A NIR-II fluorescent J-aggregate with enhanced aggregation-induced emission efficiency and its application

Abstract

The present invention provides a NIR-II fluorescent J-aggregate with enhanced aggregation-induced emission efficiency and its applications. AS5Br(δ) can easily form J-aggregates in an aqueous medium without a complex preparation process and has the characteristic of aggregation-induced enhancement of fluorescence efficiency. After being transformed into J-aggregate nanoparticles AS5Br(δ) NPs, the highest quantum yield ever reported for NIR-II fluorescent J-aggregates is achieved, with the NIR-II fluorescence quantum yield reaching 5.2%. The fluorescence quantum yield of AS5Br(δ) NPs even exceeds that of AS5Br(δ) in the best solvent, overcoming the challenges faced in transforming NIR-II fluorescent J-aggregates into biocompatible nanoparticles. In NIR-II imaging of blood vessels and tumors in mice and tumor resection experiments, its potential as a super-bright NIR-II fluorophore is demonstrated.

Description

Technical Field

[0001] The invention belongs to the field of near-infrared second-zone fluorescence imaging and relates to the design, synthesis and application of a near-infrared second-zone fluorescence probe. Background Art

[0002] Near-infrared II (NIR-II, defined as the spectral range of 1000-1700 nm or 900-1700 nm) fluorescence imaging technology has shown great potential in disease diagnosis and treatment. This technology not only retains the advantages of traditional fluorescence imaging in the near-infrared I (NIR-I, 700-900nm) window, such as high sensitivity, fast feedback, minimal invasiveness, and non-ionizing radiation, but also surpasses NIR-I imaging in terms of signal-to-noise ratio and spatiotemporal resolution of deep tissue imaging. This improvement is attributed to the significant reduction of photon scattering and autofluorescence in the NIR-II window, which has obvious advantages over the NIR-I window. The construction of photostable organic NIR-II fluorophores with ultrahigh brightness has always been an important research direction in the field of NIR-II fluorescence imaging. This is because ultrahigh brightness helps to use low injection doses and minimum excitation light power, thereby reducing dose- and power-related toxicity. The NIR-II brightness of a fluorophore is determined by the product of its extinction coefficient (ε) at the excitation wavelength and the NIR-II fluorescence quantum yield (QY). Although remarkable progress has been made in the development of NIR-II fluorophores based on various organic materials, fluorophores with ultrahigh NIR-II brightness are still scarce.

[0003] Due to the extended π-conjugated structure and planar conformation, small molecules with a fused ring core and two electron-deficient units exhibit a variety of properties, including narrow band gaps, high extinction coefficients, and high photostability, which make them very suitable as NIR-II fluorophores. Some of these molecules with similar structural features show the property of enhanced aggregation-induced fluorescence efficiency, which allows them to exhibit high fluorescence quantum yields in the aggregated state. Although the mechanism behind it is still unclear, this property is very advantageous for the preparation of high-brightness NIR-II nanofluorophores because aggregation is inevitable during the formation of nanoparticles. However, compared with monomers, nanofluorophores derived from these molecules usually show obvious blue-shifted absorption bands, which may be due to the presence of a large number of H-aggregates. The presence of H-aggregates will weaken their potential as ultrabright NIR-II fluorophores because H-aggregates have blue-shifted absorption and significantly reduced fluorescence quantum yields.

[0004] The use of J-aggregates as NIR-II fluorophores has become an important research area. J-aggregates are arranged in a head-to-tail staggered stacking manner, promoting the cooperative coupling of excited-state transition dipoles. The photochemical and photophysical properties of J-aggregates, including red-shifted absorption / emission spectra, narrowed spectral bandwidths, enhanced extinction coefficients, and higher photostability, have been demonstrated to have significant advantages in in vivo NIR-II fluorescence imaging. To date, various organic fluorophores (such as cyanine dyes, squaraine dyes, and BODIPY dyes) have been developed for the formation of NIR-II fluorescent J-aggregates through structural modification, carrier assistance, or experimental condition optimization. However, existing NIR-II fluorescent J-aggregates face some inherent problems when prepared into nanoparticles to enhance biocompatibility and applicability. First, the preparation of J-aggregates into nanoparticles results in a significant decrease in fluorescence quantum yield, thereby reducing brightness, both of which are significantly lower than those of the corresponding monomers in suitable solvents. Second, the complex preparation process required to maintain the J-aggregate conformation within the nanoparticles leads to limited reproducibility and low encapsulation efficiency. These problems severely limit the potential of J-aggregates as NIR-II fluorophores. Therefore, exploring novel fluorescent materials that can easily form J-aggregates and be prepared into nanoparticles without complex preparation processes, while maintaining high NIR-II brightness, has great potential but also faces major challenges. Summary of the Invention

[0005] The technical problem solved by the present invention is: The present invention discloses an NIR-II fluorescent J-aggregate with aggregation-enhanced luminescence efficiency as an NIR-II fluorescent probe, and reports a small molecule AS5Br(δ), which can easily form J-aggregates in an aqueous medium. Independently or with DSPE-PEG 2000Co-assembly forms ultra-bright NIR-II fluorescent J-aggregates. The prepared J-aggregate nanoparticles (AS5Br(δ) NPs) combine the inherent properties of J-aggregates and the unique phenomenon of aggregation-induced enhanced fluorescence efficiency, achieving significant fluorescence quantum yields (QYs) of 20.6%, 16%, and 5.2% at wavelengths exceeding 800 nm, 900 nm, and 1000 nm, respectively. These values represent the highest fluorescence quantum yields reported so far for NIR-II fluorescent J-aggregates. Notably, compared with all reported NIR-II fluorescent J-aggregates, the AS5Br(δ) J-aggregates exhibit unique fluorescence enhancement characteristics: their fluorescence quantum yields not only do not decrease due to aggregation, but instead are significantly enhanced compared to their monomeric state in the optimal solvent. This phenomenon appears for the first time in the NIR-II fluorescent J-aggregate system, highlighting the uniqueness of AS5Br(δ) in molecular design and aggregation state regulation. The combination of high fluorescence quantum yield and high extinction coefficient makes AS5Br(δ) NPs the brightest NIR-II fluorescent J-aggregate nanoparticles currently. Their ultra-high NIR-II brightness, combined with a simple preparation process, effectively solves the inherent limitations of existing NIR-II fluorescent J-aggregates when prepared into biocompatible nanoparticles. AS5Br(δ) NPs provide a new NIR-II fluorescence imaging method that can be excited using only low-power ambient white light, demonstrating the potential of AS5Br(δ) NPs as ultra-bright NIR-II fluorophores through NIR-II fluorescence image-guided tumor surgery.

[0006] To solve the above technical problems, the technical solution proposed by the present invention is: a compound, and the structural formula of compound AS5Br(δ) is as follows:

[0007] 。

[0008] To solve the above technical problems, another technical solution proposed by the present invention is: the synthesis route of compound AS5Br(δ) is as follows:

[0009]

[0010] Reagents and conditions used in the synthesis route of AS5Br(δ): (i) pyridine, chloroform, 56 °C, 12 h.

