Near-infrared compound with aggregation-induced luminescence characteristics and preparation method and application thereof

Abstract

The present invention discloses a near-infrared compound with aggregation-induced luminescence properties, a preparation method thereof, and three-photon bioimaging applications. The near-infrared compound has high-brightness near-infrared emission, excellent near-infrared three-photon absorption performance, and a large Stokes shift. It can be used for deep and high-resolution three-photon fluorescence bioimaging of mouse brain blood vessels. The material synthesized by the present invention exhibits the property of aggregation-induced luminescence, while traditional fluorescent dyes have the phenomenon of aggregation-induced fluorescence quenching under high concentration conditions, thereby effectively overcoming the defects of traditional fluorescent dyes.

Description

Technical Field

[0001] The present invention relates to the field of biological imaging, and more particularly to a near-infrared compound with aggregation-induced luminescence properties, a preparation method thereof, and biological applications. The subject material of the present invention mainly relates to a design strategy and its application in developing fluorescent dyes with strong near-infrared emission, excellent near-infrared three-photon absorption, large Stokes shift, and excellent three-photon fluorescence biological imaging capabilities. Background Art

[0002] Three-photon fluorescence imaging (3PFM) technology can convert the dye excitation wavelength to a longer near-infrared second region (NIR-II) wavelength, thereby achieving higher imaging depth and signal-to-noise ratio (SBR). This is because three-photon fluorescence (3PF) is a nonlinear optical process based on three-photon excitation, in which the fluorophore simultaneously absorbs three low-energy photons and then emits a high-energy photon. 3PFM imaging with a three-photon excitation wavelength in NIR-II b (1500-1700nm) is expected to penetrate the brain and focus on deep biological tissues, which is potentially helpful for laboratory research and clinical applications. In addition, 3PFM imaging has higher optical sectioning capabilities and better spatial resolution than two-photon fluorescence imaging (2PFM). All these advantages make 3PFM imaging technology a promising imaging tool for observing brain structures in vivo. Of course, achieving high-quality 3PFM imaging under NIR-II b region excitation always depends on suitable and efficient fluorescent probes, and the lack of probes with large three-photon absorption cross-sections and high fluorescence efficiency has become the main limitation of 3PFM imaging.

[0003] In recent years, various organic light emitters with aggregation-induced emission (AIE) properties have been designed and synthesized for bioimaging and therapy, and they have excellent brightness and photostability in the aggregated state. However, some AIE materials developed for 3PFM bioimaging are mainly based on tetraphenylethylene (TPE) or phenyl bridge skeletons, which have moderate three-photon absorption activity due to their short conjugation length. In view of this, the design and development of AIE materials with high-brightness near-infrared emission and strong three-photon absorption properties are of great practical significance for biomedical imaging and diagnosis and therapy. Summary of the invention

[0004] In this invention, an AIE molecule DCBT with near-infrared emission, strong near-infrared three-photon absorption and large Stokes shift was synthesized. And this molecule was applied to in vivo deep-layer high-resolution three-photon fluorescence bioimaging of cerebral blood vessels through the skull, providing a new probe for biomedical imaging and diagnosis and treatment.

[0005] The technical solution adopted by the present invention to solve the technical problem is to provide a near-infrared aggregation-induced fluorescent material with strong three-photon absorption properties, having a structure shown in the following formula:

[0006]

[0007] wherein R1 and R2 are independently selected from H, alkyl, unsaturated alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, alkyl-NCS, alkyl-N3 and alkyl-NH2.

[0008] Preferably, the near infrared compound is DCBT.

[0009] The present invention also provides a method for preparing a near-infrared aggregation-induced fluorescent material with strong three-photon absorption properties, comprising the following steps:

[0010]

[0011] Compound B is obtained by reacting compound A with a diphenylamine derivative; and

[0012]

[0013] Compound B reacts with electron-withdrawing acceptor C via Knoevenagel condensation to generate the target compound.

[0014] The invention also provides an application of a near-infrared compound with aggregation-induced luminescence properties in biological imaging.

