Fig.1 Schematic illustration of building-block, compact, and “zero-on” NIR-II PA dyes based on dimethyl-dihydroacridine for sensitive and high-fidelity molecular bioimaging. (a) Building-block molecule design of a dye library constructed on a dimethyl-dihydroacridine-based scaffold, including representative examples (DPA1-3) and their corresponding absorption maxima. (b) Schematic illustration of the highly compact molecule of DPA(1–3)Cl dyes, defined as a large ratio of the maximum absorption wavelength (λmax, abs) to molecular weight (MW). (c) Schematic illustration of “zero-on” NIR-II PA probes exhibiting a “zero” state with probe background signals statistically indistinguishable from the pure water baseline (p > 0.05).#

Fig.2 Synthesis route, compactness evaluation and photophysical property of DPA(1–3). (a) Synthesis of DPA(1–3). (I) DMSO, KOH, 40°C, 12 h; (II) Pd2(dba)3, NaOtBu, XPhos, 1,4-Dioxane, 100°C, 24 h; (III) Pd (PPh3)4, K2CO3, toluene, 105°C, 24 h. (b-d) HPLC chromatograms of (b) DPA1, (c) DPA2 and (d) DPA3 (10 µM) recorded after reaction with HClO at concentrations of 0, 20 and 60 µM. All measurements were performed at a flow rate of 0.5 mL min−1. Normalized UV–vis absorption spectra of (e) DPA1-3 and (f) DPA(1–3)Cl. (g) Molar extinction coefficients (ε) of DPA(1-3)Cl at 1064 nm and at their respective absorption maxima. (h) Compactness analysis of DPA(1–3)Cl compared with commonly used NIR probes, using the standard criterion of the ratio of maximum absorption wavelength to molecular weight. Probe numbers correspond to structures and properties listed in Table S1. (i) The absorption redshift of DPA(1–3) compared with commonly used NIR probes before and after reaction with different analytes. Probe numbers correspond to structural details and parameters in Table S3.#

如图3所示，量子化学密度泛函理论（DFT）计算揭示了分子响应前后的电子结构演变。激活前分子的最高占据轨道（HOMO）与最低未占据轨道（LUMO）分别定域于共轭骨架与苄基侧链，能隙较宽；而在次氯酸作用下发生环化后，分子的HOMO-LUMO能隙大幅缩窄至1.710 eV，分子内电荷转移（ICT）效应大幅增强，从理论层面解释了极大吸收红移与强光声响应的物理机制。

Fig.3 Density functional theory (DFT) of DPA(1–3) and their HClO-activated products. (a) Optimized ground state (S0) geometries of DPA1-3 and DPA(1-3)Cl. (b) LUMO and HOMO orbital surfaces of DPA(1–3) and DPA(1-3)Cl in the geometrically optimized structure using PBE0/6-31+G(d). LUMO: lowest unoccupied molecular orbital; HOMO: highest occupied molecular orbital.#

如图4所示，体外光学及光声特性评估进一步印证了“零背景”特性。在未激活状态下，DPA3在1064 nm波长下的光声信号与纯水基线无统计学差异（p > 0.05），定义了真正的“零背景”状态。加入次氯酸后，DPA3的光声信号呈现出高达89.8倍的增强，次氯酸检测限低至252 nM，且对其他活性氧及生物分子表现出极高的选择性与pH稳定性。

Fig.4 In vitro optical characterization of the DPA3. (a) Absorption spectrum of DPA3 (10 µM) in a mixed solution of 60% PBS (pH = 7.4) and ethanol upon addition of HClO at various concentrations. (b) A quantitative comparison of the DPA3 absorption spectra at 1064 nm was conducted following exposure to 0 and 40 µM HClO, in comparison with PBS (pH = 7.4) at the same wavelength. (c) Absorption intensity of DPA3 at 1064 nm as a function of HClO concentration. The red line represents the linear fit between absorbance and HClO concentration. (d) The time-dependent absorption of DPA3 at a wavelength of 1064 nm in the presence of HClO. (e) In a mixture of 60% PBS (pH = 7.4) and ethanol, the effect of DPA3 (10 µM) on the absorption of HClO (40 µM) and other biologically relevant substances (100 µM) was studied. (f) Schematic diagram and corresponding PA imaging at 1064 nm: DPA3 activated by 0 µM and 60 µM HClO, respectively, with PBS (pH = 7.4) as the control. (g) Quantification of the NIR-II PA intensity in (f). (h) NIR-II PA images of DPA3 at different HClO concentrations (0–60 µM). DPA3 (15 µM) was placed in a silicon tube and subjected to NIR-II PA imaging at a wavelength of 1064 nm. (i) The corresponding quantified NIR-II PA signal intensity and detection curve in (h). The red line represents the linear fit from 0 to 60 µM. (j) Effect of different pH values on the PA intensity of DPA3 at 1064 nm. (k) Effect of DPA3 (10 µM) on the PA intensity of hypochlorous acid (HClO, 40 µM) and other biologically relevant substances (100 µM) in a 60% PBS (pH = 7.4) and ethanol mixture. (l) Quantification of the NIR-II PA intensity in (k). Statistical analysis was conducted with a two-tailed Student’s t-test.#

