CN113713123B

CN113713123B - Fluorescent conjugated polymer nano-probe for imaging brain lymphatic system and blood vessels

Info

Publication number: CN113713123B
Application number: CN202110988621.5A
Authority: CN
Inventors: 熊丽琴; 李玉巧
Original assignee: Shanghai Jiaotong University
Current assignee: Shanghai Jiaotong University
Priority date: 2021-08-26
Filing date: 2021-08-26
Publication date: 2022-09-23
Anticipated expiration: 2041-08-26
Also published as: CN113713123A

Abstract

The invention discloses an application of a fluorescent conjugated polymer nano probe in preparing a contrast agent for imaging a brain lymphatic system and blood vessels. The invention realizes the visible light area fluorescence imaging with good imaging effect on the brain lymphatic system and the blood vessel by adopting a simple and convenient body type fluorescence microscope imaging means, and particularly realizes the high-brightness and high-resolution visible light area rapid fluorescence imaging on the meninges lymphatic vessel and the brain blood vessel.

Description

Fluorescent conjugated polymer nano-probe for imaging brain lymphatic system and blood vessels

Technical Field

The invention belongs to the technical field of lymphatic vessel imaging, and relates to a fluorescent conjugated polymer nano probe for imaging a lymphatic system and blood vessels of a brain.

Background

Brain disease is considered one of the world's leading health challenges in the 21 st century. In 2016, through the discussion of the department of science and technology of the Chinese Nature Foundation, the Chinese brain program, brain science and brain-like guideline technology, was proposed to better address the challenges posed by brain diseases.

The brain is traditionally thought to be an "immune-privileged" organ with limited immune surveillance for pathogens and tumors. As research progresses, scientists snoop on the traces of the lymphatic system in the central nervous system. In 2015, 2 reports confirmed the presence of meningeal lymphatic vessels. The discovery of the brain lymphatic vessels can answer many questions in the clinic. For example, some patients with cervical inflammation and tumor will have symptoms of headache, nausea and vomiting after lymph node cleaning and radiation therapy, and even brain edema in severe cases. More importantly, the existence of the cerebral lymphatic vessels opens up a new way for treating diseases related to the central nervous system, and the cure of various brain diseases becomes possible.

Because the lymphatic vessels are thin (the diameter is usually only a tenth of that of a vein), the walls of the vessels are transparent, the lumens are usually in a collapse state, and most of the lymphatic fluid in the lymphatic vessels is colorless and transparent, does not contain red blood cells, and is not easy to identify compared with blood vessels. The traditional lymphatic vessel research method generally adopts animal autopsy to perform lymphatic vessel perfusion or immunohistochemical staining on isolated tissues, and cannot perform real-time high-resolution imaging on lymphatic structure and functions in a living body (in vivo) state. Compared with the in vitro and in vitro detection methods, the molecular imaging technology as an in vivo detection method can continuously, quickly, remotely and nondestructively obtain two/three-dimensional images on the molecular cell level. Advanced molecular probes and molecular imaging technologies are applied to develop real-time, rapid and ultrahigh-resolution imaging technologies and methods for lymphatic vasculature, which are helpful for early and accurate diagnosis of conditions such as lymphatic structure and dysfunction, and provide important imaging data for clinical accurate staging and treatment scheme formulation. However, the lymphatic vessel imaging probes such as indocyanine green ICG and methylene blue can leak, which causes the pollution of the dye in the operation area and can not meet the requirement well.

In addition, the lymphatic vessels in the brain are located deeper than the peripheral lymphatic vessels, and have higher requirements for the operating method, imaging probes, and imaging methods. Moreover, the fluid dynamics in the brain are extremely complex, leading to a lack of profound understanding of the formation and maintenance of meningeal lymphatic systems and a lack of complete understanding of the global lymphatic network that connects the primary lymphatic vessels in the central nervous system and the cervical lymph nodes. Currently, there is no report of in vivo fluorescence imaging of the lymphatic vasculature of the brain.

On the other hand, a minute change in the fine structure of cerebral vessels plays a very important role in the onset of cerebrovascular diseases. With the continuous progress of science and technology, more and more brain blood vessel imaging technologies are developed. For example, CT angiography can provide important additional information about cerebral hemodynamics, and perfusion CT can rapidly assess both cerebrovascular physiology and hemodynamics qualitatively and quantitatively, but both provide only cerebrovascular structural information and are radioactive. Positron emission tomography PET can provide brain moving images, but it is costly to use and requires the injection of radiolabeled probes. Compared to the radioactivity of CT and PET, magnetic resonance imaging MRI of the brain does not use harmful ionizing radiation, but is not suitable for patients wearing metal instruments such as cardiac pacemakers, and the resolution is not sufficient to image tiny blood vessels.

The brain blood vessel fluorescence imaging has the characteristics of high imaging speed, high resolution, high signal-to-noise ratio, strong operability and the like, and has wide application prospect in the field of brain science. However, for obtaining high-resolution imaging data in brain vascular fluorescence imaging, it is often necessary to use expensive (millions of dollars in price) imported imaging systems, such as a rotating-disk confocal microscope, a multi-photon confocal microscope, a light sheet microscope, etc., in combination, which requires a long sample preparation time, a long data acquisition time, etc. At present, there is no report on in vivo fluorescence imaging of whole brain microvascular networks.

Therefore, there is a need in the art for fluorescence imaging techniques of the lymphatic and blood vessels of the brain, particularly meningeal lymphatic vessels and cerebral blood vessels, that can achieve good imaging results with low-cost imaging equipment.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides a fluorescence imaging technology for the lymphatic system and blood vessels of the brain. The fluorescent conjugated polymer nanoprobe with a specific structure is applied to imaging of a brain lymphatic system and blood vessels, particularly meninges lymphatic vessels and brain blood vessels, so that visible light area fluorescent imaging with good imaging effect on the brain lymphatic system and the blood vessels by using a simple, convenient and low-cost body type fluorescent microscope is realized, and particularly high-brightness and high-resolution visible light area rapid fluorescent imaging on the meninges lymphatic vessels and the whole cerebral blood vessels is realized.

Specifically, the invention provides application of a fluorescent conjugated polymer nanoprobe in preparing a contrast agent for imaging blood vessels and a brain lymphatic system, wherein the fluorescent conjugated polymer nanoprobe comprises a fluorescent conjugated polymer, the brain lymphatic system is selected from one or more of meningeal lymphatic vessels, facial lymph nodes, neck lymph nodes and neck lymphatic vessels, and the blood vessels are selected from one or more of brain blood vessels, eye blood vessels, extraocular muscle blood vessels, kidney blood vessels, liver blood vessels, spleen blood vessels, lung blood vessels, intestinal blood vessels and blood vessels in white adipose tissues.

In one or more embodiments, the contrast agent is an aqueous solution, physiological saline solution, or buffer solution of the fluorescent conjugated polymer nanoprobe, and the concentration of the fluorescent conjugated polymer nanoprobe of the contrast agent is 0.01-1 mg/mL.

In one or more embodiments, the hydrated particle size of the fluorescent conjugated polymer nanoprobe is 30-100nm, preferably 40-60 nm.

In one or more embodiments, the fluorescent conjugated polymer is selected from one or both of 9, 9-dioctylfluorene-2, 1, 3-benzothiadiazole copolymer and poly [ 2-methoxy-5- (2-ethylhexyloxy) -1, 4-phenylenevinylene ], preferably 9, 9-dioctylfluorene-2, 1, 3-benzothiadiazole copolymer.

In one or more embodiments, the fluorescent conjugated polymer nanoprobes do not comprise surface ligands.

In one or more embodiments, the fluorescent conjugated polymer nanoprobe comprises only a fluorescent conjugated polymer.

In one or more embodiments, the fluorescent conjugated polymer nanoprobe further comprises a surface ligand.

In one or more embodiments, the surface ligand is selected from one or more of polystyrene grafted carboxyl terminated polyethylene oxide, amino terminated polymethyl methacrylate, and styrene-maleic anhydride copolymer, preferably polystyrene grafted carboxyl terminated polyethylene oxide.

In one or more embodiments, in the fluorescent conjugated polymer nanoprobe, the mass ratio of the surface ligand to the fluorescent conjugated polymer is 0: 1 to 2.5: 1, e.g. 0.8: 1 to 1.2: 1.

in one or more embodiments, the imaging is in vivo imaging or ex vivo imaging.

In one or more embodiments, the imaging is bulk fluorescence microscopy imaging.

In one or more embodiments, the imaging is brain lymphatic system imaging. Preferably, the lymphatic system of the brain is meningeal lymphatic vessels. Preferably, the contrast agent is an aqueous solution, a physiological saline solution or a buffer solution of the fluorescent conjugated polymer nanoprobe, and the concentration of the fluorescent conjugated polymer nanoprobe of the contrast agent is 0.1-1 mg/mL. Preferably, the fluorescent conjugated polymer is a 9, 9-dioctyl fluorene-2, 1, 3-benzothiadiazole copolymer. The fluorescent conjugated polymer nanoprobes may comprise surface ligands. Preferably, the surface ligand may be selected from one or more of polystyrene grafted carboxyl terminated polyethylene oxide, amino terminated polymethyl methacrylate and styrene-maleic anhydride copolymer, preferably polystyrene grafted carboxyl terminated polyethylene oxide. Preferably, in the fluorescent conjugated polymer nanoprobe, the mass ratio of the surface ligand to the fluorescent conjugated polymer is 0: 1 to 2.5: 1, e.g. 0.8: 1 to 1.2: 1. preferably, the hydrated particle size of the fluorescent conjugated polymer nanoprobe is 30-100nm, preferably 40-60 nm. Preferably, the imaging is non-craniotomy imaging or craniotomy imaging. Preferably, the imaging is in vivo imaging. Preferably, the imaging is bulk fluorescence microscopy imaging.

In one or more embodiments, the imaging is brain vessel imaging.

In one or more embodiments, the cerebral blood vessels are whole cerebral blood vessels including whole cerebral blood vessels before meningeal dissection and whole cerebral blood vessels after meningeal dissection or partial cerebral blood vessels including cerebral blood vessels, cerebellar blood vessels, and meningeal blood vessels.

In one or more embodiments, the cerebral blood vessels are of a normal subject, an elderly subject, and/or a stroke subject.

