Fig.1 Molecular engineering of long-wavelength xanthenoid dyes.#

Fig.2 Convergent synthesis of (top) 2-dialkylaminoanthracene and (bottom) ESi7. Reaction conditions: (a) Pd(dppf)Cl2, K3PO4, Dioxane/H2O, 100 °C, 10 h. (b) POCl3, DMF, dichloroethane, 75 °C, 12 h. (c) CH3SO3H, CH2Cl2, r. t., 2 h. (d) 2-bromo-4-methylphenyl magnesium chloride, THF, 0 °C, 3 h. (e) HI, AcOH, 120 °C, 2 h. (f) (1) nBuLi, THF, −78 °C, 30 min. (2) Me2SiCl2, THF, −78 °C, 30 min. (3) KMnO4, MeCN, −10 °C, 2 h. (g) NBS, AIBN, CHCl3, 70 °C, 6 h. (h) (1) 1,1-dimethylallylic bromide, KI, DMF, r. t., 12 h. (2) H2SO4, CH2Cl2, −10 °C, 30 min (i) (BPin)2, Pd(dppf)Cl2, Cs2CO3, Dioxane, 85 °C, 12 h. (j) (1) 2,6-dimethylphenyl lithium reagent, THF, 0 °C, 30 min. (2) CF3COOH, CH2Cl2, 0 °C, 30 min.#

Fig.3 Spectral studies of ESi7. (a) UV–vis absorption and fluorescence emission spectra of ESi7 (10 μM) in CH2Cl2 and their deconvolutional analysis. (b) HOMO/LUMO orbitals of ESi7. (c) fs-transient absorption spectra of ESi7 (10 μM) in CH2Cl2. (d) Kinetics traces at 920 and 1220 nm and fitting for lifetime. (e) Comparison of reorganization energies of EC7 and ESi7.#

光谱与光物理测试表明，ESi7在二氯甲烷中的吸收峰位于1176纳米（摩尔吸光系数达$2.38 \times 10^5\text{ M}^{-1}\text{cm}^{-1}$），荧光发射峰位于1275纳米。其荧光量子产率达到0.19%，相比碳桥连前体EC7提升了近4倍，荧光亮度达到$452\text{ M}^{-1}\text{cm}^{-1}$；瞬态吸收光谱测得其激发态寿命延长至30皮秒（高于EC7的20皮秒）。在光稳定性和化学稳定性测试中，ESi7在1064纳米激光连续照射60分钟后无光漂白现象，对活性氧（过氧亚硝酸根）的耐受性明显优于EC7；细胞毒性实验显示其在10微摩尔/升浓度下细胞存活率维持在95%以上。

Fig.4 PEAK evaluation of the performance of ESi7 in in vivo imaging. (a) Evaluation of the “Penetration depth” of ESi7 in 1% intralipid phantom. ICG, ESi5, IR1061, and EC7 were used as benchmark dyes for side-by-side comparison. Imaging parameters of ICG: 808 nm/100 mW•cm–2/7 ms/1000 nm LP; ESi5: 880 nm/100 mW•cm–2/20 ms/1100 LP; IR1061, EC7, and ESi7: 1064 nm/100 mW•cm–2/50 ms/1400 nm LP. (b–e) Evaluation of the “Contrast” of ESi7 (2.5 mg/kg) in BALB/c mice. ESi7 was encapsulated by DSPE-mPEG5000 and tail vein-injected. Whole-body angiography images were taken at three different positions, i.e., (b) ventral, right side, and dorsal with injection. Imaging parameters: 1064 nm/120 mW•cm–2/500 ms/1400 nm LP. Scale bar: 5 mm. (c) Stereomicroscopic imaging of the fem0ral vein and artery through a 0.64× objective with different long-pass (LP) filters at 1200, 1300, and 1400 nm, respectively. Imaging parameters: 1064 nm/60 mW•cm–2. Scale bar: 2 mm. (d) Evaluation of the “Exposure time” of ESi7 (2.5 mg/kg, encapsulated by DSPE-mPEG5000) by stereomicroscopic imaging of the hindlimb vasculature at different exposure times from 50 to 800 ms. Imaging parameters: 1064 nm/60 mW•cm–2/1400 nm LP. Scale bar: 2 mm. (e) Evaluation of the “Acquisition duration” of ESi7 by monitoring the in vivo clearance of tail vein-injected ESi7 (2.5 mg/kg, encapsulated by DSPE-mPEG5000) in a period of up to 240 h. Imaging parameters: 1064 nm/120 mW•cm–2/500 ms/1400 nm LP. Scale bar: 5 mm.#

在活体与组织模拟验证阶段，采用1% 脂肪乳模型测定穿透深度，ESi7的穿透深度达到10.9毫米，显著超越同等条件下的EC7（9.5毫米）、IR1061（7.0毫米）、ESi5（3.7毫米）和临床经典染料吲哚菁绿ICG（2.6毫米）。经磷脂聚乙二醇纳米包裹后，研究人员开展了小鼠全身血管造影与立体显微成像：在1064纳米激发和1400纳米长通滤光下，股静脉成像的信号背景比高达8.3；成像曝光时间可缩短至50毫秒以实现高质量实时视频采集；体内代谢监测显示其成像有效采集时长可长达10天。此外，结合短波长的ESi5，团队成功完成了小鼠肠道与血管的无串扰双通道活体成像。

Fig.5 Evaluation of the potential of ESi7 in multiplexed in vivo bioimaging. (a) Absorption spectra of ESi5 and ESi7 in CH2Cl2. (b) Fluorescence images of ESi5 (6 μM) and ESi7 (2 μM) in CH2Cl2 under 880 nm/100 mW•cm–2/20 ms/1200 nm LP and 1064 nm/100 mW•cm–2/50 ms exposure time/1400 nm LP, respectively. (c) Schematic illustration of the dual-channel imaging procedure for the mice’s intestine (880 nm) and vasculature (1064 nm). (d–f) Small intestine, whole body vasculature, and the overlay images of BALB/c mice. Imaging parameters of ESi5: 880 nm/100 mW•cm–2/150 ms/1200 nm LP, at 1.5 mg/kg; ESi7 nanoparticles: 1064 nm/120 mW•cm–2/500 ms/1400 nm LP, at 2.5 mg/kg. Scale bar: 5 mm. (j-l) Linescan intensities of three selected different regions of Figure 5f,i.#