NIR-II fluorescent J-aggregate capable of aggregating and enhancing luminous efficiency and application of NIR-II fluorescent J-aggregate

Abstract

The invention provides an NIR-II fluorescent J-aggregate capable of aggregating and enhancing luminous efficiency and application of the NIR-II fluorescent J-aggregate. AS5Br (delta) can easily form a J-aggregate in a water medium, a complicated preparation process is not needed, and the AS5Br (delta) has the characteristic of enhancing aggregation-induced fluorescence efficiency. After the J-aggregate nanoparticles AS5Br (delta) NPs are converted into J-aggregate nanoparticles AS5Br (delta) NPs, the record of the highest quantum yield in the NIR-II fluorescent J-aggregate reported so far is realized, and the quantum yield of NIR-II fluorescence reaches 5.2%. The fluorescence quantum yield of AS5Br (delta) NPs even exceeds the fluorescence quantum yield of AS5Br (delta) NPs in an optimal solvent, the problem that NIR-II fluorescent J-aggregate is converted into biocompatible nanoparticles is solved, and the potential of the AS5Br (delta) NPs as a superbright NIR-II fluorophore is shown in NIR-II imaging and tumor excision experiments on blood vessels and tumor parts of mice.

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, which promotes cooperative coupling of excited-state transition dipoles. The photochemical and photophysical properties of J-aggregates, including red-shifted absorption / emission spectra, narrowed spectral bandwidth, enhanced extinction coefficient, and higher photostability, have been shown to have significant advantages in in vivo NIR-II fluorescence imaging. To date, a variety of 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 they are prepared into nanoparticles for enhanced biocompatibility and applicability. First, the preparation of J-aggregates into nanoparticles leads to a significant decrease in fluorescence quantum yield and thus brightness, both of which are significantly lower than the performance 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 issues severely limit the potential of J-aggregates as NIR-II fluorophores. Therefore, exploring new fluorescent materials that can easily form J-aggregates and can 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 a NIR-II fluorescent J-aggregate with enhanced luminescence efficiency as a NIR-II fluorescent probe, and reports a small molecule AS5Br(δ), which can easily form J-aggregates in an aqueous medium. 2000The co-assembly formed ultrabright NIR-II fluorescent J-aggregates. The thus prepared J-aggregate nanoparticles (AS5Br(δ) NPs) combined the intrinsic properties of J-aggregates with the unique phenomenon of aggregation-induced fluorescence efficiency enhancement, achieving significant fluorescence quantum yields (QY) of 20.6%, 16% and 5.2% at wavelengths above 800 nm, 900 nm and 1000 nm, respectively. These values ​​represent the highest fluorescence quantum yields among NIR-II fluorescent J-aggregates reported to date. It is worth noting that compared with all reported NIR-II fluorescent J-aggregates, AS5Br(δ) J-aggregates exhibit unique fluorescence enhancement properties: their fluorescence quantum yields not only do not decrease due to aggregation, but are significantly improved compared to their monomeric state in the most suitable solvent. This phenomenon is the first to occur 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 available. Its ultra-high NIR-II brightness, coupled with a simple preparation process, effectively solves the inherent limitations of existing NIR-II fluorescent J-aggregates when they are 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] In order to solve the above technical problems, the technical solution proposed by the present invention is: a compound, the structural formula of the compound AS5Br(δ) is as follows:

[0007] .

[0008] In order 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] The reagents and conditions used in the synthetic route of AS5Br(δ) are as follows: (i) pyridine, chloroform, 56°C, 12h.

[0011] In order to solve the above technical problems, another technical solution proposed by the present invention is: the near-infrared second-zone fluorescent J-aggregates prepared by the compound, AS5Br(δ) can independently form J-aggregates in an aqueous environment, or by reacting with DSPE-PEG 2000The co-assembly formed J-aggregates, which benefited from the hydrophobic steric hindrance and π-bromine interaction of AS5Br(δ). The fluorescence quantum yield of the formed J-aggregates exceeded that of AS5Br(δ) in chloroform solvent; in addition, AS5Br(δ) also had unique aggregation-induced fluorescence efficiency enhancement characteristics.