[0015] Specifically, the near-infrared aggregation-induced fluorescent material with strong three-photon absorption can be prepared into nanoparticles for in vivo deep-layer high-resolution three-photon fluorescence imaging through the skull, which has the advantages of high brightness, high photostability and high contrast.

[0016] Preferably, the in vivo test is performed by injecting the AIE nanomaterial into the retroocular vein, and the light source for the three-photon fluorescence imaging is a 1550nm femtosecond laser.

[0017] The implementation of the present invention can achieve the following beneficial effects:

[0018] The present invention proposes a new strategy for designing and synthesizing near-infrared aggregation-induced fluorescent materials with strong three-photon absorption, achieving a perfect balance between near-infrared emission and aggregation-induced luminescence of this type of molecules, and can be widely used in fields such as biological imaging; the material synthesized by the present invention exhibits the property of aggregation-induced luminescence, a solid fluorescence wavelength of 648nm, and a fluorescence quantum yield of 13.6%; the material synthesized by the present invention exhibits the characteristic of near-infrared luminescence; the material synthesized by the present invention exhibits the characteristic of aggregation-induced luminescence; the material synthesized by the present invention exhibits the characteristic of strong three-photon excited fluorescence; the material synthesized by the present invention exhibits the characteristic of being able to be used for deep-layer high-resolution three-photon fluorescence imaging through the skull in vivo. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The following are: A) the synthesis route and molecular structure of DCBT; B) the absorption spectrum of DCBT in THF; C) the HOMO and LUMO molecular orbital diagrams of DCBT calculated based on the single crystal structure under B3LYP / 6-31G; D) the emission spectra of DCBT in solvents of different polarities; inset: fluorescence photographs of DCBT in solvents of different polarities under 365nm UV light; E) PL spectra of DCBT in acetone / water mixtures with different moisture contents, [DCBT] = 10μM; inset: fluorescence photographs of DCBT (10μM) in acetone / water mixtures with different moisture contents under 365nm UV light; F) the relationship between the relative PL intensity (I / I0) and the emission wavelength and the composition of the acetone / water mixture of DCBT; G) the normalized PL spectrum of DCBT in the solid state; inset: fluorescence photograph of DCBT powder under 365nm UV light.

[0020] Figure 2 This is the hydrogen nuclear magnetic resonance spectrum of the compound DCBT.

[0021] Figure 3 This is the carbon NMR spectrum of compound DCBT.

[0022] Figure 4 This is the high-resolution mass spectrum of compound DCBT.

[0023] Figure 5A) Schematic diagram of DCBT dots prepared by nanoprecipitation method. B) DLS and TEM images of DCBT dots (inset); C) Absorption (black) and PL (red) spectra of DCBT dots in water; the inset shows a photo of the suspension of DCBT dots under 365nm UV light; D) Schematic diagram of 3PF process; E) 3PF spectrum of DCBT dots in water; the inset describes the relationship between the logarithm of the 3PF intensity of DCBT dots and the 1550nm fs laser power; F) Three-photon absorption cross section of DCBT in different states; G) X-ray crystal structure of DCBT (CCDC: 2107702) and top view of dimer molecules in the crystal; H) Antiparallel dimers of DCBT are stacked through π-π interactions; Atomic colors: H = green; C = gray; N = blue; S = yellow; I) Schematic diagram of the stacking structure of DCBT molecules.

[0024] Figure 6 3PFM images of cerebral vessels marked with DCBT dots in mice: A) Schematic diagram of 3PFM imaging through the cranial window in mice: BK) 3PFM images of cerebral vessels at different vertical penetration depths: from 0 to 1010μm; L0) FWHM images of capillaries at depths of 200, 500, 700 and 1010μm as shown by yellow lines in images C, F, H and K, respectively; all images have the same scale bar: 100μm; excitation: 1550nm fs laser; power: up to 100mW in front of the objective lens.

[0025] Figure 7 Schematic diagram of the three-photon microscope system.