[0011] To solve the above technical problems, another technical solution proposed by the present invention is: the near-infrared second-region fluorescent J-aggregates prepared from the compound, AS5Br(δ) can independently form J-aggregates in an aqueous environment, or by combining with DSPE-PEG 2000Co-assembly forms J-aggregates, which benefits from the hydrophobic steric hindrance and π-bromine interaction of AS5Br(δ). The fluorescence quantum yield of the formed J-aggregates exceeds that of AS5Br(δ) in chloroform solvent; in addition, AS5Br(δ) also has the unique property of aggregation-induced fluorescence efficiency enhancement.

[0012] To solve the above technical problems, another technical solution proposed by the present invention is: preparing the compound AS5Br(δ) into AS5Br(δ) NPs as a near-infrared second-region fluorescence probe.

[0013] Preferably, the compound AS5Br(δ) is encapsulated in an amphiphilic polymer by the nano co-precipitation method to prepare water-soluble AS5Br(δ) NPs. The amphiphilic polymer includes DSPE-PEG and Pluronic F-127, and the molecular weight of PEG is 1000-10000.

[0014] Preferably, AS5Br(δ) NPs are synthesized by the nano co-precipitation method. Dissolve the compound AS5Br(δ) (1 mg) and DSPE-PEG 2000 (5 mg) in 1 mL of tetrahydrofuran; then inject the obtained solution into 10 mL of ultrapure water and perform ultrasonic treatment for 2 minutes with a probe sonicator. At room temperature, after the organic solvent volatilizes, filter the solution through a 0.22 μm filter head and concentrate it using a filter membrane with a molecular weight of 30 kDa to obtain AS5Br(δ) NPs.

[0015] To solve the above technical problems, another technical solution proposed by the present invention is: the fluorescence probe AS5Br(δ) NPs provides a novel NIR-II fluorescence imaging method that can be excited only by low-power ambient white light.

[0016] Preferably, the fluorescence probe AS5Br(δ) NPs are used to prepare a near-infrared second-region fluorescence imaging probe. The fluorescence probe AS5Br(δ) NPs have excellent biocompatibility and can achieve significant accumulation at the tumor site. With the help of NIR-II fluorescence imaging technology, mouse blood vessel visualization and tumor resection surgery can be achieved.

[0017] The NIR-II fluorescence probe AS5Br(δ) NPs effectively overcomes the problems faced in converting NIR-II fluorescence J-aggregates into biocompatible nanoparticles. Through in vitro brightness comparison with ICG in FBS, NIR-II imaging in the blood vessels and tumor sites of mice, and tumor resection experiments, the in vivo efficacy of AS5Br(δ) nanoparticles as excellent NIR-II fluorophores is demonstrated.

[0018] The present invention designs and synthesizes a compound AS5Br(δ). Due to the extended π-conjugated structure and planar conformation, small molecules with a fused-ring core and two electron-deficient units exhibit various properties, including a narrow bandgap, a high extinction coefficient, and high photostability, which make them very suitable as NIR-II fluorophores. Some molecules with similar structural features exhibit the property of enhanced fluorescence efficiency upon aggregation, which results in a high fluorescence quantum yield in the aggregated state. Although the mechanism behind this is not yet clear, this property is very beneficial for the preparation of highly bright NIR-II nanophosphors because aggregation is inevitable during the formation of nanoparticles. However, compared with the monomers, the nanophosphors derived from these molecules usually exhibit a significantly blue-shifted absorption band, which may be due to the presence of a large number of H-aggregates. The presence of H-aggregates weakens their potential as ultra-bright NIR-II fluorophores because H-aggregates have a blue-shifted absorption and a significantly reduced fluorescence quantum yield.

[0019] To inhibit the formation of H-aggregates and induce the formation of J-aggregates, we designed and synthesized the small molecule AS5Br(δ), which has long-chain branched side chains on the fused-ring core and a bromine atom at the δ-position of each acceptor unit. This is based on the principle that the long-chain branched side chains provide steric hindrance to prevent the H-aggregation of AS5Br(δ), while the presence of bromine atoms provides π-bromine interactions, which are beneficial for inducing the formation of J-aggregates. To further clarify the molecular design principle of AS5Br(δ), two control compounds AS5H and AS5Br(γ) were designed for comparison, where AS5H lacks bromine substituents and AS5Br(γ) has a bromine atom at the γ-position.

[0020] The structural formula of the compound AS5Br(γ) is as follows:

[0021]

[0022] The structural formula of the compound AS5H is as follows:

[0023]

[0024] The beneficial effects of the present invention:

[0025] The present invention discloses an NIR-II fluorescent J-aggregate with enhanced luminescence efficiency upon aggregation and its application. Compared with the existing NIR-II fluorescent J-aggregates, the J-aggregate based on AS5Br(δ) exhibits several significant advantages.

[0026] First, AS5Br(δ) can be independent or combined with DSPE-PEG in an aqueous medium 2000The co-assembly to form J-aggregates was performed using a simple nanoprecipitation method without complicated experimental steps. In addition, the formation of these J-aggregates was less concentration-dependent, which significantly reduced the restrictions on experimental conditions and improved reproducibility and practicality. The experimental results showed that the high tendency of J-aggregate formation was attributed to the synergistic effect of hydrophobic steric interactions and enhanced π···Br interactions.

[0027] Secondly, AS5Br(δ) NPs exhibited the highest fluorescence quantum yield of 20.6% among the reported NIR-II fluorescent J-aggregates to date in the full spectrum and 5.2% in the NIR-II range. Comparative experiments showed that this high fluorescence quantum yield was achieved by combining the advantages of J-aggregates and the enhanced properties of aggregation-induced fluorescence efficiency. Notably, due to their high extinction coefficient and fluorescence quantum yield, the NIR-II brightness of AS5Br(δ) NPs was 37.5 times higher than that of ICG / FBS at the same molar concentration under 808 nm excitation, highlighting the great potential of AS5Br(δ) nanoparticles as ultrabright NIR-II fluorescent probes. AS5Br(δ) NPs demonstrated their potential as ultrabright NIR-II fluorophores in comparison with ICG in FBS in vitro, NIR-II imaging of vascular and tumor sites in mice, and tumor resection experiments.

[0028] Third, AS5Br(δ) NPs exhibit a unique property that has not been observed in other NIR-II fluorescent J-aggregates: the fluorescence quantum yield of AS5Br(δ) NPs even significantly exceeds that of AS5Br(δ) in the most suitable solvent. This indicates that during the preparation of J-aggregates into nanoparticle form, the fluorescence quantum yield of the resulting J-aggregate nanoparticles not only did not decrease, but increased, which is contrary to the behavior of current NIR-II fluorescent J-aggregates.

[0029] Overall, the simple preparation process combined with the high fluorescence quantum yield of AS5Br(δ) NPs effectively addresses the challenges faced by existing NIR-II fluorescent J-aggregates when prepared into biocompatible nanoparticles, laying the foundation for their application as high-performance NIR-II fluorophores in the biological field. In addition, AS5Br(δ) NPs exhibit excellent photostability and biocompatibility, making them highly suitable for long-term in vivo NIR-II fluorescence imaging. Their potential as ultra-bright NIR-II fluorophores was demonstrated in NIR-II imaging and tumor resection experiments on blood vessels and tumor sites in mice. In this study, for the first time, by combining the advantages of J-aggregates and the property of enhanced fluorescence efficiency by aggregation-induced emission, an ultra-bright NIR-II fluorophore was constructed, providing a novel and highly effective approach for the development of ultra-bright NIR-II fluorophores. Brief Description of the Drawings

[0030] The present invention will be further described below in conjunction with the accompanying drawings.