In one or more embodiments, the imaging is brain vessel imaging. The imaging is body type fluorescence microscope imaging; preferably, the contrast agent is an aqueous solution, a physiological saline solution or a buffer solution of the fluorescent conjugated polymer nanoprobe, and the concentration of the fluorescent conjugated polymer nanoprobe of the contrast agent is 0.01-0.1 mg/mL; preferably, the fluorescent conjugated polymer is one or two selected from 9, 9-dioctyl fluorene-2, 1, 3-benzothiadiazole copolymer and poly [ 2-methoxy-5- (2-ethylhexyloxy) -1, 4-phenylene vinylene ], and is preferably 9, 9-dioctyl fluorene-2, 1, 3-benzothiadiazole copolymer. The fluorescent conjugated polymer nanoprobe may comprise only the fluorescent conjugated polymer, or further comprise a surface ligand. Preferably, the surface ligand is selected from one or more of polystyrene grafted carboxyl-terminated polyethylene oxide, amino-terminated polymethyl methacrylate and styrene-maleic anhydride copolymer, preferably polystyrene grafted carboxyl-terminated polyethylene oxide; preferably, in the fluorescent conjugated polymer nanoprobe, the mass ratio of the surface ligand to the fluorescent conjugated polymer is 0: 1 to 2.5: 1, e.g. 0.8: 1 to 1.2: 1; preferably, the hydrated particle size of the fluorescent conjugated polymer nanoprobe is 30-100nm, preferably 40-60 nm; preferably, the imaging is non-craniotomy imaging or craniotomy imaging; preferably, the imaging is in vivo imaging or ex vivo imaging.

In one or more embodiments, the imaging is of ocular vessels, extraocular intramusculary vessels, renal vessels, hepatic vessels, spleen vessels, pulmonary vessels, intestinal vessels, and/or white adipose tissue intravascular vessels. Preferably, the contrast agent is an aqueous solution, a physiological saline solution or a buffer solution of the fluorescent conjugated polymer nanoprobe, and the concentration of the fluorescent conjugated polymer nanoprobe of the contrast agent is 0.01-0.1 mg/mL; preferably, the fluorescent conjugated polymer is a 9, 9-dioctyl fluorene-2, 1, 3-benzothiadiazole copolymer; preferably, the surface ligand is polystyrene grafted carboxyl terminated polyethylene oxide; preferably, in the fluorescent conjugated polymer nanoprobe, the mass ratio of the surface ligand to the fluorescent conjugated polymer is 0: 1 to 2.5: 1, e.g. 0.8: 1 to 1.2: 1; preferably, the hydrated particle size of the fluorescent conjugated polymer nanoprobe is 30-100nm, preferably 40-60 nm; preferably, the imaging is in vivo imaging or ex vivo imaging; preferably, the imaging is bulk fluorescence microscopy imaging.

Drawings

FIG. 1 shows fluorescent conjugated polymer nanoprobes PFBT, PFBT-COOH and PFBT-NH ₂ Ultraviolet absorption spectrum (left), fluorescence emission spectrum (middle), and hydrated particle size distribution map (right).

FIG. 2 shows fluorescent conjugated polymer nanoprobes PPV, PPV-COOH, PPV-NH ₂ Ultraviolet absorption spectrum (left), fluorescence emission spectrum (middle), and hydrated particle size distribution map (right).

FIG. 3 is a fluorescence diagram of in vitro penetration experiment of the fluorescent conjugated polymer nanoprobe PFBT, and FIG. 3 shows the experiment results when the penetration depth is 0mm (upper left), 1mm (upper right), 2mm (lower left) and 3mm (lower right).

Fig. 4 is a brightfield (left) and fluorescence (right) image of meningeal lymphatic vessels after lateral ventricle injection of PFBT-COOH without opening the skull.

Fig. 5 is a brightfield image (left) of meningeal lymphatic vessels imaged on opening the skull after lateral ventricle injection of PFBT-COOH and fluorescence images (middle, right) imaged at different magnifications.

FIG. 6 is a brightfield (left) and fluorescence (right) image of lymphatic vessels in the skull layer after PFBT-COOH injection in the medullary canal of the cerebellum.

FIG. 7 is a brightfield (left) and fluorescence (right) image of facial superficial lymphatic vessels in the skull layer after PFBT-COOH injection in the medullary canal of the cerebellum.

FIG. 8 is a bright field image (left), a fluorescence image (center) and a bright field and fluorescence overlay image (right) of the deep cervical lymph nodes and lymphatic vessels imaged after PFBT-COOH injection in the medullary oblongata pool of the cerebellum.

Fig. 9 is a fluorescence map of the fluorescent conjugated polymer nanoprobe PFBT-COOH for in vivo imaging of ocular vessels (left) and ex vivo imaging of ocular vessels (right).

Fig. 10 is a fluorescence imaging graph (left), a bright field imaging graph (middle) and a superposition graph (right) of fluorescence imaging and bright field imaging of the fluorescence conjugated polymer nano probe PFBT-COOH on isolated extraocular intramuscular blood vessels of normal mice.

Fig. 11 is a fluorescence image of ex vivo imaging ocular vessels with a fluorescent conjugated polymer nanoprobe PFBT-COOH.

FIG. 12 is a fluorescence image of PFBT-COOH imaged blood vessels in kidney (A), spleen (B), lung (C), liver (D), intestine (E) and white adipose tissue (F) of normal mice.

FIG. 13 is a fluorescence image of PFBT imaging blood vessels at different magnifications in brain and cerebellar sites of normal mice when the skull is not opened.

FIG. 14 is a fluorescence image of PFBT imaging blood vessels at different magnifications in the olfactory bulb, brain and cerebellum of normal mice when the skull is opened but the meninges are retained.

FIG. 15 is a fluorescence image of PFBT imaged at different magnifications in the olfactory bulb, brain and cerebellum sites of normal mice when the skull is opened and the meninges is stripped.

FIG. 16 is a fluorescence image of PFBT imaging normal rat cerebral vessels at different magnifications.

FIG. 17 is a fluorescence image of PFBT-COOH imaged the olfactory bulb, brain and cerebellar sites of normal mouse brain at different magnifications when the skull is not opened.

FIG. 18 is a fluorescence image of PFBT-COOH imaging whole brain cerebral vessels of a normal mouse when the skull is opened (left) and a fluorescence image of vessels at a brain part position (middle, right).

FIG. 19 is a fluorescence image of PPV-COOH at different magnifications imaging blood vessels at the olfactory bulb, brain and cerebellar positions of a normal rat brain without opening the skull.

FIG. 20 is a fluorescence image of PPV-COOH imaged whole brain cerebral vessels (upper left) and vessels at a brain part position (upper right, lower left, lower right) in normal mice with the skull opened.

FIG. 21 is a fluorescence image of PFBT-COOH at different magnifications imaging blood vessels at brain and cerebellar sites of an aged mouse when the skull is opened.

FIG. 22 is a fluorescence image of PFBT-COOH imaging blood vessels at the cerebral and cerebellar sites of a stroke model mouse when the skull is opened (left) and a fluorescence image of imaging blood vessels near the ischemic site of the cerebral of a stroke model mouse (right).

FIG. 23 is a fluorescence image of PFBT-COOH imaged blood vessels at brain and cerebellar sites of a stroke model mouse after opening the skull and stripping the meninges.

FIG. 24 is a fluorescence image of vessels on meninges of PFBT-COOH imaged stroke model mice.

Detailed Description

To make the features and effects of the present invention comprehensible to those skilled in the art, general description and definitions are made below with reference to terms and expressions mentioned in the specification and claims. 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 theory or mechanism described and disclosed herein, whether correct or incorrect, should not limit the scope of the present invention in any way, i.e., the present disclosure may be practiced without limitation to any particular theory or mechanism.

The numerical ranges described herein should be considered to have covered and specifically disclosed all possible subranges and any individual numerical value within the range. As used herein, "comprising," "including," "containing," and similar words encompass the meaning of "consisting essentially of … …" and "consisting of … …," for example, when "a comprises B and C," it is to be understood that "a consists essentially of B and C components" and "a consists of B and C" are disclosed herein.

Herein, when embodiments, examples or examples are described, it should be understood that they are not intended to limit the invention to these embodiments, examples or examples. On the contrary, all alternatives, modifications, and equivalents of the methods and materials described herein are intended to be included within the scope of the invention as defined by the appended claims. Those of skill in the art will recognize many methods and materials similar or equivalent to those described herein that can be used in the practice of the present invention.

In this context, for the sake of brevity, not all possible combinations of individual features in the various embodiments, examples or examples are described. Therefore, the respective features in the respective embodiments, examples or examples may be arbitrarily combined as long as there is no contradiction between the combinations of the features, and all possible combinations should be considered as the scope of the present specification.

Herein, the object to be imaged includes animals such as mammals, mice, and humans.

Patent documents CN108653751A and CN112007173A, the entire contents of which are incorporated herein by reference, relate to fluorescent conjugated polymer nanoprobes and methods for their preparation.

The invention aims to develop a method for realizing imaging of a lymphatic system and a blood vessel by using a fluorescent conjugated polymer nano probe. The invention realizes the imaging of the lymphatic system and the blood vessels of the brain with good imaging effect, and the good imaging effect can be reflected in high brightness, high resolution and/or high penetrability. The method can realize high-brightness, high-resolution and rapid in-vivo imaging on the lymphatic system and blood vessels of the brain, particularly the lymphatic vessels of the brain and the whole brain microvascular network by using the simple and convenient integral fluorescence microscope with low cost. The imaging method is simple and effective, has low cost, can effectively distinguish the brain microvascular density of the old subject and the young subject, distinguish the whole cerebral vascular stroke area and the normal blood supply area of the stroke subject, and can carry out high-resolution rapid imaging on the vascular network of eyeballs, the vascular network of extraocular muscles and the vascular network of kidney, liver, spleen, lung, intestine and white adipose tissue, and the imaging effect is obviously better than the existing reports in the literature.

The fluorescent conjugated polymer nanoprobes used in the present invention include fluorescent conjugated polymers.

In the present invention, the fluorescent conjugated polymer is a polymer having two or more conjugated pi bonds in a structural unit and capable of emitting fluorescence under excitation of light. Fluorescent conjugated polymers suitable for use in the present invention include 9, 9-dioctylfluorene-2, 1, 3-benzothiadiazole copolymer (abbreviated as PFBT in the English name) and poly [ 2-methoxy-5- (2-ethylhexyloxy) -1, 4-phenylenevinylene ] (abbreviated as MEH-PPV in the English name).