[0012] In order 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 fluorescent probe.

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

[0014] Preferably, AS5Br(δ) NPs are synthesized by nanocoprecipitation method. 1 mL of tetrahydrofuran is used to dissolve the compound AS5Br(δ) (1 mg) and DSPE-PEG 2000 (5 mg); the resulting solution was then injected into 10 mL of ultrapure water and sonicated for 2 min using a probe sonicator. After the organic solvent evaporated at room temperature, the solution was filtered through a 0.22 μm filter head and concentrated using a 30 kDa filter membrane to obtain AS5Br(δ) NPs.

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

[0016] Preferably, the fluorescent probe AS5Br(δ) NPs is used to prepare a near-infrared II fluorescence imaging probe. The fluorescent probe AS5Br(δ) NPs has 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 fluorescent probe AS5Br(δ) NPs effectively overcomes the difficulty of converting NIR-II fluorescent J-aggregates into biocompatible nanoparticles. The in vivo efficacy of AS5Br(δ) nanoparticles as an excellent NIR-II fluorophore was demonstrated by comparing the brightness with ICG in FBS in vitro, NIR-II imaging in vascular and tumor sites in mice, and tumor resection experiments.

[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 multiple properties, including a narrow band gap, a high extinction coefficient and high photostability, which make them very suitable as NIR-II fluorophores. Some of the molecules with similar structural characteristics exhibit the characteristics of enhanced aggregation-induced fluorescence efficiency, which makes them exhibit high fluorescence quantum yield 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 exhibit 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 its potential as an ultra-bright NIR-II fluorophore because H-aggregates have blue-shifted absorption and 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 cyclic 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 AS5Br(δ) from H-aggregation, while the presence of bromine atoms provides π-bromine interactions, which is conducive to the induction of J-aggregate formation. To further clarify the molecular design principle of AS5Br(δ), two control compounds AS5H and AS5Br(γ) were designed for comparison, where AS5H lacks a bromine substituent 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] Beneficial effects of the present invention:

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

[0026] First, AS5Br(δ) can be synthesized in aqueous medium either independently or by reacting with DSPE-PEG 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 solves the challenges faced by existing NIR-II fluorescent J-aggregates in the preparation of biocompatible nanoparticles, laying the foundation for its application as a high-performance NIR-II fluorescent agent in the biological field. In addition, AS5Br(δ) NPs exhibit excellent photostability and biocompatibility, making them very suitable for long-term in vivo NIR-II fluorescence imaging. Its potential as an ultrabright NIR-II fluorophore was demonstrated in NIR-II imaging of blood vessels and tumor sites in mice, as well as tumor resection experiments. This study is the first to construct an ultrabright NIR-II fluorophore by combining the advantages of J-aggregates and the characteristics of aggregation-induced fluorescence efficiency enhancement, providing a novel and extremely effective approach for the development of ultrabright 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 The AS5Br(δ) in Example 2 of the present invention is different in the THF-water mixture. w Dynamic light scattering results at 200 MHz and 800 MHz.

[0032] Figure 2 AS5Br(δ) in Example 2 of the present invention at different f w Absorption spectra of THF-water mixtures at 2.5 Å.

[0033] Figure 3 AS5Br(δ) in Example 2 of the present invention at different f w Fluorescence spectra in THF-water mixtures at 2.5 Å.

[0034] Figure 4 In Example 2 of the present invention, w =90% using different concentrations of AS5Br(δ) to prepare the AS5Br(δ) aggregates.

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

[0036] Figure 6a is the UV-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 entire 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 AS5H in Example 2 of the present invention at different f w Absorption and fluorescence spectra of AS5Br(γ) in THF-water mixtures at different f values; b is the absorption and fluorescence spectra of AS5Br(γ) in THF-water mixtures at different f values; w Absorption and fluorescence spectra in THF-water mixtures at 2.5 Å.