[0031] Figure 1 Dynamic light scattering results of AS5Br(δ) in THF-water mixtures with different f w values in Example 2 of the present invention.

[0032] Figure 2 Absorption spectra of AS5Br(δ) in THF-water mixtures with different f w values in Example 2 of the present invention.

[0033] Figure 3 Fluorescence spectra of AS5Br(δ) in THF-water mixtures with different f w values in Example 2 of the present invention.

[0034] Figure 4 Absorption spectra of AS5Br(δ) aggregates prepared with different concentrations of AS5Br(δ) at f w = 90% in Example 2 of the present invention.

[0035] Figure 5 B / A ratio of AS5Br(δ) as a function of f w (where B is the fluorescence integrated area and A is the absorption at 780 nm) in Example 2 of the present invention.

[0036] Figure 6a is the ultraviolet-visible-near-infrared absorption spectrum of AS5Br(δ) at different concentrations in THF in Example 2 of the present invention; b is the fluorescence spectrum of AS5Br(δ) in THF under excitation at 780 nm; c is the relationship function between the integrated fluorescence intensity of AS5Br(δ) in the whole spectrum and the absorbance at 780 nm based on the measured values in (a) and (b).

[0037] Figure 7 a is the normalized absorption spectrum of AS5Br(δ) in different solvents in Example 2 of the present invention; b is the fluorescence spectrum of AS5Br(δ); c is the fluorescence quantum yield of AS5Br(δ) in different solvents.

[0038] Figure 8 a is the absorption and fluorescence spectra of AS5H in THF-water mixture at different f w values in Example 2 of the present invention; b is the absorption and fluorescence spectra of AS5Br(γ) in THF-water mixture at different f w values.

[0039] Figure 9 a is the ultraviolet-visible-near-infrared absorption spectrum of AS5Br(δ) at different concentrations in chloroform in Example 4 of the present invention; b is the relationship graph between the absorbance of AS5Br(δ) at 766 nm and the concentration of AS5Br(δ) in chloroform; c is the absorption spectrum of AS5Br(δ) NPs in water; d is the corresponding absorption spectrum of AS5Br(δ) NPs in chloroform (n = 3).

[0040] Figure 10 a is the absorption spectrum of AS5Br(δ) NPs at different concentrations in water in Example 5 of the present invention; b is the fluorescence spectrum of AS5Br(δ) NPs under excitation at 780 nm; c is the relationship graph between the fluorescence integral area and the absorbance intensity at 780 nm; d is the relationship graph between the fluorescence integral area exceeding 1000 nm and the absorbance intensity at 780 nm based on the measurement in (c).

[0041] Figure 11 is the comparison of the photostability of AS5Br(δ) NPs and ICG / FBS in Example 5 of the present invention, where I 0 and I respectively represent the fluorescence intensities before and after laser irradiation.

[0042] Figure 12 is the cell survival rate after co-incubation of L02 cells with AS5Br(δ) NPs at different concentrations in Example 6 of the present invention (the results are expressed as mean ± standard deviation, n = 4).

[0043] Figure 13Hemolysis percentage of red blood cells after treatment with different concentrations of AS5Br(δ) NPs for 24 hours in Example 6 of the present invention. Triton X-100 (1%) and PBS were used as positive and negative controls, respectively.

[0044] Figure 14 Serum biochemical analysis of PBS (control group) and mice treated with AS5Br(δ) NPs on the 1st day and 7th day in Example 6 of the present invention. The indicators include: a alanine aminotransferase (ALT), b aspartate aminotransferase (AST), c total protein (TP), d albumin (ALB), e blood urea nitrogen (BUN), f creatinine (CR), g globulin (GLB), and h albumin / globulin (A / G) ratio (data are expressed as mean ± standard deviation, n = 3).

[0045] Figure 15 H&E staining of major organs (heart, liver, spleen, lung, and kidney) on the 1st day and 7th day after intravenous injection of PBS (control) and AS5Br(δ) NPs in Example 7 of the present invention.

[0046] Figure 16 a Fluorescence signals in the NIR-II region presented by AS5Br(δ) NPs, ICG dissolved in water, and a 50 μM solution of ICG dissolved in fetal bovine serum (FBS) when excited by a shadowless lamp were observed under different imaging windows of a 900 nm to 1400 nm long-pass filter in Example 7 of the present invention; b NIR-II fluorescence signals after reducing the concentration of the three solutions under the same imaging window (1200 nm LP) when excited by a shadowless lamp.

[0047] Figure 17 Comparison of fluorescence quantitative values of the three solutions under the 1200 nm long-pass filter window in Example 7 of the present invention.

[0048] Figure 18 NIR-II fluorescence imaging was performed by exciting with a shadowless lamp after injecting ICG and AS5Br(δ) NPs into the abdominal wall blood vessels of mice in Example 8 of the present invention.

[0049] Figure 19 In Example 8 of the present invention, for Figure 18 Precise fluorescence analysis of the marked blood vessels.

[0050] Figure 20 Fluorescence signals in the NIR-II region presented by AS5Br(δ) NPs, ICG dissolved in water, and a 10 μM solution of ICG dissolved in fetal bovine serum (FBS) were observed under different imaging windows of a 900 nm to 1400 nm long-pass filter when excited by an ambient white light lamp in Example 9 of the present invention.

[0051] Figure 21 This is the right field of view and fluorescence imaging of the mouse CT26 subcutaneous tumor model after injecting AS5Br(δ) NPs in Example 9 of the present invention.

[0052] Figure 22 a shows the innovative surgical paradigm of NIR-II fluorescence excited by white light and preoperative localization of the tissue to be resected under the shadowless lamp in Example 9 of the present invention. b shows precise resection during the operation through the luminescence effect of AS5Br(δ) NPs. c relies on the NIR-II fluorescence of AS5Br(δ) NPs for double-checking after resection.

[0053] Figure 23 This is the imaging of the blood vessels on the surface of the mouse brain using a NIR-II fluorescence confocal microscope at an ultra-low excitation power in Example 10 of the present invention.

[0054] Figure 24 This is the cerebrovascular image at different depths (100 μm, 200 μm, 300 μm, 400 μm, 500 μm) below the pia mater of the mouse in Example 10 of the present invention.

[0055] Figure 25 This is from Figure 24 The fluorescence-normalized intensity profile (black dots) of the cerebrovascular cross-section and its Gaussian fit (blue line) obtained at a position with a depth of 500 μm (along the green line) in Example 10 of the present invention.

[0056] Figure 26 This is the top view of the three-dimensional reconstruction image of the cerebrovasculature in Example 10 of the present invention. Detailed implementation mode

[0057] Example 1

[0058] The synthetic routes of AS5Br(δ), AS5H, and AS5Br(γ) are as follows.