The structural formula of PFBT is:

wherein n is the degree of polymerization. The weight average molecular weight of the PFBT suitable for the present invention is preferably 10000-52000.

The structural formula of MEH-PPV is:

wherein n is the degree of polymerization. The MEH-PPV suitable for use in the present invention preferably has a weight average molecular weight of 150000-250000.

The fluorescent conjugated polymer nanoprobes used in the present invention may or may not include surface ligands. In some embodiments, the fluorescent conjugated polymer nanoprobes of the present invention comprise only fluorescent conjugated polymers. In some embodiments, the fluorescent conjugated polymer nanoprobes of the invention are comprised of a fluorescent conjugated polymer and a surface ligand.

In the present invention, the surface ligand is an amphiphilic polymer. In the fluorescent conjugated polymer nano probe, the surface ligand is coated on the surface of the fluorescent conjugated polymer, so that the biocompatibility of the probe is improved. Surface ligands suitable for use in the present invention include polystyrene grafted carboxyl end-capsPolyethylene oxide (abbreviated as PS-PEG-COOH in English), amino-terminated polymethyl methacrylate (abbreviated as PMMA-NH in English) ₂ ) And styrene-maleic anhydride copolymer (abbreviated as PSMA in english name).

The structural formula of PS-PEG-COOH is as follows:

wherein n represents the polymerization degree of Polystyrene (PS), and m represents the polymerization degree of grafted polyethylene oxide (PEG). The PS moiety of the PS-PEG-COOH suitable for use in the present invention preferably has a weight average molecular weight of 6500-21700, and the PEG moiety preferably has a weight average molecular weight of 1200-4600.

PMMA-NH ₂ The structural formula of (A) is:

wherein n represents the degree of polymerization. PMMA-NH suitable for use in the present invention ₂ The weight average molecular weight of (1) is preferably 150000-250000.

PSMA can be a polystyrene-polymaleic anhydride diblock copolymer. The weight average molecular weight of PSMA suitable for use in the present invention is preferably 1000-10000.

In some embodiments, the hydrated particle size of the fluorescent conjugated polymer nanoprobes used in the present invention is from 30 to 100nm, such as from 40 to 90nm, from 40 to 60nm, 50 nm. In some embodiments, the fluorescent conjugated polymer nanoprobes used in the present invention have a maximum absorption wavelength of 350-550nm, such as 370nm, 510nm, preferably 350-400nm, and a maximum emission wavelength of 500-650nm, such as 540nm, 590nm, preferably 520-560 nm.

In the fluorescent conjugated polymer nanoprobe used in the invention, the mass ratio of the surface ligand to the fluorescent conjugated polymer can be 0: 1 to 2.5: 1, e.g., 0: 1 to 2.2: 1. 0.8: 1 to 1.2: 1. 1.8: 1 to 2.2: 1. 1: 1. or 2: 1, preferably 0.8: 1 to 1.2: 1.

in some embodiments, the fluorescent conjugated polymer nanoprobes used in the present invention comprise PFBT and/or PPV, but do not contain surface ligands. In some embodiments, the fluorescent conjugated polymer nanoprobes used in the present invention are PFBT. The invention finds that the fluorescent conjugated polymer nanoprobe containing PFBT and/or PPV (particularly PFBT) but not containing surface ligands can realize high-resolution microscopic imaging of the cerebral vessels of a subject in a visible region under the condition of craniotomy or no craniotomy, has higher penetration depth, and has the advantage of simple formula compared with the fluorescent conjugated polymer nanoprobe containing the surface ligands.

In some embodiments, the fluorescent conjugated polymer nanoprobes used in the invention are those in which the fluorescent conjugated polymer is PFBT and the surface ligand is selected from the group consisting of PS-PEG-COOH and PMMA-NH ₂ One or two of them, for example, PS-PEG-COOH, the mass ratio of the surface ligand to the fluorescent conjugated polymer is 0.8: 1 to 1.2: 1. in some embodiments, the fluorescent conjugated polymer nanoprobes used in the present invention comprise PFBT and PS-PEG-COOH. In some embodiments, the fluorescent conjugated polymer nanoprobes used in the present invention, wherein the fluorescent conjugated polymer is MEH-PPV and the surface ligand is selected from the group consisting of PS-PEG-COOH and PMMA-NH ₂ One or two of them, for example, PS-PEG-COOH, the mass ratio of the surface ligand to the fluorescent conjugated polymer is 0.8: 1 to 1.2: 1. in some embodiments, the fluorescent conjugated polymer nanoprobes used in the present invention comprise MEH-PPV and PS-PEG-COOH. The invention finds use with a fluorescent conjugated polymer comprising a polymer selected from PFBT and MEH-PPV and a polymer selected from PS-PEG-COOH and PMMA-NH ₂ The fluorescent conjugated polymer nanoprobe of the surface ligand can realize visible region high-resolution microscopic imaging on the lymphatic vessels of the brain, the blood vessels of the eye, the blood vessels in the extraocular muscles, the blood vessels of the kidney, the blood vessels of the liver, the blood vessels of the spleen, the blood vessels of the lung, the blood vessels of the intestine and the blood vessels in white adipose tissues of a subject.

The fluorescent conjugated polymer nano probe used by the invention is prepared by adopting a method comprising the following steps:

(1) providing a stock solution of a fluorescent conjugated polymer and optionally a stock solution of a surface ligand, wherein the stock solution of the fluorescent conjugated polymer and the stock solution of the surface ligand are formed by respectively dispersing the fluorescent conjugated polymer and the surface ligand in an organic solvent; the organic solvent may be tetrahydrofuran or chloroform;

(2) mixing a stock solution of the fluorescent conjugated polymer and a stock solution of an optional surface ligand, adding an organic solvent for dilution according to needs, and performing ultrasonic treatment to obtain a first mixed solution; the concentration of the first mixed solution can be 0.1-0.5 mg/mL; the sonication time may be 2-5 minutes, e.g. 3 minutes;

(3) adding the first mixed solution into water, and performing ultrasonic treatment to obtain a second mixed solution; the volume ratio of the first mixed liquid to the water may be 1: 2 to 1: 10, e.g. 1: 5; the sonication power may be 8-20%, e.g. 10%, may be 2-4 seconds per sonication 4-6 seconds, e.g. 3 seconds per sonication 5 seconds, and the total sonication time may be 0.5-2 minutes, e.g. 1 minute;

(4) introducing nitrogen into the second mixed solution, and removing the organic solvent in the second mixed solution to obtain an aqueous solution of the fluorescent conjugated polymer nanoprobe; nitrogen may be introduced under heating conditions, for example, at 45-55 ℃ and 50 ℃.

After the aqueous solution of the fluorescent conjugated polymer nanoprobe is prepared, the aqueous solution can be adjusted to the required concentration by adding water for dilution or concentrating by using an ultrafiltration tube.

The contrast agent of the present invention includes a fluorescent conjugated polymer nanoprobe. The contrast agent can contain a carrier or an excipient which is commonly used for the contrast agent besides the fluorescent conjugated polymer nano probe. For example, the carrier or excipient may be water, physiological saline, buffer solutions, various organic macromolecules, or various types of nanostructures formed of inorganic materials, and the like. The buffer solution may be phosphate buffered saline (PBS buffer). The contrast agent may also contain a suitable amount of a co-solvent. In some embodiments, the contrast agent is an aqueous solution, physiological saline solution, or buffer solution of the fluorescent conjugated polymer nanoprobe. In the contrast agent in the form of a solution, the concentration of the fluorescent conjugated polymer nanoprobe may be 0.01-1mg/mL, for example, 0.01-0.5mg/mL, 0.02-0.5 mg/mL.

The present invention uses the contrast agent of the present invention to image the lymphatic system and blood vessels of the brain. In the invention, the imageable cerebral lymphatic system comprises meningeal lymphatic vessels, facial lymph nodes, neck lymph nodes and neck lymphatic vessels, the imageable blood vessels comprise cerebral blood vessels, ocular blood vessels, extraocular intramuscular blood vessels, renal blood vessels, hepatic blood vessels, splenic blood vessels, pulmonary blood vessels, intestinal blood vessels and white adipose tissue blood vessels, the imaging of cerebral blood vessels can be full cerebral blood vessel imaging or partial cerebral blood vessel imaging, the full cerebral blood vessels comprise full cerebral blood vessels before and after the meninges is stripped, the partial cerebral blood vessels comprise cerebral position blood vessels, cerebellum position blood vessels and meningeal blood vessels, and the ocular blood vessels comprise iris blood vessels, iris artery annuli, iris artery macrocycle, retrociliary artery, scleral venous sinus and choroid blood vessels. In some embodiments, the contrast agent used to image the lymphatic system of the brain comprises a fluorescent conjugated polymer nanoprobe comprising PFBT and PS-PEG-COOH. In some embodiments, the contrast agent used to image the blood vessel comprises a fluorescent conjugated polymer nanoprobe that is PFBT, or comprises PFBT and PS-PEG-COOH, or comprises MEH-PPV and PS-PEG-COOH.

In the present invention, the imaging mode may be in vivo imaging and ex vivo imaging. In the present invention, the imaging is preferably bulk fluorescence microscopy imaging. For example, the imaging may be in vivo or ex vivo fluorescence microscopy imaging. In some embodiments, the invention uses the contrast agents of the invention for in vivo fluorescence microscopy imaging of the lymphatic system of the brain. In some embodiments, the invention uses the contrast agents of the invention to image brain vessels, eye vessels, extraocular intramusculary vessels, renal vessels, hepatic vessels, spleen vessels, pulmonary vessels, intestinal vessels, and/or white adipose tissue vessels for in vivo or ex vivo fluorescence microscopy imaging. In the present invention, imaging the lymphatic system or the blood vessels of the brain may be craniotomy imaging or non-craniotomy imaging. In the present invention, imaging the cerebral blood vessels may be imaging cerebral blood vessels of a normal subject, an elderly subject, and/or a stroke subject. In the present invention, a normal subject refers to a subject who has not entered the elderly and has not suffered a stroke, as generally understood in the art.