[0026] Figure 8 Three-dimensional reconstructed 3PFM images of cerebral blood vessels at different depths; A) 0-200, B) 200-400, C) 400-600, D) 600-800, and E) 800-1010; F, G) Three-dimensional reconstructed 3PFM images of cerebral blood vessels at a depth of 0-1010μm; all images have the same scale: 100μm; excitation: 1550nm fs laser; power: up to 100mW in front of the objective lens.

[0027] Fig. 9 Transcranial 3PFM images of DCBT dot-marked cerebral vessels in mice: A) Schematic diagram of transcranial 3PFM imaging of cerebral vessels in living mice; BG) 3PFM images of the vascular system at different vertical depths of 0–500μm under the skull; H) FWHM analysis of 3PFM images of cerebral vessels at 400μm; I, J) 3D reconstructed 3PFM images of cerebral vessels at a depth of 0-500μm. ; All images are the same scale bar: 100μm; excitation: 1550nm fs laser; power: up to 120mW in front of the objective lens. DETAILED DESCRIPTION

[0028] In order to have a clearer understanding of the technical features, purposes and effects of the present invention, the specific embodiments of the present invention are now described in detail with reference to the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments described in this specification, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0029] The present invention provides a near-infrared aggregation-induced fluorescent material with strong three-photon absorption, which has a structure shown in the following formula:

[0030]

[0031] wherein R1 and R2 are independently selected from H, alkyl, unsaturated alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, alkyl-NCS, alkyl-N3 and alkyl-NH2.

[0032] The present invention also provides a method for preparing a near-infrared aggregation-induced fluorescent material with strong three-photon absorption properties, and the method for preparing the near-infrared aggregation-induced fluorescent material with strong three-photon absorption properties comprises the following steps:

[0033]

[0034] Compound A and a diphenylamine derivative are reacted to obtain compound B, and;

[0035]

[0036] Compound B reacts with electron-withdrawing acceptor C via Knoevenagel condensation to generate the target compound.

[0037] More preferably, the near-infrared aggregation-induced fluorescent material with strong multiphoton absorption includes the compound DCBT.

[0038] The present invention also provides the application of the near-infrared compound with aggregation-induced emission property in biological imaging.

[0039] Specifically, the near-infrared aggregation-induced fluorescent material with strong three-photon absorption can be prepared into nanoparticles for in vivo deep-layer high-resolution three-photon fluorescence imaging through the skull, and has the advantages of high brightness, high light stability and high contrast.

[0040] Preferably, the in vivo test is performed by injecting the AIE nanomaterial into the retroocular vein, and the light source for the three-photon fluorescence imaging is a 1550nm femtosecond laser.

[0041] The implementation of the present invention can achieve the following beneficial effects:

[0042] The present invention proposes a new strategy for designing and synthesizing near-infrared aggregation-induced fluorescent materials with strong three-photon absorption, achieving a perfect balance between near-infrared emission and aggregation-induced luminescence of such molecules, and can be widely used in fields such as biological imaging.

[0043] The material synthesized by the present invention exhibits the property of aggregation-induced luminescence, with a solid fluorescence wavelength of 648nm and a fluorescence quantum yield of 13.6%;

[0044] The material synthesized by the present invention exhibits the characteristic of near-infrared luminescence;

[0045] The material synthesized by the present invention exhibits the characteristic of aggregation-induced luminescence;

[0046] The material synthesized by the present invention exhibits the characteristics of strong three-photon excited fluorescence;

[0047] The material synthesized by the present invention exhibits the property of being applicable to deep-layer high-resolution three-photon fluorescence imaging through the skull in vivo;

[0048] Taking the DCBT compound as an example, the preparation of the near-infrared aggregation-induced fluorescent compound with strong three-photon absorption and its biological imaging are described in detail below.

[0049] The synthetic route of DCBT is as follows: Figure 1 .

[0050] Synthesis of Compound DCBT To a solution of 7-(diphenylamino)-9-ethyl-9H-carbazole-2-carboxaldehyde (390 mg, 1.0 mmol) and benzothiazole-2-acetonitrile (209 mg, 1.2 mmol) in acetonitrile (8 mL) were added two drops of piperidine. The resulting solution was stirred at 40° C. for 8 hours. After the reaction mixture was cooled, the red precipitate formed was collected by filtration and washed with cold ethanol. The crude product was recrystallized in ethanol / dichloromethane to obtain red crystalline DCBT (0.50 g, yield: 91%).