[0059]

[0060] (1) Synthesis of AS5Br(δ). Dissolve compound 1 (120 mg, 0.08 mmol) and compound 3 (140 mg, 0.5 mmol) in chloroform (30 mL), add pyridine (1 mL) under nitrogen protection for reaction. Stir the mixture at 56 °C for 12 hours. After cooling to room temperature, pour the reaction mixture into methanol to precipitate the crude product, which is then collected by filtration. The crude product is then purified using silica gel column chromatography (eluent: petroleum ether / dichloromethane = 7 / 3), and then evaporated to dryness under vacuum conditions to obtain the blue-black compound AS5Br(δ) (yield 76.7%, 0.12 mg).1 H NMR (400 MHz, CDCl 3 , δ) 9.17 (s, 2H), 8.84 (d, 2H), 7.84 (dd, 2H), 7.77 (d, 2H), 4.71 (d, 6H), 3.22 (t, 4H), 2.38 - 2.32 (m, 1H), 2.07 - 1.97 (m, 2H), 1.90 - 1.83 (m, 4H), 1.54 - 1.48 (m, 4H), 1.45 - 1.33 (m, 12H), 1.31 - 1.21 (m, 32H), 1.17 - 0.92 (m, 56H), 0.83 - 0.75 (m, 24H). 13 C NMR (101 MHz, CDCl 3 , δ) 187.48, 159.64, 153.84, 145.08, 141.50, 138.04, 137.16, 136.70, 135.98, 135.56, 133.77, 133.56, 130.06, 130.02, 128.20, 124.54, 119.75, 115.41, 114.87, 112.07, 68.24, 59.79, 55.37, 40.43, 39.04, 32.06, 32.02, 31.93, 31.42, 30.49, 30.03, 29.96, 29.84, 29.78, 29.73, 29.66, 29.60, 29.54, 29.46, 29.31, 28.45, 25.52, 24.00, 23.03, 22.81, 22.72, 14.25, 14.14, 10.57.

[0061] (2) Synthesis of AS5H. Compounds 1 (120 mg, 0.08 mmol) and 2 (100 mg, 0.5 mmol) were dissolved in chloroform (30 mL), and pyridine (1 mL) was added under nitrogen protection for reaction. The mixture was stirred at 56 °C for 12 hours. After cooling to room temperature, the reaction mixture was poured into methanol to precipitate the crude product, which was then collected by filtration. The crude product was subsequently purified by silica gel column chromatography (eluent: petroleum ether / dichloromethane = 7 / 3), and then evaporated to dryness under vacuum to obtain the blue - black compound AS5H (yield 75.9%, 0.11 mg). 1 H NMR (400 MHz, CDCl 3, δ) 9.17(s, 2H), 8.72 (dd, 2H), 7.93 (dd, 2H), 7.78-7.71 (m, 4H), 4.71 (d, 6H), 3.23(t, 4H), 2.36-2.32 (m, 1H), 2.07-1.98 (m, 2H), 1.91-1.82 (m, 4H), 1.54-1.48(m, 4H), 1.45-1.36 (m, 12H), 1.28-1.20 (m, 32H), 1.18-0.90 (m, 56H), 0.88-0.74 (m, 24H). 13 C NMR (101 MHz, CDCl 3 , δ) 188.56, 161.25, 153.25, 144.81,140.18, 138.00, 137.03, 136.16, 136.00, 135.24, 134.95, 134.16, 133.60,133.30, 129.48, 125.25, 123.53, 120.41, 115.66, 115.26, 111.89, 67.70, 59.77,55.39, 40.44, 39.04, 32.03, 32.00, 31.92, 31.37, 30.52, 30.01, 29.91, 29.83,29.77, 29.71, 29.65, 29.60, 29.52, 29.44, 29.30, 28.47, 25.53, 24.03, 23.02,22.78, 22.70, 14.21, 14.11, 10.57.

[0062] (3) Synthesis of AS5Br(γ). Dissolve compound 1 (120 mg, 0.08 mmol) and compound 4 (140 mg, 0.5 mmol) in chloroform (30 mL), add pyridine (1 mL) under nitrogen protection and react. Stir the mixture at 56 °C for 12 hours. After cooling to room temperature, pour the reaction mixture into methanol to precipitate the crude product, which is then collected by filtration. The crude product is subsequently purified by silica gel column chromatography (eluent: petroleum ether / dichloromethane = 7 / 3), and then evaporated to dryness under vacuum to obtain the blue-black compound AS5Br(γ) (yield 70.3%, 0.11 mg). 1 H NMR (400 MHz, CDCl 3, δ) 9.17 (s, 2H), 8.57 (dd, 2H), 8.02 (s, 2H), 7.86 (d, 2H), 4.73 (d, 6H), 3.22 (t, 4H), 2.39 - 2.32 (m, 1H), 2.07 - 1.99 (m, 2H), 1.91 - 1.83 (m, 4H), 1.54 - 1.48 (m, 4H), 1.45 - 1.33 (m, 12H), 1.31 - 1.21 (m, 32H), 1.16 - 0.94 (m, 56H), 0.86 - 0.76 (m, 24H). 13 C NMR (101 MHz, CDCl 3 , δ) 187.00, 160.19, 153.80, 145.06, 138.62, 138.43, 137.96, 137.55, 136.67, 135.99, 135.57, 133.84, 133.53, 130.08, 129.35, 126.69, 126.40, 119.68, 115.55, 115.19, 112.03, 67.77, 59.79, 55.41, 40.44, 39.14, 32.03, 32.00, 31.94, 31.40, 30.61, 30.54, 30.01, 29.94, 29.87, 29.76, 29.71, 29.67, 29.64, 29.58, 29.50, 29.45, 29.32, 28.47, 25.61, 24.03, 23.03, 22.79, 22.72, 14.22, 14.11, 10.57.

[0063] Example 2

[0064] Measurement of the optical properties of AS5Br(δ) in a mixed solvent of tetrahydrofuran / water (THF - H 2 O). To explore the tendency of AS5Br(δ) to form J - aggregates in an aqueous medium, we initially carried out UV - Vis - NIR spectroscopy studies of AS5Br(δ) in a mixture of tetrahydrofuran (THF) and water, examining different volume fractions of water (f w s). Before measuring their absorption spectra, we performed dynamic light scattering (DLS) measurements, and the results showed that once f w exceeds 20%, aggregation begins (as shown in Figure 1as shown). In pure THF, AS5Br(δ) exhibits characteristic monomer absorption peaks at 741 nm. As f w increases from 0 to 40%, the absorption intensity initially decreases slightly and then significantly. When f w is further increased, the absorption maximum gradually shifts towards longer wavelengths while the absorption intensity increases. When f w reaches 90%, two distinct red-shifted absorption bands appear at 826 nm and 864 nm respectively, indicating that these two absorption bands are caused by different stacking patterns (as Figure 2 shown).

