of tetrahydrofuran to obtain a PFO solution having a concentration of 1000 ppm. Weighing 10mg of Coumarin6 dye dissolved in 10mL of tetrahydrofuran gave a 1000ppm Coumarin6 solution. 10mg of styrene maleic anhydride copolymer was weighed and dissolved in 10mL of tetrahydrofuran to obtain a PSMA solution having a concentration of 1000 ppm.

(2) And adding 100 mu L of the solution of LPFO, 1 mu L of the solution of Coumarin6 and 20 mu L of the solution of LPSMA into 879 mu L of tetrahydrofuran, and uniformly mixing to obtain a precursor solution.

(3) And injecting the precursor solution into 10mL of deionized water under the ultrasonic oscillation condition, and keeping for 2min under the ultrasonic oscillation condition to obtain the ultrasonic solution.

(4) The sonicated solution was placed on a 90 ℃ heating table and a stream of nitrogen was passed through to remove excess tetrahydrofuran. When 5mL of the sonicated solution remained, it was removed from the hot plate and allowed to stand to room temperature to give a residue.

(5) Filtering the residue with 0.22 μm water system syringe filter, removing large-size aggregates in the preparation process, and obtaining PFO-Coumarin6Pdots with uniform size distribution.

Example 2

(1) 10mg of poly [ 2-methoxy-5- (2-ethylhexyloxy) -1,4- (1-cyanoethylene-1, 4-phenyl) ] are dissolved in 10mL of tetrahydrofuran to give a 1000ppm CNPPV solution. 10mg of 2,9,16, 23-T-tert-butyl-29H, 31H phthalocyanine was weighed out and dissolved in 10mL of tetrahydrofuran to give a 1000ppm NIR695 solution. 10mg of styrene maleic anhydride copolymer was weighed and dissolved in 10mL of tetrahydrofuran to obtain a PSMA solution having a concentration of 1000 ppm.

(2) 100 mu L of CNPPV solution, 0.5 mu L of NIR695 solution and 20 mu L of LPSMA solution are added into 879.5 mu L of tetrahydrofuran to be uniformly mixed to obtain precursor solution.

(4) The sonicated solution was placed on a 90 ℃ heating table and a stream of nitrogen was passed through to remove excess tetrahydrofuran solution. When 5mL of the sonicated solution remained, it was removed from the hot plate and allowed to stand to room temperature to give a residue.

(5) The residue was filtered with a 0.22 μm aqueous syringe filter to remove large-sized aggregates during the preparation process, and CNPPV-NIR695Pdots with uniform size distribution were obtained.

Example 3

(1) 10mg of poly [ 2-methoxy-5- (2-ethylhexyloxy) -1,4- (1-cyanoethylene-1, 4-phenyl) ] was dissolved in 10mL of dimethyl sulfoxide to obtain a 1000ppm CNPPV solution. 10mg of 2,9,16, 23-T-tert-butyl-29H, 31H phthalocyanine was weighed out and dissolved in 10mL of dimethyl sulfoxide to give a 1000ppm NIR695 solution. 10mg of styrene maleic anhydride copolymer was weighed and dissolved in 10mL of dimethyl sulfoxide to obtain a PSMA solution having a concentration of 1000 ppm.

(2) 100 mu L of CNPPV solution, 0.5 mu L of NIR695 solution and 50 mu L of PSMA solution are added into 849.5 mu L of dimethyl sulfoxide and mixed evenly to obtain precursor solution.

(3) And injecting the precursor solution into 5mL of deionized water under the ultrasonic oscillation condition, and keeping for 2min under the ultrasonic oscillation condition to obtain the ultrasonic solution.

(4) The sonicated solution was dialyzed to remove dimethyl sulfoxide to give a residue.

Example 4

(1) 10mg of poly [9, 9-dioctylfluorenyl-2, 7-diyl) ] was dissolved in 10mL of dimethylformamide to obtain a PFO solution having a concentration of 1000 ppm. Weighing 10mg of Coumarin6 dye dissolved in 10mL of dimethylformamide gave a 1000ppm Coumarin6 solution. 10mg of styrene maleic anhydride copolymer was weighed and dissolved in 10mL of dimethylformamide to obtain a PSMA solution having a concentration of 1000 ppm.