[0039] Fig. 9 a is the UV-visible-near infrared absorption spectrum of AS5Br(δ) at different concentrations in chloroform in Example 4 of the present invention; b is the relationship 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] Fig.10 a is the absorption spectrum of AS5Br(δ) NPs in Example 5 of the present invention at different concentrations in water; b is the fluorescence spectrum of AS5Br(δ) NPs under 780 nm excitation; c is the relationship between the fluorescence integrated area and the absorbance intensity at 780 nm; d is the relationship between the fluorescence integrated area over 1000 nm and the absorbance intensity at 780 nm based on the measurement of (c).

[0041] Fig.11 This is a comparison of the photostability of AS5Br(δ) NPs and ICG / FBS in Example 5 of the present invention. I0 and I represent the fluorescence intensity before and after laser irradiation, respectively.

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

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

[0044] Fig.14 Serum biochemical analysis of mice treated with PBS (control group) and AS5Br(δ) NPs on day 1 and day 7 in Example 6 of the present invention. 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] Fig.15 H&E staining of major organs (heart, liver, spleen, lung and kidney) in Example 7 of the present invention at 1 day and 7 days after intravenous injection of PBS (control) and AS5Br(δ) NPs.

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

[0047] Fig.17 The fluorescence quantitative values ​​of the three solutions in Example 7 of the present invention are compared under the 1200 nm long-pass filter window.

[0048] Fig.18 In Example 8 of the present invention, after ICG and AS5Br(δ) NPs were injected into the abdominal wall blood vessels of mice, NIR-II fluorescence imaging was performed by excitation with a shadowless lamp.

[0049] Fig.19 For Example 8 of the present invention Fig.18 Precise fluorescence analysis of labeled blood vessels in vivo.

[0050] Fig. 20 The fluorescence signals in the NIR-II region of AS5Br(δ) NPs, ICG dissolved in water, and 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 in Example 9 of the present invention when excited by ambient white light.

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

[0052] Fig. 22 a is the innovative surgical paradigm using white light-excited NIR-II fluorescence and preoperative positioning of the tissue to be removed under a shadowless lamp in Example 9 of the present invention. b is the precise removal during surgery achieved by the luminescent effect of AS5Br(δ) NPs. c is the double check of postoperative resection based on the NIR-II fluorescence of AS5Br(δ) NPs.

[0053] Fig.23 In Example 10 of the present invention, the surface blood vessels of the mouse brain are imaged using a NIR-II fluorescence confocal microscope at ultra-low excitation power.

[0054] Fig.24 3 are images of cerebral blood vessels at different depths (100 μm, 200 μm, 300 μm, 400 μm, and 500 μm) below the pia mater of a mouse in Example 10 of the present invention.

[0055] Fig.25 In Example 10 of the present invention, Fig.24 Normalized fluorescence intensity profile (black dots) and its Gaussian fit (blue line) of a cross-section of a cerebral vascular vessel acquired at a depth of 500 μm (along the green line).

[0056] Fig.26 This is a top view of the three-dimensional reconstructed image of cerebral blood vessels in Example 10 of the present invention. DETAILED DESCRIPTION

[0057] Example 1

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

[0059]