[0065] Meanwhile, we also studied the fluorescence properties of AS5Br(δ) in THF-H 2 O mixtures. In pure THF, AS5Br(δ) exhibits two emission bands at 812 nm and 914 nm respectively (as Figure 3 shown). As f w increases, the high-energy emission band gradually redshifts to 864 nm, while the position of the low-energy emission band remains almost unchanged at 914 nm. Notably, when f w reaches 90%, the high-energy emission band suddenly disappears completely, leaving only the low-energy emission band, which is characterized by a sharp spectrum, narrow full width at half maximum (FWHM: 80 nm) and small Stokes shift (46 nm). The observed spectral properties of AS5Br(δ) at f w = 90%, including redshifted absorption / emission spectra, enhanced absorption band at 864 nm, reduced FWHM and smaller Stokes shift, indicate that the absorption band at 864 nm is attributed to the successful formation of J-aggregates. In addition, by using different concentrations of AS5Br(δ) (2 to 40 μg mL -1 ) in tetrahydrofuran / water (THF-H 2 O) mixed solvents, with water fraction f w = 90%, absorption bands related to J-aggregates can be observed (as Figure 4 shown). Therefore, the concentration dependence of J-aggregate formation is extremely low, indicating that AS5Br(δ) can easily form J-aggregates, thus significantly relaxing the experimental condition limitations required for the formation of J-aggregates.

[0066] Many studies have shown that although J-aggregates can cause redshift of absorption and emission spectra, they do not always result in higher fluorescence efficiency. Therefore, we evaluated the fluorescence efficiency of AS5Br(δ) under different f w s conditions by analyzing the ratio of brightness to absorbance (B / A) (as Figure 5As shown, this ratio can roughly evaluate the change pattern of the fluorescence quantum yield (QY). The luminance and absorbance in B / A are the integral of the fluorescence intensity over the entire emission spectrum and the absorbance at the excitation wavelength (780 nm), respectively. The observed change pattern of the B / A value can be divided into two different stages. The first stage is when f w s increases from 0 to 20%, the B / A value decreases, mainly affected by the solvent polarity because AS5Br(δ) is in a dissolved state. The second stage is when f w s increases from 30% to 90%, the B / A value recovers, indicating that AS5Br(δ) is in an aggregated state, as confirmed by the DLS results (as Figure 1 shown). This observation indicates that AS5Br(δ) has the ability to exhibit aggregation-induced enhancement of fluorescence efficiency. In addition, it is worth noting that at f w = 90%, the B / A value is 1.9 times higher than that at f w = 0%. By considering the fluorescence QY (8.8%) of AS5Br(δ) in THF (as Figure 6 shown), as well as the B / A values and the solvent refractive index of AS5Br(δ) at f w s = 0 and 90%, the fluorescence QY of AS5Br(δ) at f w = 90% can be approximately calculated to be 21.5%. This value exceeds the fluorescence QY of AS5Br(δ) observed in an ideal solvent, such as the QY of 13.4% in chloroform ( Figure 7 describes the QY of AS5Br(δ) in different solvents). This phenomenon has not been observed in other NIR-II fluorescent molecules, including NIR-II fluorescent aggregation-induced emission materials, whose fluorescence efficiency in the aggregated state is still significantly lower than that in the best solvents (e.g., cyclohexane, toluene). In addition, it is worth noting that compared with the dissolved state of AS5Br(δ), the J-aggregate has a larger proportion of the emission spectrum in the NIR-II region, thus further enhancing the potential of the J-aggregate as an NIR-II phosphor.

[0067] Previous studies have shown that small molecules with the same conjugated backbone do not form J-aggregates when they have shorter side chains or no bromine atoms on the acceptor unit, but tend to form H-aggregates. To emphasize the importance of the extended side chains on the conjugated backbone and the bromine substituents on the acceptor unit, we performed the same experiments on compounds AS5H and AS5Br(γ) as on AS5Br(δ), and studied their optical properties in a THF-water mixture. First, compared with previously reported similar molecules with shorter side chains, the aggregates of AS5H, AS5Br(γ), and AS5Br(δ) did not show any absorption bands attributable to H-aggregates. This observation indicates that introducing longer branched side chains onto the conjugated backbone provides steric hindrance, which is not conducive to face-to-face stacking, thus effectively suppressing the formation of H-aggregates ( Figure 8 ). Second, compared with AS5H, both AS5Br(γ) and AS5Br(δ) showed significant redshifts when transitioning from the dissolved state to the aggregate state, indicating that the presence of bromine atoms increases the effective conjugation length upon aggregation, probably due to additional π···Br interactions between adjacent molecules. Third, under comparable conditions, AS5H did not form J-aggregates, as evidenced by the broadened absorption peak and the lack of significant redshift of the absorption peak. In contrast, both AS5Br(γ) and AS5Br(δ) could form J-aggregates. Given that previously reported similar molecules with shorter side chains, bromine substituents, or both did not exhibit any significant J-aggregate characteristics, the ability of AS5Br(γ) and AS5Br(δ) to form J-aggregates should be attributed to the synergistic effect of hydrophobic steric hindrance and enhanced π···Br interactions. In addition, the J-aggregates of AS5Br(δ) showed more significant redshifted absorption bands than those of AS5Br(γ), indicating that the bromine atom at the δ position promotes the sliding arrangement of molecules compared to the γ position. Fourth, both AS5H and AS5Br(γ) exhibited the characteristic of aggregation-induced enhancement of fluorescence efficiency, similar to AS5Br(δ). However, it was observed that the emission spectra of the aggregates of AS5H and AS5Br(γ) were mainly concentrated in the shorter wavelength region, thus weakening their potential as NIR-II fluorophores. Therefore, subsequent studies mainly focused on the properties of AS5Br(δ).

[0068] Example 3

[0069] Preparation method of AS5Br(δ) NPs. Considering the limited stability and large size of J-aggregates generated using the above techniques, their applicability in biological applications is affected. Therefore, we used the nano-precipitation method to encapsulate them within amphiphilic polymers with good biocompatibility, such as DSPE-PEG (PEG length adjustable, PEG molecular weight of 1000 - 10000), Pluronic F-127, etc. They were transformed into nanoparticles that can be stably dispersed in aqueous solutions while retaining the morphology and fluorescence characteristics of the above J-aggregates.

[0070] Taking DSPE-PEG 2000 as the matrix as an example to prepare nanoparticles, the operating steps are as follows: Weigh 1 mg of AS5Br(δ) and 5 mg of DSPE-PEG 2000 , dissolve them in 1 mL of THF, and then quickly inject the THF solution of AS5Br(δ) into 10 mL of ultrapure water, and perform ultrasonic treatment for 2 minutes using a probe sonicator. Stir overnight to completely volatilize the tetrahydrofuran, filter the remaining aqueous solution through a 0.22 μm filter head, and concentrate it using a 30 kDa filter membrane to obtain water-soluble AS5Br(δ) NPs for further experiments. The concentration of AS5Br(δ) NPs was determined by UV-vis-NIR measurement.

[0071] Example 4

[0072] Measurement of the extinction coefficient of AS5Br(δ) NPs in water. The method for determining the extinction coefficient of AS5Br(δ) NPs in water includes three steps:

[0073] (i) Measure the mass extinction coefficient of AS5Br(δ) in chloroform. Briefly, use a chloroform stock solution of AS5Br(δ) with a concentration of 100 μg mL -1 to prepare samples with different concentrations (1, 2, 3, 4, and 5 μg mL -1 ) by diluting chloroform. Then use these samples to measure the absorption spectrum ( Figure 9 a), and thus determine the extinction coefficient of AS5Br(δ) in chloroform by plotting the relationship between the absorption intensity and concentration of AS5Br(δ) at a specific wavelength. Figure 9 The slope of b corresponds to the mass extinction coefficient of AS5Br(δ) at 766 nm in chloroform (108.1 L g -1 cm -1 ).