(2) And adding 100 mu of LPFO solution, 1 mu of LCoumarin6 solution and 5 mu of LPSMA solution into 894 mu of dimethylformamide for uniform mixing to obtain a precursor solution.

(3) And injecting the precursor solution into 20mL of deionized water under the ultrasonic oscillation condition, and keeping for 2min under the ultrasonic oscillation condition to obtain the ultrasonic solution.

(4) The sonicated solution was dialyzed to remove excess dimethylformamide to give a residue.

Comparative example 1

(1) 10mg of poly [9, 9-dioctylfluorenyl-2, 7-diyl) ] was dissolved in 10mL of tetrahydrofuran to obtain a PFO solution having a concentration of 1000 ppm. 10mg of styrene maleic anhydride copolymer was weighed and dissolved in 10mL of tetrahydrofuran to obtain a PSMA solution having a concentration of 1000 ppm.

(2) And adding 100 mu of LPFO solution and 20 mu of LPSMA solution into 880 mu of tetrahydrofuran for uniform mixing to obtain precursor solution.

(5) The residue was filtered with a 0.22 μm aqueous syringe filter to remove large-sized aggregates during the preparation process, and PFO Pdots having a uniform size distribution were obtained.

Comparative example 2

(1) 10mg of poly [ 2-methoxy-5- (2-ethylhexyloxy) -1,4- (1-cyanoethylene-1, 4-phenyl) ] are dissolved in 10mL of tetrahydrofuran to give a 1000ppm CNPPV solution. 10mg of styrene maleic anhydride copolymer was weighed and dissolved in 10mL of tetrahydrofuran to obtain a PSMA solution having a concentration of 1000 ppm.

(2) 100 mu L of 1000ppm CNPPV solution and 20 mu L PSMA solution are added into 880 mu L tetrahydrofuran to be mixed evenly, and precursor solution is obtained.

(5) The residue was filtered with a 0.22 μm aqueous syringe filter to remove large-sized aggregates during the preparation process, and CNPPV Pdots having a uniform size distribution were obtained.

Comparative example 3

(1) 10mg of polystyrene was weighed and dissolved in 10mL of tetrahydrofuran to obtain a PS solution with a concentration of 1000 ppm. 10mg of styrene maleic anhydride copolymer was weighed and dissolved in 10mL of tetrahydrofuran to obtain a PSMA solution having a concentration of 1000 ppm. Weighing 10mg of Coumarin6 dye dissolved in 10mL of tetrahydrofuran gave a 1000ppm Coumarin6 solution.

(2) And adding 100 mu of LPS solution, 1 mu of LCoumarin6 solution and 20 mu of LPSMA solution into 879 mu of tetrahydrofuran, and uniformly mixing to obtain a precursor solution.

(5) And filtering the residue by using a 0.22 mu m water-based syringe filter to remove large-size aggregates in the preparation process, thereby obtaining PS-Coumarin6Pdots with uniform size distribution.

Comparative example 4

(1) 10mg of polystyrene was weighed and dissolved in 10mL of tetrahydrofuran organic solvent to obtain a PS solution with a concentration of 1000 ppm. 10mg of styrene maleic anhydride copolymer was weighed and dissolved in 10mL of tetrahydrofuran solution to obtain a PSMA solution having a concentration of 1000 ppm. Weighing 10mg of 2,9,16, 23-T-tert-butyl-29H, 31H phthalocyanine dissolved in 10mL tetrahydrofuran solution gives a 1000ppm NIR695 solution.

(2) 100 mu L of PS solution, 0.5 mu L of LNIR695 solution and 20 mu L of PSMA solution are added into 879.5 mu L of tetrahydrofuran to be mixed evenly, and precursor solution is obtained.

(5) The residue was filtered with a 0.22 μm aqueous syringe filter to remove large-sized aggregates during the preparation process, yielding PS-NIR695Pdots with uniform size distribution.