如图5所示，在小鼠对乙酰氨基酚（APAP）诱导的早期肝损伤模型中，实时体内光声成像结果表明，注射DPA3的健康小鼠肝脏区域几乎检测不到背景信号，避免了探针富集造成的假阳性干扰。而在肝损伤小鼠体内，随着对乙酰氨基酚剂量的增加，小鼠肝脏光声信号在90分钟内实现了50.32倍的显著提升，且信号强度变化与血清转氨酶指标及组织学炎症浸润高度吻合。

Fig.5 Real-time NIR-II PA imaging of the liver in mice with liver injury using DPA3. (a) Schematic diagram of intraperitoneal injection of APAP (liver injury drug) (300or 600 mg kg−1) and NAC (HClO scavenger), along with intravenous injection of DPA3 (1 mg mL−1) for real-time NIR-II PA imaging. The in vivo study was performed via tail vein injection with the following parameters: an injected dose of 1 mg mL−1 in a total volume of 200 µL, using 10% DMSO/PBS (v/v) as the vehicle. (b) NIR-II PA images of liver injury in mice. (c) Quantification of PA signal in mouse liver as a function of time following DPA3 injection in (b). (d) PBS, 0 mg kg−1 APAP + DPA3, 300 mg kg−1 APAP + DPA3, 600 mg kg−1 APAP + DPA3, NAC + 600 mg kg−1 APAP + DPA3, 90 min PA intensity divided by 0 min PA intensity (PA90min/PA0min). (e) HE staining of liver tissue (Top) and immunohistochemistry of myeloperoxidase (MPO) analysis (Bottom) of neutrophil infiltration in liver tissue (scale bar: 50 µm, dashed-line area indicates the injured area). Control group: Healthy mouse liver tissue; Liver injury group: Intraperitoneal injection of APAP (300 mg kg−1) and (600 mg kg−1) for 24 h; Repair group: Intraperitoneal injection of APAP (600 mg kg−1) and NAC for 24 h. Statistical analysis was conducted with a two-tailed Student’s t-test. ***p<0.001.#

如图6所示，研究团队开展了一项严谨的盲法动物诊断研究，对比了DPA3探针与模拟传统微弱背景探针（30%预激活的30% DPA3）的诊断效果。诊断人员在对40只小鼠的组别完全不知情的情况下进行成像诊断。结果显示，基于“零背景”探针DPA3建立的诊断阈值成功识别出所有早期肝损伤小鼠，诊断准确率达到100%；而传统微弱背景探针由于组织累积导致的背景重叠，产生了假阴性判断，准确率仅为85%。这一实验有力证明了“零背景”设计在提高深部组织诊断忠实度方面的决定性优势。

Fig.6 A blinded study for screening animal patients with liver injury. (a) Design strategy for “off-on” probe (30% DPA3) group and “zero-on” probe (DPA3) group. Schematic depiction of the distinct in vivo imaging performance enabled by “zero-on” versus “off-on” probes, emphasizing the superior reliability afforded by the zero-background design. (b) Healthy mice (n = 5) received an intravenous injection of the “off–on” probe (30% DPA3 + HClO). PA60min/PA0min levels in the liver and diagnostic thresholds were measured 60 min post-injection. (c) Healthy mice (n = 5) received an intravenous injection of the “zero-on” probe (DPA3 1 mg mL−1). PA60min/PA0min levels in the liver and diagnostic thresholds were measured 60 min post-injection. (d) Schematic diagram of screening for each group of mice. (e) NIR-II PA imaging results of mice in the “zero-on” (DPA3) group and the “off–on” (30%DPA3) group in the blind method experiment. (f) Based on the diagnostic thresholds established by the “zero-on” (DPA3) group, mice were grouped by comparing PA60min/PA0min values following intravenous administration of DPA3 in 20 mice. (g) Based on the diagnostic thresholds established by the “off–on” (30%DPA3) group, mice were grouped by comparing PA60min/PA0min values following intravenous administration of 30% DPA3 + HClO in 20 mice. (h) H&E staining of liver tissue from two groups of mice in the blind study (scale bar: 50 µm, dashed-line area indicates the injured area).#