[0074] (ii) Measure the absorption spectra of AS5Br(δ) NPs in water and samples prepared by dissolving the freeze-dried AS5Br(δ) NPs in chloroform. Initially, divide the aqueous solution of AS5Br(δ) NPs into four samples of the same volume. One sample is used to measure the absorption spectrum ( Figure 9 c), while the remaining three samples are freeze-dried and vacuum-dried. The resulting powder is then dissolved in chloroform in a volume equal to that of the original aqueous solution, and the absorption spectra of these solutions are measured ( Figure 9 d). According to the extinction coefficient of AS5Br(δ) in chloroform described in step (i), the actual concentration of AS5Br(δ) in the above four water samples can be determined according to the Beer-Lambert law: A = εcl. Here, A represents the average absorbance of the freeze-dried powder redissolved in chloroform, c represents the actual concentration of AS5Br(δ) in the above four water samples, and l is the optical path length (1 cm).

[0075] (iii) Calculate the extinction coefficient of AS5Br(δ) NPs in water. Specifically, using the absorption spectrum data of AS5Br(δ) NPs in water and the actual concentration of AS5Br(δ) described in step (ii), the mass extinction coefficient of AS5Br(δ) NPs in water can be determined according to the Beer-Lambert law. In addition, the molar extinction coefficient of AS5Br(δ) NPs at a specific wavelength can be calculated by multiplying the mass extinction coefficient by the molecular weight of AS5Br(δ). The results show that AS5Br(δ) NPs exhibit enhanced absorption ability in the near-infrared region, with an extinction coefficient of 53.4 L g -1 cm -1 or 104900 L mol -1 cm -1 at 808 nm, indicating its strong light-trapping ability.

[0076] Example 5

[0077] The fluorescence quantum yield (QY) of AS5Br(δ) NPs is determined by the following method, using ICG as a reference (QY = 6.0% in DMSO, wavelength above 900 nm). ICG is diluted with DMSO to make a series of samples with an absorbance intensity not exceeding 0.1 at 780 nm. Then, the emission spectrum is integrated into the region from 800 to 1500 nm. The same treatment is carried out on AS5Br(δ) NPs in water. Plot the obtained emission integral values against the absorbance intensity ( Figure 10 a, Figure 10 b, Figure 10 c, Figure 10 d), and fit them into a linear relationship. The calculation formula for QY is as follows:

[0078] Among them, ST and X represent the standard sample and the test sample respectively, QY represents the fluorescence quantum yield, Grad is the gradient obtained from the integral fluorescence intensity and absorbance graph, η represents the refractive index of the solvent, ref represents ICG, and sam represents AS5Br(δ).

[0079] We observed that the absorption and emission spectral characteristics of AS5Br(δ) NPs were highly consistent with those of the above J-aggregates, indicating that the J-aggregate structure was effectively retained in AS5Br(δ) NPs. The overall fluorescence quantum yield of AS5Br(δ) NPs was 20.6%, which was close to the QY value of the J-aggregates formed in a THF-water mixed solvent (90% f w ), further confirming the stability of the J-aggregates in the nanoparticles. Notably, the fluorescence quantum yield of AS5Br(δ) NPs in the NIR-II region reached 5.2%, which was the highest value reported for NIR-II fluorescent J-aggregates to date, highlighting its significant advantage in the field of NIR-II fluorescence imaging. Taking the reported commercially available NIR-II fluorescent dye ICG / FBS as a reference, the NIR-II brightness of AS5Br(δ) NPs increased by more than 37.5 times at the same molar concentration, indicating that AS5Br(δ) NPs are a highly bright NIR-II fluorescent dye. In addition, AS5Br(δ) NPs exhibited excellent photostability under continuous laser irradiation, which is a necessary property for long-term fluorescence imaging (as shown in Figure 11 ). Furthermore, all previous studies have shown that the QY of NIR-II fluorescent J-aggregates decreases significantly when prepared in the form of nanoparticles, even for J-aggregates with aggregation-induced emission characteristics. However, when converting J-aggregates into nanoparticles using a simple nanoprecipitation method, the QY of AS5Br(δ) did not show any decrease, which has not been observed in other NIR-II fluorescent J-aggregates. This advantage will greatly relax the experimental condition constraints for achieving highly bright J-aggregate nanoparticles, thereby improving the reproducibility and practicality of the experiments.

[0080] Example 6

[0081] In vitro and in vivo biosafety characterization of AS5Br(δ) NPs prepared in Example 3. Before verifying the bioimaging ability of AS5Br(δ) NPs, we first evaluated the cytotoxicity of AS5Br(δ) NPs in L02 cells using Cell Counting Kit-8 (CCK-8). As Figure 12 shown, at 0 to 30 μg mL -1In the concentration range, AS5Br(δ) NPs had no significant effect on the viability of L02 cells. Next, we conducted a hemolysis test to evaluate the hemolytic activity of AS5Br(δ) NPs. In this experiment, Triton X-100 and AS5Br(δ) NPs in PBS were used as positive and negative controls, respectively. The results showed that at all tested concentrations, AS5Br(δ) NPs did not cause any significant hemolysis, while the positive control showed obvious hemolysis ( Figure 13 ) Figure 13 Pictures showing direct observation of hemolysis are presented.

[0082] To further investigate the in vivo safety of AS5Br(δ) NPs, we injected AS5Br(δ) NPs into mice via the tail vein and collected serum samples for component analysis 1 day and 7 days after injection. All indicators were within the normal range ( Figure 14 ) ). At the end of the experiment, we collected samples from the major organs (heart, liver, spleen, lung, kidney) for histological analysis. Hematoxylin and eosin (H&E) staining showed that no morphological abnormalities were observed in the mice injected with AS5Br(δ) NPs compared with the PBS control group ( Figure 15 ) . These experimental results indicate that AS5Br(δ) NPs have good biocompatibility.

[0083] Example 7

[0084] NIR-II fluorescence imaging excited by a shadowless lamp. Given the relatively broad absorption spectrum of AS5Br(δ) NPs in the range of 400 nm to 900 nm and the significantly increased NIR-II brightness compared with other NIR-II fluorescent probes, we considered using a shadowless lamp (commonly used in operating rooms) as the excitation light source to achieve high-quality NIR-II fluorescence imaging of AS5Br(δ) NPs under low-power density excitation. Initially, we prepared solutions of AS5Br(δ) NPs, ICG, and ICG / FBS complex at a concentration of 50 μM and excited them in vitro using a shadowless lamp (10 mW cm - ²) to evaluate the NIR-II brightness of AS5Br(δ) NPs. Fluorescence signals were collected using various long-pass filters with a wavelength range from 900 nm to 1400 nm (see Figure 16a). It can be observed that compared with ICG and ICG / FBS at the same molar concentration, the AS5Br(δ) NPs solution can be excited by shadowless lamp light to emit significant NIR-II fluorescence. Even in the long-pass window up to 1400 nm, this fluorescence can still be observed. In contrast, under the same conditions, the fluorescence signals of ICG or ICG / FBS can hardly be detected. Subsequently, we systematically reduced the concentrations of the three solutions to 40 μM, 30 μM, 20 μM, and 10 μM to determine whether distinguishable signals could still be observed within the long-wavelength detection window through a 1200 nm long-pass filter (see Figure 16 b). The results show that even when the solution concentration is reduced to 10 μM, the fluorescence intensity of AS5Br(δ) NPs can still be detected. To more intuitively show the differences in their fluorescence intensities, we quantitatively calculated the fluorescence intensities of the five concentrations under the same wavelength window (1200 nm LP) and normalized them based on the brightness of ICG (see Figure 17 ). After quantification, the fluorescence intensity of AS5Br(δ) NPs is significantly stronger than that of the other two solutions.