[0060] (1) Synthesis of AS5Br(δ). Compound 1 (120 mg, 0.08 mmol) and compound 3 (140 mg, 0.5 mmol) were dissolved in chloroform (30 mL), and pyridine (1 mL) was added under nitrogen protection to react. 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 then purified using silica gel column chromatography (eluent: petroleum ether / dichloromethane = 7 / 3), and then evaporated to dryness under vacuum conditions to obtain a blue-black compound AS5Br(δ) (yield 76.7%, 0.12 mg).1 H NMR (400 MHz, CDCl3,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, CDCl3, 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. Compound 1 (120 mg, 0.08 mmol) and compound 2 (100 mg, 0.5 mmol) were dissolved in chloroform (30 mL), and pyridine (1 mL) was added under nitrogen protection to react. 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 then purified using silica gel column chromatography (eluent: petroleum ether / dichloromethane = 7 / 3), and then evaporated to dryness under vacuum conditions to obtain a blue-black compound AS5H (yield 75.9%, 0.11 mg). 1H NMR (400 MHz, CDCl3, 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, CDCl3, 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). Compound 1 (120 mg, 0.08 mmol) and compound 4 (140 mg, 0.5 mmol) were dissolved in chloroform (30 mL), and pyridine (1 mL) was added under nitrogen protection to react. 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 then purified using silica gel column chromatography (eluent: petroleum ether / dichloromethane = 7 / 3), and then evaporated to dryness under vacuum conditions to obtain a blue-black compound AS5Br(γ) (yield 70.3%, 0.11 mg). 1H NMR (400 MHz, CDCl3,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, CDCl3, 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 tetrahydrofuran / water (THF-H2O) mixed solvents. To investigate the tendency of AS5Br(δ) to form J-aggregates in aqueous media, we initially conducted UV-Vis-NIR spectroscopy studies of AS5Br(δ) in tetrahydrofuran (THF) and water mixtures, examining the water fractions (f w s). Before measuring their absorption spectra, we performed dynamic laser scattering (DLS) measurements, which showed that once f w Above 20%, aggregates will begin to form (e.g. Figure 1As shown in Figure 2). In pure THF, AS5Br(δ) exhibits a unique monomer absorption peak at 741 nm. w As the ratio increases from 0 to 40%, the absorption intensity decreases slightly at first and then decreases significantly. w When f w When the mass fraction reaches 90%, two obvious red-shifted absorption bands appear, located at 826 nm and 864 nm, respectively, which indicates that the two absorption bands are caused by different stacking modes (e.g. Figure 2 shown).

[0065] At the same time, we also studied the fluorescence properties of AS5Br(δ) in THF-H2O mixtures. In pure THF, AS5Br(δ) exhibits two emission bands, located at 812 nm and 914 nm (e.g. Figure 3 As f w As f 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. w When it reaches 90%, the high-energy emission band suddenly disappears completely, leaving only the low-energy emission band, which is characterized by a sharp spectrum, a narrow full width at half maximum (FWHM: 80 nm) and a small Stokes shift (46 nm). w = 90%, including the red-shifted absorption / emission spectra, enhanced absorption band at 864 nm, decreased FWHM, and smaller Stokes shift, indicating that the absorption band at 864 nm is attributed to the successful formation of J-aggregates. -1 ) in a tetrahydrofuran / water (THF-H2O) mixed solvent, water fraction f w = 90%, absorption bands associated with J-aggregates can be observed (e.g. Figure 4 Therefore, the concentration dependence of J-aggregate formation is extremely low, indicating that AS5Br(δ) can readily form J-aggregates, thereby significantly relaxing the experimental conditions required for the formation of J-aggregates.

[0066] Many studies have shown that although J-aggregates can lead to red-shifts in the absorption and emission spectra, they do not always result in higher fluorescence efficiency. Therefore, we evaluated the fluorescence performance of AS5Br(δ) at different f by analyzing the brightness to absorbance (B / A) ratio. w Fluorescence efficiency under s conditions (such as Figure 5The ratio can roughly evaluate the change pattern of fluorescence quantum yield (QY). The brightness and absorbance in B / A are the integral of the fluorescence intensity of the entire emission spectrum and the absorption intensity 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 f w The B / A value decreases when s increases from 0 to 20%, which is mainly affected by the polarity of the solvent because AS5Br(δ) is in a dissolved state. w s increased from 30% to 90%, the B / A value recovered, which indicated that AS5Br(δ) was in an aggregate state, as confirmed by the DLS results (e.g. Figure 1 This observation indicates that AS5Br(δ) has the ability to aggregate and induce enhanced fluorescence efficiency. In addition, it is worth noting that in f w = 90%, the B / A value is f w = 0% is 1.9 times higher. By considering the fluorescence QY of AS5Br(δ) in THF (8.8%) (e.g. Figure 6 As shown), and AS5Br(δ) at f w The B / A values ​​at s = 0 and 90% and the solvent refractive index can be used to approximately calculate the AS5Br(δ) at f w = 90% is 21.5%. This value exceeds the observed fluorescence QY of AS5Br(δ) in ideal solvents, such as 13.4% in chloroform ( Figure 7 The QY of AS5Br(δ) in different solvents is described. 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 aggregate state is still significantly lower than that in the optimal solvent (e.g., cyclohexane, toluene). In addition, it is noteworthy that the J-aggregate has a larger proportion of the emission spectrum in the NIR-II region compared to the dissolved AS5Br(δ), further enhancing the potential of the J-aggregate as a NIR-II fluorophore.