The fluorescent probes obtained in example 1, example 2 and comparative examples 1 to 4 were examined below. It should be noted that the size distribution of the fluorescent probes obtained in the examples and comparative examples herein were measured by using a dynamic light scattering system (DLS) (Malvern NANOZS). The absorption spectrum of the fluorescent probe was measured with an ultraviolet-visible spectrophotometer (Agilent Cary 60). The fluorescence spectra of the fluorescent probes were determined using a Horiba FluoroMax-4 fluorescence spectrophotometer.

Herein, single particle fluorescence spectroscopy is performed using a microscopic micro-area spectroscopy test system. The system is specifically configured as follows: an Andon Shamrock SR500i-D2 spectrometer was placed at the left optical port of an inverted microscope of Orobas IX71, and a 395nm or 490nm fiber coupled LED (Thorabs, M395F3, M490F3) was directed by a collimator (Thorabs, F240SMA-532) at the back pupil of an objective lens (Orobas, UPLSAPO40X, NA: 0.9). For PFO-Coumarin6Pdots, a single-edge standard inverted fluorescence spectrometer (Semrock,

FF409-Di03-25x36) to reflect the excitation light and transmit the emission light. Subsequently, the emission light received through a 409nm long pass emission filter (Semrock, FF02-409/LP-25) is directed into a spectrometer and projected onto an EMCCD (Andor, Newton920, UK). For CNPPV-NIR695Pdots, 490nm LED excitation, 495nm dichromatic color chips (Semrock, FF495-Di03-25x36) and 515nm long-pass filters (Semrock, FF01-515/LP-25) are adopted.

Single particle fluorescence intensity tracking experiments and subcellular structure imaging were performed in the following systems: the microscope was a Nikon N-STORM system equipped with a 100xoiltIRF objective (Nikon, NA: 1.49, Japan). A405 nm laser (Chroma, OBIS, 405nm, 200mw, USA) and a 488nm Sapphire laser (Chroma, Sapphire, 488nm, 300mw, USA) are coupled to illuminate the rear pupil of the objective lens at a fiber-coupled TIRF illuminator (Nikon, TIRF-E, Japan). Multichannel dichroic filters (Chroma, ZT405/488/561/640rpcv2) were used to distinguish between excitation light and fluorescence. Bandpass emission filters (Chroma, ET525/30m and Semrock, FF01711/25-25) were configured for PFO-Coumarin6Pdots and CNPPV-NIR695Pdots, respectively. The focus system (PFS) is then used to lock the focus. An extra 1.5 magnification was used for imaging to produce 106nm per pixel imaging on an EMCCD (Andor, DU897, UK).

Herein, both single particle and sub-cytoskeleton imaging sequence plots were processed in Matlab 2016b (Mathworks inc., USA) with drift correction from the written Matlab code based on a sub-pixel drift correction function. Single particle fluorescence intensity scintillation data was extracted from self-compiled Matlab code. The mutual cumulant SOFI analysis of the single particle and the subcellular skeleton imaging sequence diagram is based on the bSOFI algorithm

Dynamic light scattering measurements were performed on PFO-Coumarin6Pdots and CNPPV-NIR695Pdots obtained in example 1 and example 2, respectively, to investigate their size distribution, and the results are shown in FIGS. 3-a and 3-b. Wherein, the figure 3-a is the particle size distribution diagram of PFO-Coumarin6Pdots, and the figure 3-b is the particle size distribution diagram of CNPPV-NIR695 Pdots. As can be seen from the figure, the particle sizes of PFO-Coumarin6Pdots and CNPPV-NIR695Pdots are 18nm and 16nm, respectively.