In the present invention, the contrast agent of the present invention is administered to a subject by means of a lateral ventricular injection, a cisterna magna injection, a cardiac perfusion or a tail vein injection. For example, when imaging the lymphatic system of the brain of a subject, the contrast agent of the present invention may be administered by means of a lateral ventricular injection or a cerebellar medullary cistern injection. In some embodiments, after injecting the contrast agent through the lateral ventricle or cisterna magna, the subject's heart is perfused with a PBS buffer solution. The invention discovers that the contrast agent can realize high-resolution microscopic imaging of a visible region of a brain lymphatic system by adopting a body type fluorescence microscope by adopting a lateral ventricle injection or cerebellum medulla oblongata injection mode to administer the contrast agent. The contrast agent of the present invention may be administered by cardiac perfusion when performing integrated fluorescence microscopy imaging of cerebral, ocular, extraocular, intramusculary, renal, hepatic, splenic, pulmonary, intestinal, and/or white adipose tissue vessels of a subject. The method of operation of cardiac perfusion may be: the chest wall is cut off along the axillary line after the diaphragm muscle of the subject is cut off, the chest wall is turned upwards to expose the heart, then the right auricle is cut off, the apex of the heart is lifted, a syringe filled with liquid (such as contrast medium) is inserted into the left ventricle and faces the direction of the aorta, and then the liquid is injected into the subject at a constant speed. The invention discovers that the contrast agent can realize visible region high-resolution microscopic imaging of blood vessels, particularly cerebral blood vessels by using a body type fluorescence microscope when the contrast agent is applied by adopting a heart perfusion mode.

The concentration of the contrast agent in the form of a solution of the present invention (content of the fluorescent conjugated polymer nanoprobe) may be 0.01-1mg/mL, such as 0.01-0.5mg/mL, 0.02-0.5 mg/mL. In some embodiments, the contrast agent of the present invention is administered at a concentration of 0.1-1mg/mL when imaging the lymphatic system of the brain of a subject, e.g., the contrast agent can have a concentration of 0.2-0.5mg/mL, e.g., 0.4 + -0.1mg/mL, when the contrast agent comprises a fluorescent conjugated polymer nanoprobe comprising PFBT and PS-PEG-COOH. In some embodiments, the contrast agent of the present invention is administered at a concentration of 0.01 to 0.1mg/mL when performing integrated fluorescence microscopy imaging of cerebral vessels, ocular vessels, extraocular intramusculary vessels, renal vessels, hepatic vessels, spleen vessels, pulmonary vessels, intestinal vessels, and/or white adipose tissue vessels in a subject, e.g., the concentration of the contrast agent can be 0.02 ± 0.01mg/mL when the contrast agent comprises a fluorescent conjugated polymer nanoprobe that is PFBT; when the fluorescent conjugated polymer nanoprobe contained in the contrast agent comprises PFBT and PS-PEG-COOH, the concentration of the contrast agent can be 0.04 +/-0.01 mg/mL; when the fluorescent conjugated polymer nanoprobe contained in the contrast agent comprises MEH-PPV and PS-PEG-COOH, the concentration of the contrast agent can be 0.08 +/-0.01 mg/mL.

In the present invention, the lymphatic system of the brain of a subject is imaged, and the contrast agent of the present invention may be administered by means of a lateral ventricular injection or a cisterna magna injection. For example, the subject may be anesthetized, the skull of the subject surgically exposed, then the contrast agent of the present invention is injected at a concentration of 0.1-1mg/mL, such as 0.2-0.5mg/mL or 0.4mg/mL into the lateral ventricle or cisterna magna, then the subject is subjected to cardiac perfusion surgery with phosphate buffered saline (PBS buffered saline), then the subject's skull is optionally opened, the subject's facial skin is dissected, or the subject's cervical skin is dissected, and then the subject's meningeal, facial, cervical and/or cervical lymphatic vessels are imaged. The imaging may be volumetric fluorescence microscopy imaging. In some embodiments, the fluorescent conjugated polymer nanoprobe comprises PFBT and PS-PEG-COOH when imaging the lymphatic system of the brain of the subject, preferably in a mass ratio of 0.8: 1 to 1.2: the hydrated particle size of the fluorescent conjugated polymer nanoprobe is preferably 40-60 nm.

In the present invention, the contrast agent of the present invention can be administered by cardiac perfusion when imaging a blood vessel of a subject by an integral fluorescence microscope. For example, a subject may be anesthetized, a contrast agent of the present invention at a concentration of 0.01-0.1mg/mL, such as 0.02mg/mL, 0.04mg/mL, or 0.08mg/mL, may be injected into the subject by cardiac perfusion, the skull of the subject may be surgically exposed, and the skull of the subject may then optionally be opened, the meninges stripped, and/or the brain tissue dissected, or the corresponding tissue organ (e.g., eyeball, extraocular muscle, kidney, liver, spleen, lung, intestine, white adipose tissue) dissected, and the blood vessels of the subject may then be subjected to integrated fluorescence microscopy. In some embodiments, when performing bulk fluorescence microscopy imaging of a blood vessel of a subject, the fluorescent conjugated polymer nanoprobe is PFBT, or comprises PFBT and PS-PEG-COOH, or comprises MEH-PPV and PS-PEG-COOH, preferably PFBT or comprises PFBT and PS-PEG-COOH, wherein the mass ratio of PS-PEG-COOH to PFBT or PS-PEG-COOH to MEH-PPV is preferably 0.8: 1 to 1.2: the hydrated particle size of the fluorescent conjugated polymer nanoprobe is preferably 40-60 nm.

The invention also includes a method of imaging the lymphatic system of the blood vessels and the brain comprising administering to a subject a contrast agent of the invention. In the imaging method of the invention, the object and site to be imaged, the mode of administering the contrast agent and the mode of imaging are as described in any of the embodiments above.

Some cerebral vascular imaging techniques in the prior art suffer from the following problems: (1) the brain blood vessel in-vivo near-infrared two-zone fluorescence imaging needs imaging equipment such as an expensive near-infrared two-zone camera, and the resolution is still not enough to image a tiny pipeline; (2) the brain blood vessel two-photon imaging needs the help of expensive two-photon femtosecond laser and other equipment, has small imaging range and long imaging time, and can not image the whole brain blood vessel. The contrast agent, the application and the imaging method overcome the problems and realize the high-resolution rapid in-vivo imaging of the visible light region of the cerebral vessels, particularly the whole cerebral vessels by adopting the low-cost body type fluorescence microscope. The contrast agent, the application and the imaging method also realize the high-resolution and rapid in-vivo imaging of the visible light region of the brain lymphatic system, particularly meningeal lymphatic vessels by adopting a low-cost body type fluorescence microscope.

The invention has the following beneficial effects:

(1) the contrast agent, the application and the imaging method realize the high-resolution rapid in-vivo fluorescence imaging of visible light regions of blood vessels (particularly cerebral blood vessels) and a cerebral lymphatic system by adopting a low-cost in-vivo fluorescence microscope, and the preparation process of the adopted fluorescence conjugated polymer nanoprobe is simple;

(2) the contrast agent, the application and the imaging method of the invention have the advantages of high-brightness fluorescence signal and high penetration depth when the skull is not opened;

(3) the contrast agent, the application and the imaging method have the advantage of high resolution when the skull is opened for imaging cerebral vessels;

(4) the contrast agent, the application and the imaging method realize that the accompanying arterial vein blood vessels can be imaged simultaneously when the cranial bones are opened to image the cerebral blood vessels;

(5) the contrast agent, the application and the imaging method realize in-vivo imaging of the eye vascular network, in-vitro imaging of the eyeball vascular network and in-vitro imaging of the extraocular intramuscular vascular network;

(6) the contrast agent, application and imaging method of the invention enable high-brightness high-resolution imaging of vascular networks within tissue organs (e.g. kidney, liver, spleen, lung, intestine, white adipose tissue) of a subject;

(7) the contrast agent, the application and the imaging method realize that the blood vessel density difference of an old subject and a young subject can be compared when the cranial imaging cerebral blood vessel is opened;

(8) the contrast agent, the application and the imaging method realize that the ischemic part and the normal blood supply part of the brain of the stroke model object can be distinguished when the cranial bone imaging cerebral blood vessel is opened;

(9) the contrast agent, the application and the imaging method realize imaging of meningeal blood vessels of normal model and stroke model objects;

(10) the contrast agent, the application and the imaging method realize imaging of meningeal lymph vessels without opening a skull or opening the skull after injecting the probe into a lateral ventricle;

(11) the contrast agent, the application and the imaging method realize imaging of meningeal lymph vessels without opening a skull after injecting the probe into a cerebellar medullary canal;

(12) the contrast agent, the application and the imaging method realize imaging of the superficial lymphatic system, the deep cervical lymph nodes and the lymphatic vessels of the face after the probe is injected into the medullary oblongata pool of the cerebellum;

(13) the contrast agent, the application and the imaging method are suitable for a domestic fluorescence microscope, and have the advantages of convenience in operation, economy, practicability and the like.

The present invention is further illustrated by the following specific examples. The scope of the present invention is not limited by the contents of the following examples. The scope of the present invention is defined only by the appended claims, and any omissions, substitutions, and changes in the form of the embodiments disclosed herein that may be made by those skilled in the art are intended to be included within the scope of the present invention.

The following examples use instrumentation conventional in the art. The experimental methods and detection methods without specifying specific conditions in the following examples are generally performed under conventional conditions or under conditions recommended by the manufacturers. The various starting materials used in the following examples, unless otherwise specified, were conventional commercially available products.

In the examples, the hydrated particle size distribution of the fluorescent conjugated polymer nanoprobe was measured using a Malvern Zetasizer Nano ZSP particle size analyzer (DLS), the ultraviolet absorption spectrum of the fluorescent conjugated polymer nanoprobe was measured using a UV-1901PC ultraviolet spectrophotometer, and the fluorescence emission spectrum of the fluorescent conjugated polymer nanoprobe was measured using a SpectraMax i3x microplate reader.

Example 1: preparation and performance characterization of fluorescent conjugated polymer nanoprobe PFBT

Accurately pipette 0.2mL of PFBT stock at 1mg/mL into 1800 μ L of filtered Tetrahydrofuran (THF), and ultrasonically mix for 3 minutes to obtain a mixture, wherein the PFBT stock is prepared from THF as a solvent. Under the ultrasonic condition, the mixed solution is rapidly added into 10mL of ultrapure water, the ultrasonic power is set to be 10%, each ultrasonic treatment lasts for 3 seconds every 5 seconds, and the total ultrasonic treatment time is set to be 1 minute. After the ultrasonic treatment is finished, THF in the solution is completely volatilized by introducing nitrogen for about 25 minutes at 50 ℃, and an aqueous solution of the PFBT fluorescent conjugated polymer nano probe with the concentration of 0.02mg/mL is prepared.