[0085] Example 8

[0086] In vivo angiography. On the premise of these in vitro experiments, we designed and constructed a small animal near-infrared II (NIR-II) fluorescence wide-field imaging system, which is excited by the white light illumination of a shadowless lamp. After injecting ICG or AS5Br(δ) nanoparticles (100 μL, 100 μM) into the mouse tail vein, we used this imaging system to detect the NIR-II fluorescence signals of blood vessels. Obviously, when ICG was injected into the mouse body, the fluorescence signal was very weak and could not be detected, and even with a 900 nm long-pass filter, only negligible background signals could be distinguished. In contrast, in the mice injected with AS5Br(δ) nanoparticles, the NIR-II fluorescence signals originating from the abdominal wall blood vessels could still be detected even within the 1200 nm long-pass window (see Figure 18 . When precisely analyzing the fluorescence of the smallest blood vessels in the mouse abdominal blood vessels, it was found that the full width at half maximum (FWHM) of the blood vessels was as narrow as 0.21 mm (see Figure 19 ), which is comparable to the value obtained using a laser as the light source.

[0087] Example 9

[0088] NIR-II Fluorescence Imaging of Tumors Excited by Low-Power Ambient White Light. The data in the previous section demonstrated that AS5Br(δ) NPs overcame the limitations of traditional laser excitation, paving the way for the application of NIR-II fluorescence imaging technology in surgery. However, there are still some limitations in the use of shadowless lamps. As a specific medical device, shadowless lamps are only equipped in operating rooms or certain laboratories. In contrast, ambient white light is ubiquitous in various scenarios and has higher accessibility. In emergency treatment scenarios where time is crucial and operating room equipment may not be immediately available, ambient white light can be quickly used as the excitation light source for NIR-II fluorescence imaging technology. Similarly, in field medicine such as on the battlefield where traditional operating room facilities are lacking, ambient white light provides a practical solution, making this imaging technology possible. In addition, in primary healthcare institutions with relatively simple equipment, the difficulty of equipping professional operating room shadowless lamps and related excitation equipment is overcome by ambient white light. Using this technology, these institutions can achieve simple and efficient fluorescence diagnosis, thus improving the overall level of primary healthcare services.

[0089] To explore the potential of AS5Br(δ) NPs as NIR-II fluorescence probes excited by ambient white light, we conducted in vitro excitation experiments. In these tests, we used low-power ambient white light (3 mW cm -2 −2) as the excitation source. Notably, even at very low concentrations (10 μM), AS5Br(δ) NPs could emit appreciable NIR-II fluorescence, observed up to 1300 LP, highlighting their excellent performance under these conditions (see Figure 20 ). In our study using a mouse model of peritoneal disseminated colorectal cancer tumors, we clearly verified the significant tumor tropism and enrichment ability of AS5Br(δ) NPs. Subsequently, NIR-II fluorescence imaging using an 808 nm laser was able to clearly show the distribution of these nanoparticles within the tumors. To go beyond the limitations imposed by traditional laser-based excitation mechanisms, we designed a wide-field NIR-II fluorescence imaging system excited by low-power ambient white light to investigate the feasibility of using it as an alternative for in vivo tumor fluorescence imaging. Five days after subcutaneous implantation of CT26 colorectal cancer tumors in mice, an AS5Br(δ) NPs solution (100 μL, 100 μM) was injected intravenously. Twenty-four hours later, NIR-II fluorescence imaging was performed using low-power ambient white light (3 mW cm -2 −2). It was found that within the 900 nm long-pass window, the fluorescence intensity in the tumors was significantly higher than that in the surrounding tissues (see Figure 21). The accurate quantification results of NIR-II fluorescence intensity strongly demonstrate that under low-level ambient white light excitation, AS5Br(δ) NPs exhibit remarkable tumor labeling ability. In addition, as the ambient white light excitation power density increases from 3 mW cm -2 gradually to 10 mW cm -2 , the fluorescence intensity ratio between the tumor and adjacent normal tissues was studied to clarify the effect of excitation intensity variation on fluorescence contrast.

[0090] AS5Br(δ) NPs provide a novel NIR-II fluorescence imaging method that can be excited using only low-power ambient white light, different from traditional methods that rely on high-power sources or lasers. In surgical applications, AS5Br(δ) NPs imply the potential to improve tumor visualization and allow for more precise resection and better patient outcomes. An innovative surgical paradigm: leveraging white light and shadowless lamps to excite the NIR-II fluorescence of NPs.

[0091] The surgical approach integrating fluorescence and white light responds to the urgent need to improve the precision of surgical procedures. Traditional surgical lighting relies on white light and has limitations in identifying subtle diseased tissues and differentiating complex anatomical structures. Integrating fluorescence and white light aims to combine the advantages of wide-field illumination of white light and the precise identification ability of fluorescence. For example, in breast cancer surgery, fluorescence-guided sentinel lymph node biopsy can improve the accuracy of lymph node dissection. In hepatobiliary surgery, identifying bile ducts through fluorescence can effectively reduce the incidence of bile duct injury during surgery. However, the technology of integrating NIR-II fluorescence and white light still faces many challenges during its development. The surgical approach combining NIR-II fluorescence and white light changes the operating procedures and visual experience of traditional surgery, posing new requirements for surgeons' operating habits. Surgeons need to simultaneously monitor the image information of fluorescence and white light and quickly switch and comprehensively judge them, which increases the complexity of the operation and the cognitive burden. Our research team previously proposed a collaboration integrating visible light and NIR-II fluorescence. To more effectively meet surgeons' demand for rapid switching between fluorescence and white light images and considering the data mentioned above, we propose an innovative surgical paradigm (as Figure 22 a). This surgical paradigm consists of three steps. The first step is to achieve surface localization by leveraging the NIR-II fluorescence of AS5Br(δ) NPs, which is excited by the white light of a shadowless lamp. For example, in the context of mouse lymph node resection, relying solely on the white light illumination provided by the shadowless lamp, the popliteal lymph nodes of the mouse can be clearly distinguished by their NIR-II fluorescence (see Figure 22a). In the second step, the surgeon carefully and methodically opens the anatomical layers. Each layer, from the epidermis, subcutaneous tissue to the deeper fascia and muscle layers, is meticulously dissected. Here, the colorimetric properties of AS5Br(δ) NPs under white light come into play. These nanoparticles exhibit strong and distinguishable color contrasts when illuminated by white light. This colorimetric effect enables the surgeon to clearly demarcate the boundaries of the target tissue, including the lymph nodes to be excised. In the experimental example, the AS5Br(δ) NPs present in the popliteal lymph nodes of mice exhibit remarkable colorimetric properties. This unique attribute plays a crucial role in significantly enhancing the clarity and efficiency of the procedures involving the exposure of anatomical layers and lymph node resection (see Figure 22 b). In the final step, after the resection surgery is completed, the surgeon can not only rely on the naked eye and professional experience to judge the completeness of the resection, but also conduct a double-check through the NIR-II fluorescence of the residual AS5Br(δ) NPs excited by the white light of the shadowless lamp. In the mouse example, after successfully performing the popliteal lymph node resection, a meticulous double-check was carried out to verify the thoroughness and accuracy of the resection (see Figure 22 c). Summarizing the above experimental results and examples, the surgical paradigm we proposed, leveraging the unique properties of AS5Br(δ) NPs, not only ensures the completeness of the resection, but also improves the safety and precision of the entire surgical process. This paradigm has the potential to improve the patient's prognosis and reduce the necessity for reoperation due to incomplete resection.