The semiconductor polymer quantum dots and the fluorescent dyes of example 1 and example 2 were respectively subjected to fluorescence analysis to obtain absorption spectra and emission spectra as shown in fig. 4-a and fig. 4-b. Wherein, the curves in fig. 4-a are absorption spectrum of PFO, emission spectrum of PFO, absorption spectrum of Coumarin6, and emission spectrum of Coumarin6 from left to right. As can be seen in fig. 4-a, there is a large spectral overlap between PFO and Coumarin6 enabling fluorescence resonance energy transfer. The curves in fig. 4-b are, from left to right, the absorption spectrum of CNPPV, the emission spectrum of CNPPV, the absorption spectrum of NIR695 and the absorption spectrum of NIR 695. As can be seen in fig. 4-b, there is a large spectral overlap between CNPPV and NIR695, enabling fluorescence resonance energy transfer. Thus, at lower doping ratios (1 wt% coumarin6 in PFO, 0.5 wt% NIR695 in CNPPV) for the donor polymer quantum dots and acceptor fluorescent dyes in examples 1 and 2, the fluorescence emission is dominated by the acceptor dye luminescence.

In order to verify whether the fluorescent dye is doped into the polymer quantum dot, single particle emission spectrum studies were respectively carried out on PFO-Coumarin6Pdots in example 1 and CNPPV-NIR695Pdots in example 2, and the results are shown in FIGS. 5-a and 5-b. Wherein, FIG. 5-a is a single particle emission spectrum of PFO-Coumarin6Pdots, and FIG. 5-b is a single particle emission spectrum of CNPPV-NIR695 Pdots. As can be seen in FIGS. 5-a and 5-b, the single particle spectra show that the fluorescence of the fluorochrome-doped polymer quantum dots at the single particle level is dominated by the emission peak of the fluorochrome acceptor. The fluorescence of the dye clusters was randomly "on" and "off indicating that the energy transfer was regulated by a random process. The emission spectrum of the single particles was compared with the fluorescence spectrum of the semiconductor polymer quantum dots doped with the fluorescent dye dispersed in water (as shown in fig. 6-a and 6-b). Wherein, FIG. 6-a is the absorption spectrum and emission spectrum of PFO-Coumarin6Pdots, and FIG. 6-b is the absorption spectrum and emission spectrum of CNPPV-NIR695 Pdots. As can be seen from a comparison of FIGS. 5-a and 5-b and FIGS. 6-a and 6-b, the single particle emission spectrum is consistent with the emission spectrum of fluorescent probes dispersed in water. This indicates that the fluorescent dye was successfully doped into the semiconducting polymer quantum dot and that efficient FRET from the donor semiconducting polymer to the acceptor fluorescent dye was achieved.

To study the cooperative blinking mechanism of energy transfer activation of the semiconductor polymer quantum dot donor and the fluorescent dye acceptor, comparative studies were performed with the different Pdots in each example and comparative example. Wherein, FIG. 7-a is a fluorescence trace diagram of pure PFO and PS-Coumarin6Pdots, FIG. 7-b is a fluorescence trace diagram of PS-NIR695Pdots and pure CNPPV, FIG. 8-a is a fluorescence trace diagram of PFO-Coumarin6, and FIG. 8-b is a fluorescence trace diagram of CNPPV-NIR 695. As can be seen in FIGS. 7-a and 7-b, the single particle intensity traces of the original pure PFO and pure CNPPV showed no fluorescence flashes, indicating that the larger size Pdots (> 15nm) consisted of more chromophores and gave relatively stable fluorescence.

Similarly, the fluorescent intensity of nanoparticles of coumarin 6-doped polystyrene and NIR 695-doped polystyrene are also flicker-free, consistent with the single particle fluorescence of dye-loaded nanospheres. The fluorescence of the nanoparticles of coumarin6 doped polystyrene and NIR695 doped polystyrene is derived from fluorescent dye molecular clusters, and fluctuation of different dye molecular clusters can be mutually compensated to ensure that the fluorescent dye molecular clusters do not have fluorescence scintillation characteristics.

As can be seen from FIGS. 8-a and 8-b, the two types of dye-doped Pdots (PFO-Coumarin6 and CNPPV-NIR695) of examples 1 and 2 have significant fluorescence scintillation upon excitation of the semiconducting polymer. The fluorescence scintillation is shown to be regulated and controlled by the cooperative activation of the semiconductor polymer quantum dot donor and the fluorescent dye acceptor.