Example 2: preparation and performance characterization of fluorescent conjugated polymer nano probe PFBT-COOH

Accurately sucking 0.2mL of PFBT stock solution with the concentration of 1mg/mL and 0.2mL of PS-PEG-COOH stock solution with the concentration of 1mg/mL into 1600 μ L of filtered Tetrahydrofuran (THF), and ultrasonically mixing for 3 minutes to obtain a mixed solution, wherein the solvents of the PFBT stock solution and the PS-PEG-COOH stock solution are both prepared by taking THF as a solvent. Under the ultrasonic condition, the mixed solution is rapidly added into 10mL of ultrapure water, the ultrasonic power is set to be 10%, each ultrasonic treatment lasts for 3 seconds every 5 seconds, and the total ultrasonic treatment time is set to be 1 minute. After the completion of the ultrasonic treatment, THF in the solution is completely volatilized by introducing nitrogen for about 25 minutes at 50 ℃, and an aqueous solution of the PFBT-COOH fluorescent conjugated polymer nanoprobe with the concentration of 0.04mg/mL is prepared.

Example 3: fluorescent conjugated polymer nano probe PFBT-NH ₂ Preparation and Performance characterization of

Accurately sucking 0.2mL of PFBT stock solution with the concentration of 1mg/mL and 0.2mL of PMMA-NH with the concentration of 1mg/mL ₂ Adding the stock solution to 1600 μ L of filtered Tetrahydrofuran (THF), and ultrasonically mixing for 3 min to obtain a mixed solution, wherein the PFBT stock solution and PMMA-NH ₂ The solvent of the stock solution is prepared by taking THF as a solvent. Under the ultrasonic condition, the mixed solution is rapidly added into 10mL of ultrapure water, the ultrasonic power is set to be 10%, each ultrasonic treatment lasts for 3 seconds every 5 seconds, and the total ultrasonic treatment time is set to be 1 minute. After the completion of the sonication, the THF in the solution was completely volatilized at 50 ℃ by introducing nitrogen gas for about 25 minutes to prepare PFBT-NH with a concentration of 0.04mg/mL ₂ An aqueous solution of a fluorescent conjugated polymer nanoprobe.

FIG. 1 shows fluorescent conjugated polymer nanoprobes PFBT, PFBT-COOH and PFBT-NH ₂ Ultraviolet absorption spectrum (left), fluorescence emission spectrum (middle), and hydrated particle size distribution map (right). From the figure, it can be seen that the three fluorescent conjugated polymer nanoprobes have maximum absorption at about 370nm and maximum emission at about 540nm, the hydrated particle size is about 50nm, and the fluorescent intensity of the fluorescent conjugated polymer nanoprobe PFBT is strongest.

Example 4: preparation and performance characterization of fluorescent conjugated polymer nanoprobe PPV

Accurately 0.4mL of a 1mg/mL MEH-PPV stock solution prepared from THF as a solvent was added to 1600 μ L of filtered Tetrahydrofuran (THF) and mixed with sonication for 3 minutes to obtain a mixture. Under the ultrasonic condition, the mixed solution is rapidly added into 10mL of ultrapure water, the ultrasonic power is set to be 10%, each ultrasonic treatment lasts for 3 seconds every 5 seconds, and the total ultrasonic treatment time is set to be 1 minute. After the completion of the ultrasonic treatment, THF in the solution was completely volatilized by introducing nitrogen gas at 50 ℃ for about 25 minutes to prepare an aqueous solution of the PPV fluorescent conjugated polymer nanoprobe with a concentration of 0.04 mg/mL.

Example 5: preparation and performance characterization of fluorescent conjugated polymer nano probe PPV-COOH

Accurately sucking 0.4mL of MEH-PPV stock solution with the concentration of 1mg/mL and 0.4mL of PS-PEG-COOH stock solution with the concentration of 1mg/mL into 1200 mu L of filtered Tetrahydrofuran (THF), and ultrasonically mixing for 3 minutes to obtain a mixed solution, wherein the MEH-PPV stock solution and the PS-PEG-COOH stock solution are both prepared by taking THF as a solvent. Under the ultrasonic condition, the mixed solution is rapidly added into 10mL of ultrapure water, the ultrasonic power is set to be 10%, each ultrasonic treatment lasts for 3 seconds every 5 seconds, and the total ultrasonic treatment time is set to be 1 minute. After the ultrasonic treatment is finished, THF in the solution is completely volatilized by introducing nitrogen for about 25 minutes at 50 ℃, and an aqueous solution of the PPV-COOH fluorescent conjugated polymer nanoprobe with the concentration of 0.08mg/mL is prepared.

Example 6: fluorescent conjugated polymer nanoprobe PPV-NH ₂ Preparation and Performance characterization of

Accurately sucking 0.4mL of MEH-PPV storage solution with the concentration of 1mg/mL and 0.4mL of PMMA-NH with the concentration of 1mg/mL ₂ Adding the stock solution to 1200 μ L of filtered Tetrahydrofuran (THF), and ultrasonically mixing for 3 min to obtain a mixed solution, wherein MEH-PPV stock solution and PMMA-NH ₂ The stock solutions were all prepared with THF as the solvent. Under the ultrasonic condition, the mixed solution is rapidly added into 10mL of ultrapure water, the ultrasonic power is set to be 10%, each ultrasonic treatment lasts for 3 seconds every 5 seconds, and the total ultrasonic treatment time is set to be 1 minute. After the completion of the sonication, THF in the solution was completely volatilized by introducing nitrogen gas at 50 ℃ for about 25 minutes to prepare PPV-NH having a concentration of 0.08mg/mL ₂ An aqueous solution of a fluorescent conjugated polymer nanoprobe.

FIG. 2 shows fluorescent conjugated polymer nanoprobes PPV, PPV-COOH, PPV-NH ₂ Ultraviolet absorption spectrum (left) fluorescence emission spectrum (middle) and hydrated particle size distribution map (right). From the figure, it can be seen that the three fluorescent conjugated polymer nanoprobes have maximum absorption at about 510nm, maximum emission at about 590nm and hydrated particle size at about 60nm, wherein the fluorescent conjugated polymer nanoprobe PPV-COOH has the strongest fluorescence intensity.

Example 7: quantum yield

And (3) determining the absolute fluorescence quantum yield of the nanoprobe by using an integral spherical fluorescence quantum yield instrument. For the nanoprobe of example 1, the wavelength of xenon lamp excitation was 370 nm; for the nanoprobes of example 2, an excitation wavelength of 509nm was chosen.

Table 1: quantum yield of nanoprobes

Nano probe	Absolute quantum yield (%)
		Example 2(PFBT-COOH)	94.2
Example 5(PPV-COOH)	11.7

Example 8: penetration depth test

The PFBT-COOH fluorescent conjugated polymer nanoprobe prepared in example 2 was concentrated to a concentration of 0.2mg/mL, 10. mu.L of 0.2mg/mL probe solution was aspirated through a glass tube having a diameter of 100. mu.m, and chicken breast slices having a thickness of 0mm, 1mm, 2mm, and 3mm were coated on the glass tube, and then the glass tube was photographed under a fluorescent microscope.

FIG. 3 is a result of a penetration depth experiment of the PFBT-COOH fluorescent conjugated polymer nanoprobe, which shows that stronger fluorescence can be seen when a 2mm meat slice is covered on a glass tube, indicating that the imaging penetration depth of the PFBT-COOH fluorescent conjugated polymer nanoprobe exceeds 2 mm.

Example 9: imaging research of meningo-lymphatic vessels of lateral ventricle injected fluorescent conjugated polymer nanoprobes PFBT-COOH when skull is not opened

After deeply anesthetizing the rat, the skull of the rat was exposed by cutting the skin of the rat head through a surgical operation, 10 μ L of an aqueous solution of the PFBT-COOH fluorescent conjugated polymer nanoprobe prepared in example 2 with a concentration of 0.4mg/mL was injected into the left ventricle of the rat, the heart perfusion operation was performed on the rat using 20mL of a 4 ℃ PBS buffer solution with a pH of 7.4, and finally the brain region of the rat was imaged under a body type fluorescence microscope, with the results shown in fig. 4.

The left and right panels in fig. 4 are a bright field and fluorescence image, respectively, of meningeal lymphatic vessels imaged in unopened skull after lateral ventricle injection of PFBT-COOH. From fig. 4, it can be seen that PFBT-COOH can clearly image meningo-lymphatic vessels when the skull is not opened.

Example 10: imaging research of meningeal lymphatic vessel of lateral ventricle injected fluorescent conjugated polymer nanoprobe PFBT-COOH when skull is opened

After deeply anesthetizing the rat, the skull of the rat was exposed by cutting the skin of the head of the rat through a surgical operation, then 10 μ L of an aqueous solution of the PFBT-COOH fluorescent conjugated polymer nanoprobe prepared in example 2 with a concentration of 0.4mg/mL was injected into the left ventricle of the rat, then the heart perfusion operation was performed on the rat with 20mL of a PBS buffer solution with a pH of 7.4 at 4 ℃, then the skin of the head of the rat was cut off through a surgical operation and the skull was opened to expose the meninges, and finally the exposed meningeal region was imaged under a stereomicroscope, with the result as shown in fig. 5.

Fig. 5 is a bright field image (left panel) and fluorescence image at different magnifications (middle panel and right panel) of meningeal lymphatic vessels imaged when the skull is opened after lateral ventricle injection of PFBT-COOH. From fig. 5, it can be seen that PFBT-COOH has better fluorescence imaging effect on meningeal lymphatic vessels in the condition of opening the skull.

Example 11: imaging research on brain lymphatic system of cerebellum medulla oblongata injected with fluorescent conjugated polymer nano probe PFBT-COOH when skull is not opened

After deeply anesthetizing the mouse, the skull of the mouse was exposed by surgical incision of the skin at the head of the mouse, 10. mu.L of an aqueous solution of the PFBT-COOH fluorescent conjugated polymer nanoprobe prepared in example 2 was injected into the medullary canal of the mouse brain at a concentration of 0.4mg/mL, the heart perfusion operation was performed on the mouse using 20mL of a PBS buffer solution at 4 ℃ and pH of 7.4, and finally the brain region of the mouse was imaged under an integral fluorescence microscope, and the results are shown in FIG. 6.