[0051] The chemical structure of the compound DCBT was characterized and the characterization data were obtained: 1H NMR (400MHz, CDCl3), δ (ppm): 8.44 (s, 1H), 8.22 (s, 1H), 8.11-8.06 (m, 2H), 7.97-7.92 (m, 2H), 7.81 (d, J = 8.2Hz, 1H), 7.57-7.53 (m, 1H), 7.46-7 .42(m,1H),7.32-7.28(m,4H),7.20-7.18(m,4H),7.11-7.06(m,3H),7. 01(dd,J=8.5,1.8Hz,1H),4.27(q,J=7.1Hz,2H),1.40(t,J=7.2Hz,3H), such as Figure 2 . 13 C NMR (100MHz, CDCl3), δ (ppm): 163.02, 153.12, 147.61, 147.57, 147.30, 142.38, 139.55, 134.26, 128.72, 127.84, 126.24, 126.19, 125. 05,123.97,123.93,122.74,122.55,122.15,121.25,120.99,119.42,116.98,116.84,116.54,109.25,102.83,102.15,37.05,13.16, such as Figure 3 HRMS:m / z calculated molecular weight is [M] + C 36 H 26 N4S 546.1878; the measured molecular weight is 546.1871, such as Figure 4 .

[0052] Preparation of DCBT dots

[0053] DCBT dots were synthesized by a reported nanoprecipitation method. First, 1 mg DCBT and 12 mg Pluronic F127 were dissolved in 1 mL tetrahydrofuran. The mixed solution was then dropped into 10 mL of stirred deionized water. The mixture was stirred at room temperature overnight until the tetrahydrofuran was completely evaporated. Afterwards, the solution was filtered with a syringe filter (200 nm) and concentrated by a centrifugal filter. The working concentration of DCBT dots can be changed by diluting the concentrated DCBT dot solution to a certain volume, and can be confirmed by measuring the absorption intensity of the DCBT dot solution and the molar absorption coefficient of the DCBT molecule.

[0054] Three-photon absorption cross section measurement

[0055] The three-photon absorption cross section was obtained by the comparison method. The three-photon absorption cross section of TPATCN NPs at 1550 nm was selected as a reference. DCBT dots and TPATCN NPs in water medium were excited by 1550 nm fs laser, their three-photon fluorescence signals were collected by photomultiplier tube (PMT), and the average intensity was calculated by Image J. The σ3 value of DCBT dots was calculated by the following formula.

[0056]

[0057] Where F is the three-photon fluorescence intensity, η is the fluorescence quantum yield, c is the molar concentration of the sample, n is the refractive index of the solvent, and subscripts 1 and 0 represent DCBT dots and TPATCN NPs, respectively. The three-photon absorption cross sections of DCBT dots at other excitation wavelengths are calculated by referring to the value of 1550 nm measured above and by the following formula.

[0058]

[0059] where σ 3,λ is the wavelength-dependent three-photon absorption cross section, σ 3，1550 is the σ3 of the DCBT point excited at 1550nm, P 1550 and P λ is the excitation power measured on the sample, τ 1550 and τ λ is the pulse width measured on the sample, F 1550 and F λ The three-photon fluorescence signals were measured under excitation at 1550 nm and other wavelengths, respectively.

[0060] The invention discloses a near-infrared aggregation-induced fluorescent material with strong three-photon absorption, and the near-infrared aggregation-induced fluorescent material with strong three-photon absorption is used for biological imaging.

[0061] The near-infrared aggregation-induced fluorescent material with strong three-photon absorption can be used for deep-layer high-resolution three-photon fluorescence imaging of living skulls. It has the advantages of high brightness, high photostability and high contrast. The near-infrared aggregation-induced fluorescent material with strong three-photon absorption can be prepared into nanoparticles for deep three-photon fluorescence imaging of tissue layers.

[0062] The chemical structure of the synthesized DCBT was characterized ( Figure 2 to Figure 4 ).