[0067] Previous studies have shown that small molecules with the same conjugated backbone but with shorter side chains or without bromine atoms on the acceptor unit do not form J-aggregates 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 as AS5Br(δ) on compounds AS5H and AS5Br(γ) and studied their optical properties in THF-water mixtures. First, in contrast to similar molecules with shorter side chains reported previously, the aggregates of AS5H, AS5Br(γ), and AS5Br(δ) did not show any absorption bands attributable to H-aggregates. This observation suggests that the introduction of longer branched side chains onto the conjugated backbone provides steric hindrance that is unfavorable for face-to-face stacking, thereby effectively suppressing the formation of H-aggregates ( Figure 8 ). Second, both AS5Br(γ) and AS5Br(δ) exhibited significant red shifts when transitioning from the dissolved state to the aggregated state compared to AS5H, suggesting that the presence of bromine atoms increases the effective conjugation length upon aggregation, likely due to additional π···Br interactions between neighboring molecules. Third, AS5H did not form J-aggregates under comparable conditions, as evidenced by the broadened absorption peaks and the lack of significant red shifts in the absorption peaks. In contrast, both AS5Br(γ) and AS5Br(δ) were able to form J-aggregates. Given that similar molecules with shorter side chains, bromine substituents, or both reported previously did not exhibit any significant J-aggregate features, the ability of AS5Br(γ) and AS5Br(δ) to form J-aggregates should be attributed to the synergistic effects of hydrophobic steric hindrance and enhanced π···Br interactions. In addition, the J-aggregates of AS5Br(δ) exhibited more significantly red-shifted absorption bands than those of AS5Br(γ), suggesting that the bromine atoms at the δ position facilitated the molecular arrangement in a sliding manner compared to the γ position. Fourth, both AS5H and AS5Br(γ) exhibited aggregation-induced fluorescence efficiency enhancement, similar to AS5Br(δ). However, it was observed that the emission spectra of AS5H and AS5Br(γ) aggregates were mainly concentrated in the shorter wavelength region, which weakened their potential as NIR-II fluorophores. Therefore, subsequent studies focused on the properties of AS5Br(δ).

[0068] Example 3

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

[0070] DSPE-PEG 2000 For example, the nanoparticles were prepared by taking the matrix as an example. The operation steps were as follows: 1 mg of AS5Br(δ) and 5 mg of DSPE-PEG were weighed. 2000 , dissolved in 1 mL THF, and then the THF solution of AS5Br(δ) was quickly injected into 10 mL of ultrapure water and sonicated for 2 minutes by a probe sonicator. Stir overnight to completely evaporate 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] Extinction coefficient measurement of AS5Br(δ) NPs in water. The method to determine 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, a concentration of 100 μg mL -1 The AS5Br(δ) chloroform stock solution was prepared by diluting chloroform to prepare samples of different concentrations (1, 2, 3, 4, and 5 μg mL -1 These samples were then used to measure the absorption spectra ( Fig. 9 a), thereby determining the extinction coefficient of AS5Br(δ) in chloroform by plotting the absorption intensity of AS5Br(δ) at a specific wavelength versus its concentration. Fig. 9 The slope of b corresponds to the mass extinction coefficient of AS5Br(δ) in chloroform at 766 nm (108.1 L g -1 cm -1 ).