The left and right panels in FIG. 6 are brightfield and fluorescence views, respectively, of lymphatic vessels imaged at the cranial layer after PFBT-COOH injection in the cisterna magna. From fig. 6, it can be seen that PFBT-COOH can clearly image meningeal lymphatic vessels when the skull is not opened.

Example 12: imaging research of injecting fluorescent conjugated polymer nano probe PFBT-COOH into cerebellum medulla oblongata pool to superficial lymphatic vessels and lymph nodes of mouse face

After deeply anesthetizing the mouse, the skull of the mouse was exposed by surgical incision of the skin on the mouse head, 10. mu.L of an aqueous solution of the PFBT-COOH fluorescent conjugated polymer nanoprobe prepared in example 2 was injected into the medullary oblongata of the mouse, the heart perfusion of the mouse was performed with 20mL of a PBS buffer solution at 4 ℃ and pH 7.4, and then the skin on the mouse face was excised and imaged under a body type fluorescence microscope, and the results are shown in FIG. 7.

The left and right images in fig. 7 are a bright field image and a fluorescence image, respectively, of the superficial lymphatic vessels of the face imaged in the skull layer after PFBT-COOH injection in the cisterna magna. From FIG. 7, it can be seen that PFBT-COOH can clearly image superficial lymphatic vessels and lymph nodes in the face of mice.

Example 13: imaging research of injecting fluorescent conjugated polymer nano probe PFBT-COOH into cerebellum medulla oblongata pool to deep lymph nodes and lymph vessels of neck

After deeply anesthetizing the mouse, the skull of the mouse was exposed by surgical incision of the skin at the head of the mouse, 10. mu.L of an aqueous solution of the PFBT-COOH fluorescent conjugated polymer nanoprobe prepared in example 2 was injected into the medullary canal of the mouse, at a concentration of 0.4mg/mL, and then the heart perfusion operation was performed on the mouse using 20mL of a PBS buffer solution at 4 ℃ and pH 7.4, the skin at the neck of the mouse was carefully excised, and then the image was imaged under a full body fluorescence microscope, and the result was as shown in FIG. 8.

The left, middle and right panels in fig. 8 are the bright field, fluorescence and bright field-fluorescence overlay of the imaged deep cervical lymph nodes and lymphatic vessels after PFBT-COOH injection in the cisterna magna, respectively. From fig. 8, it can be seen that PFBT-COOH can clearly image deep cervical lymph nodes and lymphatic vessels.

Example 14: imaging research of fluorescent conjugated polymer nanoprobe PFBT-COOH on in-vivo and in-vitro eye blood vessels of 4-week-old normal mice

After deeply anesthetizing the mouse, 30mL of the aqueous solution of the PFBT-COOH fluorescent conjugated polymer nanoprobe prepared in example 2 with the concentration of 0.04mg/mL is sucked by an injector, the diaphragm of the mouse is cut off, the chest wall is quickly cut off along the anterior axillary line, the chest wall is turned upwards to expose the heart, then the right auricle is cut off by scissors, the apex of the heart is lifted, the prepared injector of the aqueous solution of the fluorescent conjugated polymer nanoprobe is inserted into the left ventricle and faces the direction of the aorta, and then the liquid is injected into the mouse at a constant speed. The eye was imaged by a bulk fluorescence microscope, and the results are shown in fig. 9.

The left and right images in fig. 9 are fluorescence images of the fluorescent conjugated polymer nanoprobe PFBT-COOH in vivo imaging of ocular vessels and ex vivo imaging of ocular vessels, respectively. As can be seen from fig. 9, the fluorescent conjugated polymer nanoprobe PFBT-COOH can clearly image ocular vessels in vivo and has high brightness.

Example 15: imaging research of fluorescent conjugated polymer nano probe PFBT-COOH on isolated extraocular intramyovascular of 4-week-old normal mice

After deeply anesthetizing the mouse, 30mL of the aqueous solution of the PFBT-COOH fluorescent conjugated polymer nanoprobe prepared in example 2 with the concentration of 0.04mg/mL is sucked by an injector, the diaphragm of the mouse is cut off, the chest wall is quickly cut off along the anterior axillary line, the chest wall is turned upwards to expose the heart, then the right auricle is cut off by scissors, the apex of the heart is lifted, the prepared injector of the aqueous solution of the fluorescent conjugated polymer nanoprobe is inserted into the left ventricle and faces the direction of the aorta, and then the liquid is injected into the mouse at a constant speed. The extracted extraocular muscle portion of the eyeball was subjected to integral fluorescence microscope imaging, and the result is shown in fig. 10.

The left, middle and right images in fig. 10 are respectively fluorescence imaging, bright field imaging and fluorescence imaging and bright field imaging superposition of the fluorescence conjugated polymer nanoprobe PFBT-COOH on the blood vessels in the extraocular muscles of normal mice. From fig. 10, it can be seen that the fluorescent conjugated polymer nanoprobe PFBT-COOH can perform high-resolution imaging on the isolated extraocular intramyovascular of normal mice.

Example 16: imaging research of fluorescent conjugated polymer nanoprobe PFBT-COOH on isolated ocular vessels of 4-week-old normal mice

After deeply anesthetizing the mouse, 30mL of the aqueous solution of the PFBT-COOH fluorescent conjugated polymer nanoprobe prepared in example 2 with the concentration of 0.04mg/mL is sucked by an injector, the diaphragm of the mouse is cut off, the chest wall is quickly cut off along the anterior axillary line, the chest wall is turned upwards to expose the heart, then the right auricle is cut off by scissors, the apex of the heart is lifted, the prepared injector of the aqueous solution of the fluorescent conjugated polymer nanoprobe is inserted into the left ventricle and faces the direction of the aorta, and then the liquid is injected into the mouse at a constant speed. The extracted eyeball was imaged by a stereomicroscope, and the result is shown in fig. 11.

Fig. 11 is a fluorescence image of ex vivo imaging ocular vessels with a fluorescent conjugated polymer nanoprobe PFBT-COOH. From fig. 11, it can be seen that the fluorescent conjugated polymer nanoprobe PFBT-COOH can be used for high-resolution imaging of isolated eyeball iris vessels, iris artery annuluses, iris artery macrocycles, posterior long arteries, schlemm's canal, and choroidal vessels of normal mice.

Example 17: imaging research of fluorescent conjugated polymer nanoprobe PFBT-COOH on tissue and organ vascular network of 4-week-old normal mice

After deeply anesthetizing the mouse, 30mL of the aqueous solution of the PFBT-COOH fluorescent conjugated polymer nanoprobe prepared in example 2 with the concentration of 0.04mg/mL is sucked by an injector, the diaphragm of the mouse is cut off, the chest wall is quickly cut off along the anterior axillary line, the chest wall is turned upwards to expose the heart, then the right auricle is cut off by scissors, the apex of the heart is lifted, the prepared injector of the aqueous solution of the fluorescent conjugated polymer nanoprobe is inserted into the left ventricle and faces the direction of the aorta, and then the liquid is injected into the mouse at a constant speed. The kidney, liver, spleen, lung, intestine, white fat of the mouse were dissected and dissected by surgery, and fluorescence imaging was performed under a stereomicroscope, and the results are shown in fig. 12.

FIG. 12 is a diagram of the fluorescence of blood vessels in a part of tissues and organs of a normal mouse imaged by a fluorescent conjugated polymer nano probe PFBT-COOH. From fig. 12, it can be seen that the fluorescent conjugated polymer nanoprobe PFBT-COOH can image the vascular network in the kidney, liver, spleen, lung, intestine and white adipose tissue of the mouse with high resolution.

Example 18: imaging research of fluorescent conjugated polymer nanoprobe PFBT on whole cerebral vessels of 4-week-old normal mice under condition of not opening skull

After deeply anesthetizing the mouse, 30mL of the aqueous solution of the PFBT fluorescent conjugated polymer nanoprobe prepared in example 1 with the concentration of 0.02mg/mL is sucked by an injector, the diaphragm of the mouse is cut off, the chest wall is quickly cut off along the anterior axillary line, the chest wall is turned upwards to expose the heart, then the right auricle is cut off by scissors, the apex of the heart is lifted, the prepared injector of the aqueous solution of the fluorescent conjugated polymer nanoprobe is inserted into the left ventricle and faces the direction of the aorta, and then the liquid is injected into the mouse at a constant speed. The skin of the rat head was surgically cut to expose the skull bone of the rat, and the exposed skull bone position was imaged with a stereo fluorescence microscope, the results of which are shown in fig. 13.

FIG. 13 is a graph showing the results of imaging of different magnification fluorescence of blood vessels at brain and cerebellum positions in normal mice without opening the skull after heart perfusion with 30mL of aqueous PFBT fluorescence conjugated polymer nanoprobe with a concentration of 0.02 mg/mL. From fig. 13, it can be seen that the fluorescent conjugated polymer nanoprobe PFBT has higher fluorescence signal brightness and can image whole cerebral vessels without opening the skull, which indicates that the cerebral vessel imaging method of the present invention has higher penetration depth.

Example 19: imaging research of fluorescent conjugated polymer nanoprobe PFBT on whole cerebral vessels of 4-week-old normal mice under condition of opening skull

After a mouse is deeply anesthetized, 30mL of aqueous solution of the PFBT fluorescent conjugated polymer nanoprobe prepared in the example 1 with the concentration of 0.02mg/mL is sucked by a syringe, the diaphragm of the mouse is cut open, the chest wall is quickly cut along the line in front of the axilla, the chest wall is turned upwards to expose the heart, then the right auricle is cut open by scissors, the apex of the heart is lifted, the prepared syringe of the aqueous solution of the fluorescent conjugated polymer nanoprobe is inserted into the left ventricle and faces the direction of the aorta, and then the liquid is injected into the mouse at a constant speed. The skull was cut open and the meninges were retained by surgical operation on the rat's head skin, and the exposed whole cerebral vascular sites were imaged with a full-body fluorescence microscope, and the results are shown in fig. 14.

FIG. 14 is a graph of the imaging results of opening the blood vessels of the whole brain and brain of a normal rat by skull imaging after heart perfusion of 30mL of PFBT fluorescent conjugated polymer nanoprobe aqueous solution with the concentration of 0.02 mg/mL. From fig. 14, it can be seen that the fluorescence conjugated polymer nanoprobe PFBT has very high fluorescence signal brightness and can clearly image olfactory bulb, brain and cerebellar vessels after opening the skull (upper left, upper right), and at the same time, it can be seen that the fluorescence conjugated polymer nanoprobe PFBT has excellent resolution in imaging cerebral vessels after opening the skull, and the measurable minimum vessel size is 2.95 μm.