[0063] Photophysical properties characterization

[0064] We designed and synthesized a push-pull compound called DCBT. Figure 1A. DCBT is synthesized by a one-step knoevenagel condensation reaction with a yield of up to 91%. Its chemical structure is confirmed by nuclear magnetic resonance ( 1 H and 13 C NMR), high-resolution mass spectrometry (HRMS) and single-crystal X-ray diffraction.

[0065] The optical properties of DCBT were studied by UV-Vis and photoluminescence (PL) spectroscopy. It was observed that the maximum absorption peak of DCBT in tetrahydrofuran was 453 nm ( Figure 1 B), which may be due to the intramolecular charge transfer (ICT) characteristics from the diphenylamine group to the benzothiazole-2-acetonitrile group. To better understand the ICT process within the DCBT molecule, we used the DCBT single crystal structure data to perform density functional theory (DFT) calculations ( Figure 1 C). The electron cloud of the highest occupied molecular orbital (HOMO) of DCBT is located on the diphenylamine group and the carbazole unit. Its lowest unoccupied molecular orbital (LUMO) is mainly contributed by the orbitals of benzothiazole-2-acetonitrile and part of the carbazole ring. The calculated band gap (ΔE) of DCBT is 2.64 eV, indicating the existence of the ICT effect. We then evaluated the effect of solvent on the emission characteristics of DCBT ( Figure 1 D). A significant positive solvatochromic effect was observed in the PL spectrum. As the solvent polarity increased from n-hexane to N,N-dimethylformamide (DMF), the PL wavelength maximum gradually increased from λ em =512nm red shifted to 710nm.

[0066] In order to further study the influence of aggregation effect on the emission characteristics of DCBT, the PL spectra (f w )Record it( Figure 1 E, F). DCBT shows moderate fluorescence intensity in pure acetone solution. w =0% gradually increases to 50% ( Figure 1 E), the PL of DCBT decreased significantly, and gradually red-shifted from 681nm to 725nm, which may be due to the enhancement of the molecular TICT effect caused by the increase in solvent polarity after adding water ( Figure 1 F). This is also consistent with the solvation effect of DCBT discussed above. w When the fluorescence increased from 60% to 90%, a significant fluorescence enhancement and fluorescence blue shift (639nm) occurred, revealing the AIE properties of DCBT. Due to its unique AIE properties, DCBT exhibits bright near-infrared solid-state fluorescence with a peak at 648nm (shoulder at 705nm) and a solid-state fluorescence quantum yield (Φ f ) is 14%( Figure 1G). These results indicate that the designed DCBT possesses AIE activity and exhibits high near-infrared brightness in the solid state.

[0067] The three-photon absorption properties of DCBT molecules in solution and in nanoparticles were evaluated by collecting three-photon fluorescence (3PF) spectra. Before recording 3PF spectra, DCBT dots were prepared by a modified nanoprecipitation method using Pluronic F127 as an encapsulation matrix to improve the stability and biocompatibility of DCBT nanoparticles ( Figure 5 A). The size distribution of DCBT dots was measured by dynamic light scattering (DLS) and the average diameter was 105 nm ( Figure 5 B). In addition, scanning electron microscopy (SEM) images showed that the DCBT dots were spherical and their sizes were similar to those of the DLS results ( Figure 5 B illustration). Figure 5 C shows the absorption and PL spectra of DCBT dots. The absorption maximum of DCBT dots is located at 470 nm, the PL peak is 642 nm, and the tail extends to 850 nm, resulting in a large Stokes shift of 172 nm. This minimizes the interference between excitation and emission, which is beneficial for high contrast and low background in biological imaging.