[0092] Example 10

[0093] An in vivo NIR-II fluorescence confocal microscope with ultra-low power excitation is used to visualize the cerebral blood vessels in mice. Fluorescence microscopes have become established tools for real-time imaging in biological research. Live cell fluorescence imaging provides key insights into dynamic processes at the cellular and tissue levels. However, exciting fluorophores in biological samples requires high-intensity excitation light, making this method inherently invasive.

[0094] We developed a NIR-II fluorescence confocal microscopy system, which was modified based on a previously constructed device. 673 nm laser was used for excitation and guided to the scanner through a dichroic mirror to achieve XY point scanning. Z-axis positioning was controlled by an electric objective lens with an accuracy of 1 μm. The beam focused by a 25× objective lens (XLPLN25XWMP-SP, NA = 1.05) irradiated the sample, and the backscattered fluorescence was recollected through the optical path. The dichroic mirror redirected the emitted light to a reflective collimator, where only the focused fluorescence was collimated for fiber coupling, effectively replacing the traditional pinhole spatial filtering. Detection was performed using a superconducting nanowire single photon detector (SNSPD), which was optimized for near-infrared quantum efficiency (> 90% at 700 - 900 nm) to ensure maximum photon capture efficiency. A craniotomy was performed under sterile conditions to expose the mouse cerebral cortex, and the cranial window was sealed with a glass coverslip. After intravenous injection of AS5Br(δ) NPs (100 μL, 100 μM), in vivo imaging was performed using a 1 μm Z-axis step size and 10 μs pixel -1 dwell time. Single-plane depth scanning was performed in a 512×512 pixel grid. In existing studies, performing NIR-II fluorescence confocal microscopy imaging typically required an excitation power of at least 15 mW. The experimental results confirmed the excellent fluorescence quantum efficiency of AS5Br(δ) NPs. This property enables AS5Br(δ) NPs to perform high-quality microscopic imaging at extremely low excitation power (about 0.5 mW). Under the excitation of such a low-power light source, the morphological details of the blood vessels on the surface of the mouse brain were clearly shown in three different fields of view (see Figure 23 ). The blood vessels exhibited a complex but highly ordered branching structure, and the fluorescence intensity distribution on the vessel walls was uniform and stable.

[0095] Confocal tomography enabled three-dimensional analysis of the mouse cerebrovascular network, breaking through the limitations of traditional two-dimensional imaging. It achieved micron-scale vascular reconstruction, providing a tool for studying the vascular pathological mechanisms of diseases such as stroke and neurodegenerative diseases. Highly representative confocal images were obtained at depths of 100 μm, 200 μm, 300 μm, 400 μm, and 500 μm respectively (see Figure 24 ). In these images, the morphology of the cerebral blood vessels was clearly distinguishable, and the fine vascular branches and overall vascular directions were clearly presented. The depth-resolved imaging ability at a penetration depth of 500 μm showed continuous high-performance indicators. Quantitative analysis of the obtained optical sections showed precise spatial resolution, demonstrated by the full width at half maximum (FWHM) measurement of 5.15 μm for the representative microvessels (green line). At the same time, the system achieved superior background suppression under the same imaging conditions, quantified by the signal-to-background ratio (SBR) of 5.93 (see Figure 25). By calculating and integrating multi-plane optical slices (depth interval of 0 - 500 μm), volumetric cerebrovascular reconstruction was achieved, as Figure 26 shown.

[0096] The present invention is not limited to the specific technical solutions described in the above embodiments. Any technical solutions formed by equivalent substitution are within the scope of protection required by the present invention.

Claims

1. A compound, characterized in that: The structural formula of compound AS5Br(δ) is as follows:

2. The method for preparing the compound according to claim 1, characterized in that: The synthetic route of compound AS5Br(δ) is as follows: The synthetic route of AS5Br(δ) Reagents and conditions used: (i) pyridine, chloroform, 56°C, 12h.

3. The near-infrared second-region fluorescent J-aggregate with enhanced fluorescence efficiency prepared from the compound according to claim 1, characterized in that: AS5Br(δ) is able to utilize hydrophobic steric interactions and π-bromine interactions in aqueous environments, independently or through interaction with DSPE-PEG 2000 Co-assembly easily forms J-aggregates; the fluorescence quantum yield of the formed J-aggregates exceeds the quantum yield of AS5Br(δ) in chloroform solvent, and AS5Br(δ) also has unique aggregation-induced fluorescence efficiency enhancement characteristics.

4. The NIR-II fluorescent probe prepared from the compound according to claim 1, characterized in that: The compound AS5Br(δ) was prepared into AS5Br(δ)NPs as a near-infrared second-region fluorescent probe.

5. The NIR-II fluorescent probe prepared from the compound according to claim 4, characterized in that: The compound AS5Br(δ) was encapsulated in an amphiphilic polymer by a nano-coprecipitation method to prepare water-soluble AS5Br(δ) NPs. The amphiphilic polymer included DSPE-PEG and Pluronic F-127, and the molecular weight of PEG was 1000-10000.

6. The NIR-II fluorescent probe prepared from the compound according to claim 5, characterized in that: AS5Br(δ)NPs were synthesized by nanocoprecipitation method. 1 mg of AS5Br(δ) and DSPE-PEG were dissolved in 1 mL of tetrahydrofuran (THF). 2000 5 mg; the resulting solution was then injected into 10 mL of ultrapure water, and ultrasonicated for 2 minutes using a probe ultrasonicator. At room temperature, the tetrahydrofuran was stirred overnight to evaporate completely, and the solution was filtered through a 0.22 μm filter head and concentrated using a 30 kDa filter membrane to obtain a water-soluble near-infrared second-zone fluorescent probe AS5Br(δ)NPs.

7. The use of the NIR-II fluorescent probe according to claim 4, characterized in that: The NIR-II fluorescent probe is used in the field of near-infrared second-zone fluorescence imaging.

8. The use of the NIR-II fluorescent probe according to claim 4, characterized in that: The NIR-II fluorescent probe can achieve significant accumulation at the tumor site and is used for preparing visualization agents in vascular imaging or tumor resection surgery.

Images