[0074] (ii) Measurement of the absorption spectra of AS5Br(δ) NPs in water and samples prepared by dissolving freeze-dried AS5Br(δ) NPs in chloroform. Initially, the aqueous solution of AS5Br(δ) NPs was divided into four samples of equal volume. One sample was used to measure the absorption spectrum ( Fig. 9 c), while the remaining three samples were freeze-dried and vacuum-dried. The resulting powders were then dissolved in chloroform in a volume equal to that of the original aqueous solution, and the absorption spectra of these solutions were measured ( Fig. 9 d). Based on 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 lyophilized 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 showed that AS5Br(δ) NPs exhibited enhanced absorption in the near-infrared region, with an extinction coefficient of 53.4 L g at 808 nm. -1 cm -1 or 104900 L mol -1 cm -1 , indicating its strong light-harvesting ability.

[0076] Example 5

[0077] The fluorescence quantum yield (QY) of AS5Br(δ) NPs was determined by the following method, using ICG as a reference (QY = 6.0% in DMSO over 900 nm). ICG was diluted with DMSO to prepare a series of samples with an absorbance intensity of no more than 0.1 at 780 nm. The emission spectrum was then integrated over the 800 to 1500 nm region. The same treatment was performed on AS5Br(δ) NPs in water. The resulting emission integral values ​​were plotted against the absorbance intensity ( Fig.10 a. Fig.10 b. Fig.10 c. Fig.10 d) and fitted into a linear relationship. The calculation formula of QY is as follows:

[0078] Wherein, ST and X represent the standard sample and the test sample, respectively, QY represents the fluorescence quantum yield, Grad represents the gradient obtained from the integrated fluorescence intensity versus 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 spectra of AS5Br(δ) NPs were highly consistent with those of the J-aggregates, indicating that the J-aggregate structure was effectively preserved in AS5Br(δ) NPs. The overall fluorescence quantum yield of AS5Br(δ) NPs was 20.6%, which was comparable to that in THF-water mixed solvent (90%). f w ), further confirming the stability of the J-aggregates in the nanoparticles. It is worth noting that the fluorescence quantum yield of AS5Br(δ) NPs in the NIR-II region reaches 5.2%, which is the highest value among the NIR-II fluorescent J-aggregates reported so far, highlighting its significant advantages 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 at the same molar concentration is increased by more than 37.5 times. This result shows that AS5Br(δ) NPs is an extremely bright NIR-II fluorescent dye. In addition, AS5Br(δ) NPs exhibit excellent photostability under continuous laser irradiation, which is a property necessary to achieve long-term fluorescence imaging (see Figure 2). Fig.11 ). In addition, all previous studies have shown that NIR-II fluorescent J-aggregates, even those with aggregation-induced emission properties, suffer from a significant decrease in QY when prepared in nanoparticle form. However, when the J-aggregates were converted into nanoparticle form 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 constraints for achieving highly bright J-aggregate nanoparticles, thereby improving the reproducibility and practicality of its experiments.

[0080] Example 6

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

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

[0083] Example 7

[0084] NIR-II fluorescence imaging excited by shadowless lamp. Given the relatively broad absorption spectrum of AS5Br(δ) NPs in the range of 400 nm to 900 nm and the significantly improved 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 complexes at a concentration of 50 μM and in vitro illuminated them using a shadowless lamp (10 mW cm - ²) for excitation to evaluate the NIR-II brightness of AS5Br(δ) NPs. The fluorescence signals were collected by using various long-pass filters ranging from 900 nm to 1400 nm (see Fig.16a). It can be observed that the AS5Br(δ) NPs solution can be excited by shadowless light to emit significant NIR-II fluorescence compared to ICG and ICG / FBS at the same molar concentration. This fluorescence can still be observed even in the long-pass window up to 1400 nm. In contrast, under the same conditions, the fluorescence signal of ICG or ICG / FBS was almost undetectable. Subsequently, we systematically reduced the concentration of the three solutions to 40 μM, 30 μM, 20 μM, and 10 μM to determine whether a discernible signal could still be observed in the long-wavelength detection window through a 1200 nm long-pass filter (see Fig.16 b). The results show that the fluorescence intensity of AS5Br(δ) NPs can still be detected even when the solution concentration is reduced to 10 μM. In order to more intuitively show the difference in their fluorescence intensity, we quantitatively calculated the fluorescence intensity of five concentrations in the same wavelength window (1200 nm LP) and normalized it based on the brightness of ICG (see Fig.17 ). After quantification, the fluorescence intensity of AS5Br(δ) NPs was significantly stronger than that of the other two solutions.