The results of the obvious single particle fluorescence scintillation characteristics using narrow-band emission filters corresponding to coumarin6 or NIR695 (Chroma, ET525/30m for receiving fluorescence from coumarin 6; Semrock, FF01711/25-25 for receiving fluorescence from NIR695) in single particle luminescence kinetics studies indicate that the fluorescence "on" state is completely due to the dye acceptor. As a control, non-fluorescent polystyrene was selected as a carrier for dye molecules to prepare nanoparticles, and the dye molecules in both PS-Coumarin6Pdots and PS-NIR695Pdots exhibited stable fluorescence intensity without scintillation (as shown in FIGS. 7-a and 7-b). This indicates that the "off" state during fluorescence blinking of the dye molecules is primarily derived from the semiconducting polymer.

The hole polarons are reported to effectively quench the fluorescence of the semiconductor polymer quantum dots, and the intensity fluctuation and the mass center displacement are generated in single particle imaging. In this example, because FRET is extremely sensitive to the distance and spectral overlap between donor and acceptor. Thus, the generation of hole polarons in the semiconducting polymer can greatly modulate FRET efficiency to the extent that FRET can be turned off completely. The mechanism is shown in Jablonski FIG. 9.

The fluorescent probe can activate the cooperative light scintillation from the dye molecule cluster based on the principle of fluorescence resonance energy transfer. The fluorescence emitted from the fluorescent dye acceptor cluster doped in the semiconductor polymer quantum dot can be effectively modulated by the FRET process to produce a discrete, single photon emission-like optical scintillation characteristic. The fluorescence of the fluorescent dye clusters is randomly "on" and "off" indicating that the energy transfer is regulated by a random process. The single particle spectrum shows that the fluorescence of the dye-doped semiconductor polymer quantum dots at the single particle level is dominated by the emission peak of the dye acceptor. The results of single particle fluorescence scintillation characteristics evident from single particle luminescence kinetics studies indicate that the fluorescent "on" state is entirely attributable to the dye acceptor. The "off" state during the fluorescent blinking process of the dye-doped semiconducting polymer quantum dots is mainly derived from the semiconducting polymer quantum dots. The scintillation of dye-containing semiconducting polymer quantum dots is activated by polymer donors and dye molecular clusters, a mechanism that is quite unique compared to previously reported optical scintillation systems. Both dye-doped semiconducting polymer quantum dots show a Power-law (Power-law) scintillation process, similar to photon statistics of single photon emission systems. This feature highlights the possibility of using FRET pathways to convert multiple dye clusters into quantitative single photon emission applications.

In order to evaluate the SOFI performance of the fluorescent probe, 1000 PFO-Coumarin6Pdots single-particle images are analyzed, and the mutual accumulation amount of different orders is calculated. FIG. 10-a, FIG. 10-b, FIG. 10-c and FIG. 10-d are respectively wide field fluorescence imaging, 2-order, 3-order and 4-order SOFI imaging of PFO-Coumarin6Pdots in example 1. Two optical diffraction-limited blur points marked with white dashed lines in a conventional wide-field image are resolved by second, third and fourth order SOFI analysis. The enhancement of spatial resolution is illustrated by plotting the intensity profile of the PFO-Coumarin6Pdots Spread Function (PSF) (as shown in FIG. 11-a). The statistical full width at half maximum (FWHM) of twelve individual particles in the wide field fluorescence image (as shown in FIG. 11-b) was 333 nm. + -. 26 nm. In second, third and fourth order SOFF analyses, FWHM was 201nm + -15 nm, 148nm + -16 nm and 117nm + -12 nm, respectively, and the resolution was improved by about 1.66 times, 2.25 times and 2.85 times, respectively, compared to conventional wide field images. These results provide a universal method for the application of fluorescent probes in super-resolution optical fluctuation imaging.

The same SOFI analysis is carried out on CNPPV-NIR695Pdots, compared with a wide-field fluorescence image, the spatial resolution is obviously enhanced, the enhancement effect is the same as that of PFO-Coumarin6Pdots, and the description is omitted.