Example 20: imaging study of fluorescent conjugated polymer nanoprobe PFBT on whole cerebral vessels of 4-week-old normal mice under conditions of opening skull and stripping meninges

After deeply anesthetizing the mouse, 30mL of the aqueous solution of the PFBT fluorescent conjugated polymer nanoprobe prepared in example 1 with the concentration of 0.02mg/mL is sucked by an injector, the diaphragm of the mouse is cut off, the chest wall is quickly cut off along the anterior axillary line, the chest wall is turned upwards to expose the heart, then the right auricle is cut off by scissors, the apex of the heart is lifted, the prepared injector of the aqueous solution of the fluorescent conjugated polymer nanoprobe is inserted into the left ventricle and faces the direction of the aorta, and then the liquid is injected into the mouse at a constant speed. The rat head skin was surgically cut open and the skull was opened and the meninges were stripped and the exposed whole cerebral vessel sites were imaged with a stereomicroscope, the results of which are shown in fig. 15.

FIG. 15 is a graph showing the results of imaging whole brain blood vessels of normal mice after opening the skull and stripping the meninges after heart perfusion with 30mL of aqueous solution of PFBT fluorescent conjugated polymer nanoprobes with a concentration of 0.02 mg/mL. From fig. 15, it can be seen that the fluorescence conjugated polymer nanoprobe PFBT has very high fluorescence signal brightness, and can clearly image olfactory bulb, brain and small cerebral vessels (upper left graph and upper right graph) after the skull is opened to strip off the meninges, and it can be seen that the fluorescence conjugated polymer nanoprobe PFBT has excellent resolution in imaging cerebral vessels after the skull is opened to strip off the meninges, and the measurable minimum blood vessel size is 2.27 μm.

Example 21: imaging research of fluorescent conjugated polymer nanoprobe PFBT on meningeal blood vessels of 4-week-old normal mice

After deeply anesthetizing the mouse, 30mL of the aqueous solution of the PFBT fluorescent conjugated polymer nanoprobe prepared in example 1 with the concentration of 0.02mg/mL is sucked by an injector, the diaphragm of the mouse is cut off, the chest wall is quickly cut off along the anterior axillary line, the chest wall is turned upwards to expose the heart, then the right auricle is cut off by scissors, the apex of the heart is lifted, the prepared injector of the aqueous solution of the fluorescent conjugated polymer nanoprobe is inserted into the left ventricle and faces the direction of the aorta, and then the liquid is injected into the mouse at a constant speed. The skull was cut open and the meninges were stripped by surgical operation to the skin of the rat head, and the stripped meninges were imaged with a stereomicroscope, and the results are shown in fig. 16.

FIG. 16 is a graph of the results of imaging normal rat brain membrane vessels after heart perfusion with 30mL of aqueous solution of PFBT fluorescent conjugated polymer nanoprobe with concentration of 0.02 mg/mL. As can be seen from FIG. 16, the fluorescent conjugated polymer nanoprobe PFBT has very high fluorescence signal brightness, and can clearly image normal mouse meningeal blood vessels.

Example 22: imaging research of fluorescent conjugated polymer nanoprobe PFBT-COOH on whole cerebral vessels of 4-week-old normal mice under condition of not opening skull

After deeply anesthetizing the mouse, 30mL of the aqueous solution of the PFBT-COOH fluorescent conjugated polymer nanoprobe prepared in example 2 with the concentration of 0.04mg/mL is sucked by an injector, the diaphragm of the mouse is cut off, the chest wall is quickly cut off along the anterior axillary line, the chest wall is turned upwards to expose the heart, then the right auricle is cut off by scissors, the apex of the heart is lifted, the prepared injector of the aqueous solution of the fluorescent conjugated polymer nanoprobe is inserted into the left ventricle and faces the direction of the aorta, and then the liquid is injected into the mouse at a constant speed. The skull of the rat was exposed by cutting the skin of the rat's head through a surgical operation, and the exposed position of the skull was imaged by a stereo fluorescence microscope, and the result is shown in fig. 17.

FIG. 17 is a graph of the results of different-magnification fluorescence imaging of vessels at olfactory bulb, brain and cerebellum positions of normal rat brain without opening skull after heart perfusion of 30mL of aqueous solution of PFBT-COOH fluorescent conjugated polymer nanoprobe with concentration of 0.04 mg/mL. From fig. 17, it can be seen that the fluorescent conjugated polymer nanoprobe PFBT-COOH has very high fluorescence signal brightness and can image whole cerebral vessels without opening the skull, which indicates that the cerebral vascular imaging method of the present invention has a high penetration depth.

Example 23: imaging research of fluorescent conjugated polymer nanoprobe PFBT-COOH on whole cerebral vessels of 4-week-old normal mice under condition of opening skull

After deeply anesthetizing the mouse, 30mL of the aqueous solution of the PFBT-COOH fluorescent conjugated polymer nanoprobe prepared in example 2 with the concentration of 0.04mg/mL is sucked by an injector, the diaphragm of the mouse is cut off, the chest wall is quickly cut off along the anterior axillary line, the chest wall is turned upwards to expose the heart, then the right auricle is cut off by scissors, the apex of the heart is lifted, the prepared injector of the aqueous solution of the fluorescent conjugated polymer nanoprobe is inserted into the left ventricle and faces the direction of the aorta, and then the liquid is injected into the mouse at a constant speed. The skin of the mouse head was first cut open by surgery to expose the skull bone of the mouse, then the skull bone of the mouse was carefully opened, and finally the exposed whole brain and a part of the position were imaged by a body type fluorescence microscope, and the results are shown in fig. 18.

FIG. 18 is a graph showing the results of imaging of blood vessels in the whole brain of a normal mouse when the skull is opened by PFBT-COOH after heart perfusion with 30mL of an aqueous solution of a PFBT-COOH fluorescent conjugated polymer nanoprobe with a concentration of 0.04mg/mL (left panel) and in the position of a part of the brain (middle panel, right panel). From fig. 18, it can be seen that the fluorescent conjugated polymer nanoprobe PFBT-COOH has very high fluorescence signal brightness and can clearly image the whole cerebral vessels after opening the skull, and at the same time, it can be seen that the fluorescent conjugated polymer nanoprobe PFBT-COOH has excellent resolution in imaging the cerebral vessels after opening the skull, and the measurable minimum vascular size is 2.91 μm.

Example 24: whole cerebral vessel imaging research of fluorescent conjugated polymer nanoprobe PPV-COOH on 4-week-old normal mice under condition of not opening skull

After a mouse is deeply anesthetized, 30mL of the aqueous solution of the PPV-COOH fluorescent conjugated polymer nano probe prepared in the embodiment 5 with the concentration of 0.08mg/mL is sucked by a syringe, the diaphragm of the mouse is cut open, the chest wall is quickly cut along the line in front of the axilla, the chest wall is turned upwards to expose the heart, then the right auricle is cut open by scissors, the apex of the heart is lifted, the prepared injector of the aqueous solution of the fluorescent conjugated polymer nano probe is inserted into the left ventricle and faces the direction of the aorta, and then the liquid is injected into the mouse at a constant speed. The skin of the rat head was surgically cut to expose the skull bone of the rat, and the exposed skull bone position was imaged with a stereo fluorescence microscope, the results of which are shown in fig. 19.

FIG. 19 is a graph of PPV-COOH imaging results at different magnifications of blood vessels at positions of olfactory bulb, brain and cerebellum of a normal rat brain without opening the skull. From the imaging result of fig. 19, it can be seen that the fluorescent conjugated polymer nanoprobe PPV-COOH has higher fluorescence brightness, and can image whole cerebral vessels without opening the skull, which illustrates that the cerebral vessel imaging method of the present invention has the advantage of good penetration. Comparing fig. 17 and 19, it can be seen that the fluorescent conjugated polymer nanoprobe PFBT-COOH has better penetration than PPV-COOH.

Example 25: whole cerebral vessel imaging research of fluorescent conjugated polymer nanoprobe PPV-COOH on 4-week-old normal mice under condition of opening skull

After deeply anesthetizing the mouse, 30mL of the aqueous solution of the PPV-COOH fluorescent conjugated polymer nanoprobe prepared in example 5 with the concentration of 0.08mg/mL is sucked by an injector, the diaphragm of the mouse is cut off, the chest wall is quickly cut off along the anterior axillary line, the chest wall is turned upwards to expose the heart, then the right auricle is cut off by scissors, the heart tip is lifted, the prepared injector of the aqueous solution of the fluorescent conjugated polymer nanoprobe is inserted into the left ventricle and faces the direction of the aorta, and then the liquid is injected into the mouse at a constant speed. The skin of the mouse head was first cut open by surgical operation to expose the skull bone of the mouse, then the skull bone of the mouse was carefully opened, and finally the exposed whole brain and a part of the position were imaged by a body type fluorescence microscope, and the result is shown in fig. 20.

FIG. 20 is a graph of experimental results of fluorescent conjugated polymer nanoprobes PPV-COOH on the cranial opening of a normal mouse for imaging of blood vessels in the whole brain (upper left) and blood vessels in a part of the brain (upper right, lower left, and lower right). From the imaging result of fig. 20, it can be seen that the fluorescent conjugated polymer nanoprobe PPV-COOH can clearly image the whole cerebral vessels after the skull is opened, and the minimum imageable blood vessel size is 3.63 μm. The experimental results of comparative example 18 and example 20 show that the fluorescent conjugated polymer nanoprobe PFBT-COOH has higher resolution than PPV-COOH.

Example 26: full-cerebral-vessel imaging research on 8-month-old aged mice by using fluorescent conjugated polymer nanoprobe PFBT-COOH under condition of opening skull

After 8-month old mice were deeply anesthetized, 30mL of the aqueous solution of the PFBT-COOH fluorescent conjugated polymer nanoprobe prepared in example 2, with a concentration of 0.04mg/mL, was drawn up with a syringe, after the diaphragm of the mouse was cut open, the chest wall was quickly cut along the anterior axillary line, and the anterior chest wall was turned up to expose the heart, then the right auricle was cut with scissors, the apex of the heart was lifted, the prepared syringe of the aqueous solution of the fluorescent conjugated polymer nanoprobe was inserted into the left ventricle and oriented toward the aorta, and then the liquid was injected into the mouse at a constant speed. The skull of the aged rat was first cut through the skin of the head of the aged rat by surgical operation, then carefully opened, and finally the exposed whole brain and a part of the position were imaged by a body-type fluorescence microscope, and the results are shown in fig. 21.