[0068] Then, the 3PF spectra of DCBT in solution and aggregated states were measured under three-photon excitation of 1550 nm fs laser ( Figure 5 D). Under the excitation of 1550nm fs laser, DCBT dots exhibited strong 3PF, which was very similar to the fluorescence spectrum obtained under single-photon excitation, indicating that the emission process from the single-photon and three-photon excited states to the ground state was consistent ( Figure 5 E). In order to verify that the observed fluorescence signal is derived from the conversion process of three-photon excitation, we obtained the logarithm of the fluorescence intensity of the DCBT dots and the logarithm of the excitation power and found that they have a good linear relationship, with a slope of 2.84. This slope undoubtedly indicates the nonlinear optical process of 3PF ( Figure 5 In order to quantitatively evaluate the 3PA of DCBT solution and DCBT dots, their 3PA cross sections (σ3) in the range of 1500-1700 nm were measured by three-photon excitation fluorescence ( Figure 5 F). The results show that the DCBT solution exhibits excellent 3PA in the range of 1500-1700nm, with a maximum σ3 of 1.57×10 -78 cm 6 s 2 photon -2, showing an excellent 3PA range. For DCBT molecules, the larger σ3 can be attributed to its strong push-pull electronic properties and long π-conjugated structure, a result that is consistent with our molecular design concept. Impressively, when DCBT molecules are aggregated into nanodots, their σ3 value is increased by 3.6 times to 5.61×10 -78 cm 6 s 2 photon -2 , which is one order of magnitude higher than the σ3 value of previously reported AIE luminophores.

[0069] In order to further understand the improvement of σ3 value and AIE behavior of DCBT in nanodots, DCBT single crystals suitable for X-ray crystallography were obtained by slow evaporation of solvent in its ethanol / dichloromethane mixed solution. Figure 5 As shown in Figure G, the dihedral angle between benzothiazole-2-acetonitrile and carbazole groups is 16.59°, indicating that they are essentially planar and conjugated, both of which give the entire molecule good π-electron delocalization. Therefore, the strong push-pull electron properties and extended π-conjugation lead to DCBT near-infrared emission and a large σ3 value. On the other hand, the twisted diphenylamine groups give DCBT unique AIE properties, avoiding the influence of the ACQ effect. When DCBT molecules aggregate, we clearly find that through close intermolecular π-π interactions, an antiparallel dimer is formed along the long molecular axis with a distance of than the typical π-π interaction distance This may be due to the presence of additional intermolecular CH···π and C···π It is obvious that in each dimer, the electron-rich diphenylamine-carbazole skeleton of one molecule and the electron-deficient benzothiazole-2-acetonitrile unit of another molecule form an intermolecular π-π interaction ( Figure 5 G,H,I). The dimer is further connected through tight intermolecular π-π interactions. Arranged in offset columnar stacks, the molecules form infinite continuous intermolecular π-π interactions in the aggregates, which is conducive to long-range electronic coupling between molecules. This should be the reason why the three-photon absorption cross section of DCBT molecules increases after they are aggregated into nanodots. In addition, in the aggregated state and solid state, multiple intermolecular interactions rigidify the molecular conformation and restrict the intramolecular motion to a greater extent. In this case, the excited state energy is greatly suppressed through the consumption of intramolecular motion, allowing the molecules to emit stronger near-infrared light.

[0070] High-resolution three-photon fluorescence imaging of deep cerebral vessels in vivo

[0071] DCBT dots with large σ3 values ​​and bright near-infrared AIE properties are promising candidates for deep tissue bioimaging. Next, we used a laboratory-modified 3PFM imaging system ( Figure 6 A, Figure 7 ) studied real-time imaging of cerebral vasculature through cranial windows in mice. The mice were anesthetized and a small area of ​​the skull was opened to serve as a cranial window. Then, DCBT was injected into the retroorbital vein (2 mg mL -1 of DCBT, 100 μL, 1× PBS) were injected into mice, and the cerebral blood vessels were imaged through the cranial window under a 3PFM bio-imaging system equipped with a 1550 nm fs laser as the excitation light. Figure 6 As shown in BK, DCBT dots achieve high-resolution and deep-tissue imaging of the cerebrovascular system from the surface to the deep cortical regions, and can even identify small capillaries with high spatial resolution. The penetration capability of 3PFM imaging can reach 1010μm ( Figure 6 K), which is one of the maximum penetration depths of in vivo 3PFM imaging achieved by AIE materials. To better evaluate the imaging quality, lines were drawn on tiny capillaries at different depths (e.g., 200, 500, 700, and 1010 μm), and the intensity of each pixel was plotted as a function of position ( Figure 6 LO). The Gaussian fit generated from the pixel intensity depicts the diameter profile of the capillaries, with full width at half maximum (FWHM) of ∼2.8, 3.3, 3.1, and 8.0 μm at depths of 200, 500, 700, and 1010 μm, respectively. These results demonstrate the high resolution of 3PFM imaging, as even small vessels with a diameter of ∼8 μm can be resolved at a depth of 1010 μm ( Figure 6 O). Subsequently, three-dimensional reconstruction images of mouse brain vessels at different vertical imaging depths (0-200, 200-400, 400-600, 600-800, 800-1010 and 0-1010 μm) were constructed, in which the main blood vessels, capillaries and connections were vividly and clearly displayed ( Figure 8 AG). The large penetration depth and high resolution of 3PFM imaging are attributed to the excellent NIR-II b three-photon activity and bright NIR-I emission of DCBT dots.