In addition, microtubules (α -tubulin) of COS-7 cells were labeled with the fluorescent probes of example 1 and example 2 coupled to streptavidin (Streptavdin) for super-resolution nano-microscopy imaging. The specific process is as follows:

(1) bioconjugation of fluorescent probes: to 1mL of Pdots solution (50. mu.g/mL) were added 20. mu.L of polyethylene glycol (PEG, MW: 4500, 5 wt%), 20. mu.L of 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES) buffer at a concentration of 1. mu.M, 60. mu.L of streptavidin (1mg/mL) and 20. mu.L of LEDC solution (5 mg/mL). Mix at room temperature for 4 hours with rotation. After the reaction is finished, unreacted streptavidin (streptavidin) in a free state in the system is removed in an ultrafiltration and centrifugation mode at the rotating speed of 3000rpm to obtain Pdots-streptavidin, and the Pdots-streptavidin is stored at the temperature of 4 ℃ for later use.

(2) Specific subcellular structural labeling: will be 5X 10⁵COS-7 cells (African green monkey kidney epithelial cells) were cultured overnight in glass culture dishes on high-sugar medium containing 10% fetal bovine serum (Gibco) and penicillin and streptomycin (Gibco). A solution containing 0.2% of polyethylene glycol octylphenyl ether (TritonX-100), 0.1M of piperazine-1, 4-diethylsulfonic acid (PIPES), 1mM of ethylene glycol bis (2-aminoethyl ether) tetraacetic acid (EGTA) and 1mM MgCl was used₂The extract of (a) was incubated for 1 minute. Then fixed with 4% Paraformaldehyde (PFA) and 0.1% Glutaraldehyde (GA) and then rinsed with Phosphate Buffered Saline (PBS). The wells were punched out by incubation with 0.25% TritonX-100 solution for 5 min. Blocking was performed for 30 minutes in a blocking solution containing 5% Bovine Serum Albumin (BSA) and 0.5% TritonX-100. Next, COS-7 cells were cultured in blocking solution at a dilution of biotin-binding alpha tubulin antibody (ab74696Abcam) of 1: 500 for 1 hour and rinsed three times with PBS. Then 6. mu.L of Pdots-streptavdin was added and cultured for 1 hour. After three washes with PBS, the cells will be available for microscopic imaging.

The fluorescence probe after the specific subcellular labeling is imaged, and the images are shown in FIGS. 12-a to 12-h. In which FIGS. 12-a and 12-c are wide field fluorescence images of PFO-Coumarin 6-labeled microtubes in COS-7 cells. FIG. 12-b and FIG. 12-d are four-stage SOFI nanoimaging of the fluorescence images of FIG. 12-a and FIG. 12-c, respectively. Fig. 12-e and 12-f are enlarged views of the area images shown by the white boxes labeled "I" in fig. 12-a and 12-b, respectively. Fig. 12-g and 12-h are enlarged views of the area images shown by the white boxes labeled "II" in fig. 12-c and 12-d, respectively. As can be seen from FIGS. 12-a to 12-h, the fluorescent probe for PFO-Coumarin6Pdots showed excellent subcellular labeling ability with high signal-to-noise ratio even under wide field fluorescence imaging.

FIG. 13-a is a graph showing a fluorescence intensity distribution of a microtube indicated by a white arrow in FIGS. 12-e and 12-f, and FIG. 13-b is a graph showing a fluorescence intensity distribution of a microtube indicated by a white arrow in FIGS. 12-g and 12-h. As can be seen from FIGS. 13-a and 13-b, the resolution of 4-order SOFI (133nm) is increased by a factor of 2.12 compared to the wide field fluorescence image (281 nm). Furthermore, after the SOFI analysis, two adjacent microtubule filaments were clearly distinguished (155 nm apart). Whereas the wide-field fluorescence image exhibits a wider bright-section profile due to optical diffraction limitations.