FIG. 21 is a graph of the results of different magnification imaging of vessels at brain and cerebellar sites of aged mice imaged PFBT-COOH after opening the skull. From the imaging result of fig. 21, it can be seen that the fluorescent conjugated polymer nanoprobe PFBT-COOH has higher fluorescence intensity, and can clearly image the whole cerebral vessels of the aged mice after opening the skull. In addition, as can be seen from fig. 18 and 21, PFBT-COOH can effectively compare the brain vascularity of the aged and normal rats.

Example 27: full cerebral vessel imaging research on apoplexy model mouse by using fluorescent conjugated polymer nanoprobe PFBT-COOH under condition of opening skull

After a stroke model mouse is deeply anesthetized, 30mL of the aqueous solution of the PFBT-COOH fluorescent conjugated polymer nanoprobe prepared in the embodiment 2 with the concentration of 0.04mg/mL is sucked by a syringe, the diaphragm of the mouse is cut open, the chest wall is quickly cut along the axillary line, the chest wall is turned upwards to expose the heart, then the right auricle is cut open by scissors, the apex of the heart is lifted, the prepared aqueous solution syringe of the fluorescent conjugated polymer nanoprobe is inserted into the left ventricle and faces the direction of the aorta, and then the liquid is injected into the mouse at a constant speed. The skull of the mouse was first exposed by surgical operation by cutting the skin of the mouse head, then carefully opening the skull of the mouse, and finally imaging the exposed whole brain and a part of the position with a body type fluorescence microscope, the result is shown in fig. 22.

Fig. 22 is a graph of the imaging results of PFBT-COOH after opening the skull for imaging blood vessels at the brain and cerebellum sites of stroke model mice (left panel) and blood vessels near the ischemic site of the brain of stroke model mice (right panel). From the imaging result of fig. 22, it can be seen that the fluorescent conjugated polymer nanoprobe PFBT-COOH has higher fluorescence intensity, and can clearly image the whole cerebral vessels of the aged mice after opening the skull. In addition, as can be seen from fig. 18 and 22, PFBT-COOH was able to compare the difference in cerebral vascularity between stroke model mice and normal mice.

Example 28: whole cerebral vessel imaging research of fluorescent conjugated polymer nanoprobe PFBT-COOH on apoplexy model mouse under conditions of opening skull and stripping meninges

After a stroke model mouse is deeply anesthetized, 30mL of the aqueous solution of the PFBT-COOH fluorescent conjugated polymer nanoprobe prepared in the embodiment 2 with the concentration of 0.04mg/mL is sucked by a syringe, the diaphragm of the mouse is cut open, the chest wall is quickly cut along the axillary line, the chest wall is turned upwards to expose the heart, then the right auricle is cut open by scissors, the apex of the heart is lifted, the prepared aqueous solution syringe of the fluorescent conjugated polymer nanoprobe is inserted into the left ventricle and faces the direction of the aorta, and then the liquid is injected into the mouse at a constant speed. The skull of the mouse was first exposed by surgical operation by cutting the skin of the mouse head, then the skull of the mouse was carefully opened and the mouse meninges were peeled off, and finally the exposed whole brain and a part of the position were imaged by a body type fluorescence microscope, and the results are shown in fig. 23.

FIG. 23 is a graph of the results of PFBT-COOH imaging blood vessels at brain and cerebellar sites of stroke model mice after opening the skull and stripping the meninges. From fig. 23, it can be seen that the fluorescent conjugated polymer nanoprobe PFBT-COOH can image the whole cerebral vessels after the brain membranes of the stroke model mouse are stripped, and can effectively distinguish the stroke area from the normal blood supply area.

Example 29: imaging research of fluorescent conjugated polymer nano probe PFBT-COOH on cerebral membrana vessels of stroke model mice

After a stroke model mouse is deeply anesthetized, 30mL of aqueous solution of the PFBT-COOH fluorescent conjugated polymer nano probe prepared in the embodiment 2 with the concentration of 0.04mg/mL is sucked by a syringe, the chest wall is quickly cut along the axillary line after the diaphragm of the mouse is cut open, the chest wall is turned upwards to expose the heart, then the right auricle is cut by scissors, the apex of the heart is lifted, the prepared fluorescent conjugated polymer nano probe aqueous solution syringe is inserted into the left ventricle and faces the direction of the aorta, and then the liquid is injected into the mouse body at a constant speed. The skull of the mouse was first exposed by surgical operation by cutting the skin of the mouse head, then carefully opening the skull and stripping the meninges, spreading the stripped meninges on a slide, and finally imaging the brain and cerebellar position and meninges with a stereomicroscope, the results of which are shown in fig. 24.

FIG. 24 is a graph of the results of PFBT-COOH imaging of blood vessels on the meninges of stroke model mice. From fig. 24, it can be seen that the fluorescent conjugated polymer nanoprobe PFBT-COOH can perform high-resolution imaging on blood vessels on the meninges of the stroke model mouse.

Claims

1. The application of the fluorescent conjugated polymer nanoprobe in preparing a contrast agent for imaging of blood vessels or a brain lymphatic system is characterized in that the fluorescent conjugated polymer nanoprobe only comprises a fluorescent conjugated polymer or consists of the fluorescent conjugated polymer and a surface ligand, the brain lymphatic system is selected from one or more of meningeal lymphatic vessels, facial lymphatic vessels and neck lymphatic vessels, the blood vessels are brain blood vessels, the fluorescent conjugated polymer is a 9, 9-dioctyl fluorene-2, 1, 3-benzothiadiazole copolymer, the surface ligand is selected from one or more of polystyrene grafted carboxyl terminated polyethylene oxide, amino terminated polymethyl methacrylate and styrene-maleic anhydride copolymers, and the imaging is visible region type fluorescence microscope imaging.

2. The use of claim 1, wherein the surface ligand is polystyrene grafted carboxy terminated polyethylene oxide.

3. The use of claim 1, wherein the fluorescent conjugated polymer nanoprobe is composed of a fluorescent conjugated polymer and a surface ligand, and the mass ratio of the surface ligand to the fluorescent conjugated polymer in the fluorescent conjugated polymer nanoprobe is 0: 1 to 2.5: 1.

4. the use of claim 3, wherein the fluorescent conjugated polymer nanoprobe comprises the surface ligand and the fluorescent conjugated polymer in a mass ratio of 0.8: 1 to 1.2: 1.

5. the use of claim 1, wherein the fluorescent conjugated polymer nanoprobe has one or more of the following characteristics:

the contrast agent is an aqueous solution, a physiological saline solution or a buffer solution of the fluorescent conjugated polymer nanoprobe, and the concentration of the fluorescent conjugated polymer nanoprobe of the contrast agent is 0.01-1 mg/mL;

the hydrated particle size of the fluorescent conjugated polymer nano probe is 30-100 nm;

the fluorescent conjugated polymer nanoprobe comprises only a fluorescent conjugated polymer.

6. The use of claim 1, wherein the hydrated particle size of the fluorescent conjugated polymer nanoprobe is 40-60 nm.

7. Use according to claim 1, wherein the imaging is in vivo or ex vivo.

8. The use of claim 1, wherein the imaging is imaging of the lymphatic system of the brain.

9. The application of claim 8, wherein the application has one or more of the following features:

the lymphatic system of the brain is meningeal lymphatic vessels;

the contrast agent is an aqueous solution, a physiological saline solution or a buffer solution of the fluorescent conjugated polymer nanoprobe, and the concentration of the fluorescent conjugated polymer nanoprobe of the contrast agent is 0.1-1 mg/mL;

the imaging is non-craniotomy imaging or craniotomy imaging;

the imaging is in vivo imaging.

10. The use of claim 8, wherein the fluorescent conjugated polymer nanoprobe is composed of a fluorescent conjugated polymer and a surface ligand, and the surface ligand is polystyrene grafted carboxyl terminated polyethylene oxide.

11. The use of claim 8, wherein the fluorescent conjugated polymer nanoprobe is composed of a fluorescent conjugated polymer and a surface ligand, and the mass ratio of the surface ligand to the fluorescent conjugated polymer in the fluorescent conjugated polymer nanoprobe is 0: 1 to 2.5: 1.

12. the use of claim 11, wherein the fluorescent conjugated polymer nanoprobe comprises a surface ligand and a fluorescent conjugated polymer in a mass ratio of 0.8: 1 to 1.2: 1.

13. the use of claim 8, wherein the hydrated particle size of the fluorescent conjugated polymer nanoprobe is 40-60 nm.

14. Use according to claim 1, wherein the imaging is brain vessel imaging.

15. The application of claim 14, wherein the application has one or more of the following features:

the cerebral blood vessels are full cerebral blood vessels or partial cerebral blood vessels, the full cerebral blood vessels comprise the full cerebral blood vessels before the meninges are stripped and the full cerebral blood vessels after the meninges are stripped, and the partial cerebral blood vessels comprise cerebral blood vessels, cerebellar blood vessels and meningeal blood vessels;

the cerebral blood vessels are cerebral blood vessels of a normal subject, an elderly subject and/or a stroke subject;

the contrast agent is an aqueous solution, a physiological saline solution or a buffer solution of the fluorescent conjugated polymer nanoprobe, and the concentration of the fluorescent conjugated polymer nanoprobe of the contrast agent is 0.01-0.1 mg/mL;

the imaging is non-craniotomy imaging or craniotomy imaging;

the imaging is in vivo imaging or ex vivo imaging.

16. The use of claim 14, wherein the fluorescent conjugated polymer nanoprobe is comprised of a fluorescent conjugated polymer and a surface ligand, the surface ligand being polystyrene grafted carboxyl terminated polyethylene oxide.

17. The use of claim 14, wherein the fluorescent conjugated polymer nanoprobe is composed of a fluorescent conjugated polymer and a surface ligand, and the mass ratio of the surface ligand to the fluorescent conjugated polymer in the fluorescent conjugated polymer nanoprobe is 0: 1 to 2.5: 1.

18. the use of claim 17, wherein the fluorescent conjugated polymer nanoprobe comprises a surface ligand and a fluorescent conjugated polymer in a mass ratio of 0.8: 1 to 1.2: 1.

19. the use of claim 14, wherein the hydrated particle size of the fluorescent conjugated polymer nanoprobe is 40-60 nm.