[0072] As a non-invasive imaging technique, transcranial brain imaging is beneficial to maintain the integrity and pressure of the skull and brain tissue, which can not only avoid interference with the physiological function of the brain, but also avoid inflammation of brain tissue. The excellent performance of DCBT dots in 3PFM imaging encouraged us to further perform in vivo cerebrovascular imaging of intact skulls in mice under 1550nm fs laser excitation ( Fig. 9 A). Fig. 9As shown in BG, different sections of the brain vessels were obtained in the vertical direction. Few 3PF signals were detected from the skull layer of the mouse, indicating that there was no accumulation and staining of DCBT points in the skull. Below the skull layer, strong 3PF signals began to appear at the DCBT points in the mouse brain vessels. As the imaging depth increased, more brain vessels could be clearly observed, such as arterial / venous vessels, junctions, and capillaries. It was calculated that at a depth of 400 μm, the FWHMs of the measured capillaries were 5.45 μm ( Fig. 9 H), which indicates good resolution and image signal-to-noise ratio. Fig. 9 As shown in G, when the depth increases to 500 μm, the fine structure of the capillaries in the mouse motor cortex can still be distinguished. However, when the imaging depth is further increased to below 518 μm, the image quality decreases significantly due to photon scattering from the skull and brain tissue. Finally, a high-resolution 3D reconstruction image of the mouse brain blood vessels was obtained, showing a clear spatial image of the main vascular system and the tiny capillary network ( Fig. 9 I and J). With the help of DCBT dots, the potential of 3PFM through cranial brain imaging was realized and exhibited the advantages of deep penetration and high spatial resolution. These results indicate that DCBT nanoprobes with strong three-photon absorption hold great promise for non-destructive biomedical studies of the brain, and the combination of NIR-II b excitation and NIR-I emission enables deep imaging in vivo.

[0073] The embodiments of the present invention are described above in conjunction with the accompanying drawings, but the present invention is not limited to the above-mentioned specific embodiments. The above-mentioned specific embodiments are merely illustrative and not restrictive. Under the guidance of the present invention, ordinary technicians in this field can make many modifications without departing from the scope of protection of the present invention and the claims, all of which belong to the scope of protection of the present invention.

Claims

1. A near-infrared compound having aggregation-induced emission properties, characterized in that: The near infrared compound is DCBT:

2. The near-infrared compound according to claim 1, characterized in that The near-infrared compound has high-brightness near-infrared fluorescence emission characteristics.

3. The near-infrared compound according to claim 1, characterized in that The near-infrared compound has aggregation-induced emission characteristics in the near-infrared region.

4. The near-infrared compound according to claim 1, characterized in that The near-infrared compound has near-infrared three-photon absorption characteristics.

5. A non-diagnostic or therapeutic application of the near-infrared compound with aggregation-induced emission properties as claimed in claim 1 in three-photon bioimaging of mouse cerebral blood vessels.

6. A non-diagnostic or therapeutic application of the near-infrared compound with aggregation-induced emission properties as claimed in claim 1 in three-photon bioimaging of transcranial cerebrovascular vessels in mice.

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