[0085] Example 8

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

[0087] Example 9

[0088] Low-power ambient white light-excited NIR-II fluorescence imaging for tumor localization. The data in the previous section showed that AS5Br(δ) NPs overcome the limitations of traditional laser excitation and pave 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, time is of the essence and operating room equipment may not be immediately available. Ambient white light can be quickly used as an excitation light source for NIR-II fluorescence imaging technology. Similarly, in field medicine such as battlefields where traditional operating room facilities are lacking, ambient white light provides a practical solution, making this imaging technology possible. In addition, in primary health care institutions with relatively simple equipment, the difficulty of equipping professional operating room shadowless lamps and related excitation equipment is overcome by ambient white light. With this technology, these institutions can achieve simple and efficient fluorescence diagnosis, thereby improving the overall level of primary health care services.

[0089] To explore the potential of AS5Br(δ) NPs as NIR-II fluorescent probes excited by ambient white light, we performed in vitro excitation experiments. In these tests, we used low-power ambient white light (3 mW cm -2 ) as the excitation source. Notably, even at very low concentrations (10 μM), AS5Br(δ) NPs were able to emit considerable NIR-II fluorescence, observed up to 1300 LP, highlighting their excellent performance under these conditions (see Fig. 20 ). In our study using a mouse peritoneally disseminated colorectal cancer tumor model, we clearly verified the remarkable 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 tumor. To transcend 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 its feasibility 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, the AS5Br(δ) NPs were excited by high-power ambient white light (3 mW cm -2 ) for NIR-II fluorescence imaging. It was found that within the 900 nm long-pass window, the fluorescence intensity in the tumor was significantly higher than that in the surrounding tissue (see Fig.21The accurate quantification of NIR-II fluorescence intensity strongly demonstrates that AS5Br(δ) NPs exhibit significant tumor labeling ability under low-level ambient white light excitation. In addition, as the ambient white light excitation power density increases from 3 mW cm -2 Gradually increase to 10 mW cm -2 , the fluorescence intensity ratio between tumor and adjacent normal tissue was studied, aiming to elucidate the effect of changes in excitation intensity on fluorescence contrast.

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

[0091] The surgical approach integrating fluorescence and white light is a response to the urgent need to improve the precision of surgical procedures. Traditional surgical illumination relies on white light, which has limitations in identifying delicate lesions and distinguishing complex anatomical structures. Integrated fluorescence and white light aims to combine the wide-area illumination advantage 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 the bile duct by 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 in its development. The surgical approach combining NIR-II fluorescence and white light changes the operating procedures and visual experience of traditional surgery, and puts forward new requirements for the operating habits of surgeons. Surgeons need to monitor the image information of fluorescence and white light at the same time, and quickly switch and comprehensively judge them, which increases the complexity and cognitive burden of the operation. Our research team previously proposed a collaboration of visible light and NIR-II fluorescence integration. In order to more effectively meet the surgeon's needs for rapid switching between fluorescence and white light images, and considering the aforementioned data, we proposed an innovative surgical paradigm (such as Fig. 22 a). This surgical paradigm consists of three steps. In the first step, surface localization is achieved by utilizing 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, the mouse popliteal lymph node can be clearly distinguished by its NIR-II fluorescence, relying solely on the white light illumination provided by the shadowless lamp (see Fig. 22a). In the second step, the surgeon carefully and methodically opens the anatomical layers. Each layer, from the epidermis and subcutaneous tissue to the deeper fascia and muscle layers, is meticulously separated. Here, the chromogenic properties of AS5Br(δ) NPs under white light come into play. These nanoparticles exhibit a strong and distinguishable color contrast when illuminated by white light. This chromogenic effect enables surgeons to clearly delineate the boundaries of target tissues, including lymph nodes that need to be removed. In the experimental example, AS5Br(δ) NPs present in the popliteal lymph nodes of mice exhibited significant chromogenic properties. This unique property played a key role in significantly improving the clarity and efficiency of the process involving exposing anatomical layers and lymph node removal (see Fig. 22 b). In the final step, after the resection is completed, the surgeon can not only rely on the naked eye and professional experience to judge whether the resection is complete, but also double-check by NIR-II fluorescence of residual AS5Br(δ) NPs excited by white light from a shadowless lamp. In the mouse example, after the successful resection of the popliteal lymph node, a meticulous double check was performed to verify the thoroughness and accuracy of the resection (see Fig. 22 c). To summarize the above experimental results and examples, our proposed surgical paradigm, utilizing the unique properties of AS5Br(δ) NPs, not only ensures the completeness of resection, but also improves the safety and precision of the entire surgical procedure. This paradigm has the potential to improve patient outcomes and reduce the need for reoperation due to incomplete resection.