A4-order SOFI analysis of microtubule structures stained with CNPPV-NIR695Pdots in COS-7 cells was also performed, and showed a 2.33-fold increase in spatial resolution.

The results all show that the FRET activated scintillation Pdots can be successfully used for super-resolution imaging of subcellular structures.

The fluorescent probes prepared in examples 1 and 2 were used for performing the super-resolution imaging of the sub-label structure, but the fluorescent probes prepared in examples 3 and 4 can also be used for performing the super-resolution imaging of the sub-label structure, and the effect is equivalent to that of examples 1 and 2, and will not be described herein again.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims

1. A fluorescent probe is characterized in that the material of the fluorescent probe comprises semiconductor polymer quantum dots and fluorescent dye, the semiconductor polymer quantum dots are donors of fluorescence resonance energy transfer, the fluorescent dye is acceptors of the fluorescence resonance energy transfer, and the fluorescent probe can generate fluorescence scintillation;

the semiconductor polymer quantum dot is poly [9, 9-dioctyl fluorenyl-2, 7-diyl) ], and the fluorescent dye is coumarin 6; or the semiconductor polymer quantum dot is poly [ 2-methoxy-5- (2-ethylhexyloxy) -1,4- (1-cyanoethylene-1, 4-phenyl) ], and the fluorescent dye is 2,9,16, 23-tetra-tert-butyl-29H, 31H phthalocyanine.

2. The fluorescent probe of claim 1, wherein the weight ratio of the semiconductor polymer quantum dots to the fluorescent dye is 100: 0.5-100: 1.

3. The fluorescent probe of claim 1, wherein the material of the fluorescent probe further comprises a styrene maleic anhydride copolymer.

4. The method for preparing the fluorescent probe according to claim 3, wherein the weight ratio of the styrene maleic anhydride copolymer to the semiconductor polymer quantum dot is 1: 2-1: 20.

5. The method of claim 4, wherein the weight ratio of the styrene maleic anhydride copolymer to the semiconductor polymer quantum dots is 1: 5.

6. A preparation method of a fluorescent probe is characterized by comprising the following steps:

injecting the precursor solution into water with a volume ratio of 5: 1-20: 1 to the precursor solution under an ultrasonic oscillation condition to obtain an ultrasonic solution;

removing the organic solvent from the sonicated solution to obtain a residue;

filtering the residue to obtain the fluorescent probe with fluorescence scintillation;

7. The method of claim 6, wherein the concentration of the semiconductor polymer quantum dots in the precursor solution is 100mg/L, and the concentration of the fluorescent dye is 0.5mg/L to 1 mg/L.

8. A super-resolution imaging method based on fluorescence resonance energy transfer regulation is characterized by comprising the following steps:

carrying out subcellular structure labeling by using a fluorescent probe to obtain a label, wherein the fluorescent probe is made of a semiconductor polymer quantum dot and a fluorescent dye, the semiconductor polymer quantum dot is a donor of fluorescence resonance energy transfer, the fluorescent dye is an acceptor of the fluorescence resonance energy transfer, and the fluorescent probe can generate fluorescence scintillation; the semiconductor polymer quantum dot is poly [9, 9-dioctyl fluorenyl-2, 7-diyl) ], and the fluorescent dye is coumarin 6; or the semiconductor polymer quantum dot is poly [ 2-methoxy-5- (2-ethylhexyloxy) -1,4- (1-cyanoethylene-1, 4-phenyl) ], and the fluorescent dye is 2,9,16, 23-tetra-tert-butyl-29H, 31H phthalocyanine;

capturing a fluorescence image of the marker;

and performing cumulant analysis on the fluorescence image.

9. The fluorescence resonance energy transfer modulation-based super-resolution imaging method according to claim 8, wherein the step of performing cumulative analysis on the fluorescence image comprises: and sequentially carrying out second-order cumulant analysis, third-order cumulant analysis and fourth-order cumulant analysis on the fluorescence image.

10. The fluorescence resonance energy transfer modulation-based super-resolution imaging method according to claim 8, wherein the number of frames for capturing the fluorescence image of the marker is 200 to 1000 frames.