[0092] Example 10

[0093] In vivo NIR-II fluorescence confocal microscopy with ultra-low power excitation is used to visualize mouse brain vasculature. Fluorescence microscopy has become an established tool for real-time imaging in biological research. Live cell fluorescence imaging provides key insights into dynamic processes at the cellular and tissue levels. However, high-intensity excitation light is required to excite fluorophores in biological samples, making this method inherently invasive.

[0094] We developed a NIR-II fluorescence confocal microscopy system based on a previously constructed device. Excitation was performed using a 673 nm laser, which was directed to a scanner via a dichroic mirror to achieve XY point scanning. Z-axis positioning was controlled by a motorized objective with 1 μm precision. The sample was illuminated by a beam focused by a 25x objective (XLPLN25XWMP-SP, NA=1.05), 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 skull window was sealed with a glass coverslip. After AS5Br(δ) NPs (100 μL, 100 μM) were injected via the tail vein, the Z-axis step size of 1 μm and the pixel size of 10 μs were used. -1 In vivo imaging was performed with a dwell time. Depth scanning in a single plane was performed with a 512×512 pixel grid. In existing studies, NIR-II fluorescence confocal microscopy imaging usually requires an excitation power of at least 15 mW. The experimental results confirmed the excellent fluorescence quantum efficiency of AS5Br(δ) NPs. This property enables high-quality microscopic imaging of AS5Br(δ) NPs at extremely low excitation power (about 0.5 mW). Under the excitation of this low-power light source, the morphological details of the surface blood vessels of the mouse brain were clearly displayed in three different fields of view (see Fig.23 ). The blood vessels showed a complex but highly ordered branching structure, and the fluorescence intensity distribution on the vessel wall was uniform and stable.

[0095] Confocal tomography enables three-dimensional analysis of the mouse brain vascular network, breaking through the limitations of traditional two-dimensional imaging. It enables 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 acquired at depths of 100 μm, 200 μm, 300 μm, 400 μm, and 500 μm (see Fig.24 ). In these images, the morphology of brain vessels is clearly discernible, with subtle vascular branching and overall vascular orientation clearly presented. Depth-resolved imaging capabilities at a penetration depth of 500 μm showed continued high performance indicators. Quantitative analysis of the acquired optical sections showed precise spatial resolution, as evidenced by a FWHM measurement of 5.15 μm for representative microvessels (green line). At the same time, the system achieved superior background suppression under the same imaging conditions, quantified by a signal-to-noise ratio (SBR) of 5.93 (see Fig.25By computationally integrating multi-plane optical slices (0-500 μm depth interval), volumetric cerebral vascular reconstruction was achieved, such as Fig.26 shown.

[0096] The present invention is not limited to the specific technical solutions described in the above embodiments, and all technical solutions formed by equivalent replacement are within the protection scope 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 reagents and conditions used in the synthetic route of AS5Br(δ) are as follows: (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 The 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 a unique aggregation-induced fluorescence efficiency enhancement property.

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 polymers 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 sonicated for 2 minutes using a probe sonicator. At room temperature, the tetrahydrofuran was completely evaporated by stirring overnight. 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