【JACS】芘官能化共价有机框架中客体诱导的振动锁定与荧光开关效应及其对全氟辛酸的高灵敏识别（检测限 2.7 μg/L）
【JACS】用于活细胞长时程结构光照明显微成像的不对称巯基取代方酸菁荧光染料
文章标题：Asymmetric Sulfhydryl-Substituted Squaraine Fluorophores for Sustained SIM Imaging in Live Cells
文章作者：Yibo Zhou ; Pin Shang ; Huiqiu Shi ; Jun Zhang ; Fan Li ; Zhihe Qing * ; Tony D. James *
研究动因
结构光照明显微成像（SIM）在时空分辨率平衡上极具优势，是研究活细胞亚细胞动态结构的重要超分辨工具；方酸菁（Squaraine）染料因近红外发射和高量子产率被广泛用于生物成像。然而，传统方酸菁多基于中心对称的氧代环丁烯二酮环与给体构建，极易发生光漂白，严重限制了其在长时程、持续光照超分辨成像中的应用。尽管已有烷基季铵盐环保护或单纯硫化修饰等改性手段，但分子骨架对称性对光稳定性的影响长期被忽视。为此，本文旨在通过构建不对称分子天线与环丁烯硫杂化的协同策略，解决方酸菁染料光稳定性差及荧光寿命受限的瓶颈，开发适用于活细胞长时程超分辨跟踪的高性能荧光探针，拓展方酸菁在活细胞动态事件监测中的实际应用。
Fig.1 Design of traditional symmetric squaraine with limited photostability (above) and asymmetric sulfhydryl-substituted squaraine with excellent photostability prepared as part of this research (below).
研究实施方案与关键实验结果
研究采用“理论设计—量化计算—体外表征—活细胞超分辨验证”的闭环技术路线。本文首次提出将分子天线的不对称化（一侧为吲哚衍生物，另一侧引入黄酮鎓盐等强给电子团）与中心氧代环丁烯二酮环的硫化取代（氧原子替换为硫原子）相协同的分子工程策略；同时沿用并拓展了酯化修饰十八烷基链以赋予线粒体靶向功能的成熟方法。其基本原理在于：打破分子对称性诱导强分子内电荷转移（ICT），有效分离电子云密度并降低最低未占分子轨道能级，从而克服“花青素极限”并增大斯托克斯位移；硫化修饰与不对称骨架协同大幅降低自旋-轨道耦合常数，阻断系间窜越至三线态的光氧化降解途径，并显著延长荧光寿命。
实验通过密度泛函理论计算电子云轨道分布、能隙、自旋-轨道耦合常数及电离能，并在体外对比了对称对照染料、不对称氧代染料以及不对称硫代方酸菁（SF–F-S）在激光连续辐照下的吸收/发射光谱衰减动力学与单线态氧生成能力。细胞实验选用活态肝癌细胞（HepG2），在结构光照明显微镜和共聚焦显微镜下开展多色共定位与细胞毒性测试。实验表明：SF–F-S能级差降至1.929 eV，自旋-轨道耦合常数降至0.07 cm⁻¹（传统对称染料为3.46 cm⁻¹），电离能提升至4.82 eV；在83 mW激光持续照射210分钟后，SF–F-S吸收保留率高达95.5%（传统对称染料降至33.2%），荧光寿命延长至2.78 ns（传统对称染料为1.08 ns），且几乎不产生单线态氧与光毒性。
在活细胞超分辨成像执行流程中，将靶向探针（Mito-SF–F-S）与活细胞共孵育后，利用640 nm激光在50 W/cm²功率下进行实时SIM采集。探针在线粒体上的共定位系数达0.85，横截面半峰全宽（FWHM）达182 nm（相比宽场成像的395 nm空间分辨率显著提升）。在持续光照拍摄模式下，商业化线粒体染料在2分钟内荧光强度骤降至13.8%，而Mito-SF–F-S荧光信号保持稳定，成功在14分钟内超分辨动态追踪了羰基氰基对氯苯腙诱导线粒体自噬过程中线粒体网络破裂（线粒体面积收缩至初始的49.8%）以及溶酶体运动汇聚的时空动态轨迹。
Fig.2 (A) Synthetic route for asymmetric squaraine derivatives. (B) Photophysical properties of the asymmetric squaraine derivatives in DMSO. a. Fluorescence quantum yields were calculated with cresyl violet as the reference (Φf = 0.54, MeOH). b. Fluorescence quantum yields were calculated with IR 780 as the reference (Φf = 0.17, EtOH). (C) UV–vis-NIR absorption spectra and normalized fluorescence emission spectra of the squaraine derivatives in DMSO.
Fig.3 (A) Molecular structure and DFT-optimized molecular orbital diagrams (LUMO and HOMO) of SF, SF-F, and SF–F-S, and the quantized energy gap between the LUMO and HOMO. (B) Optimized structures of SF, SF-F, and SF–F-S, and the RMSD of different molecular structures during excited-state geometry relaxation. (C) Calculated spin–orbit coupling (SOC) and ΔEST of SF, SF-F, and SF–F-S at their S1 state. (D) Ionization potentials of SF, SF-F, and SF–F-S at the T1 state, respectively.
Fig.4 UV–vis-NIR absorption spectra of (A) SF (5 μM), (B) SF-F (5 μM), and (C) SF–F-S (5 μM) in trifluoroethanol under laser illumination (83 mW) at different time points (0, 15, 30, 45, 60, 75, 90, 105, 120, 135, 150, 165, 180, 195, and 210 min). (D) Changes of relative absorption for the three squaraine derivatives at different time points. Fluorescence emission spectra of (E) SF (5 μM), (F) SF-F (5 μM), and (G) SF–F-S (5 μM) in trifluoroethanol under laser illumination (83 mW) at different time points (0, 15, 30, 45, 60, 75, 90, 105, 120, 135, 150, 165, 180, 195, and 210 min). (H) Fluorescence ratio changes of squaraine derivatives at different time points. UV–vis absorption spectrum of ABDA (15 μM) containing (I) SF (5 μM), (J) SF-F (5 μM), and (K) SF–F-S (5 μM) under laser illumination (83 mW) at different time points (0, 5, 10, 15, 20, 25, 30, 35, and 40 min). (L) Relative changes of absorbance at 380 nm of ABDA containing squaraine derivatives under laser illumination. Error bars were calculated from three repeated measurements.
Fig.5 SIM images of live cells incubated with (A) SF (10 μM), (B) SF-F (10 μM), and (C) SF–F-S (10 μM) under laser illumination (50 W/cm2) at different time points. λex = 640 nm, λem = 663–738 nm. Scale bars: 10 μm. (D) Changes of relative fluorescence intensity of live cells incubated with squaraine derivatives under continuous imaging mode. (E) Confocal fluorescence images of live cells incubated with squaraine derivatives and after being treated with Hoechst 33342 (5 μM, λex = 405 nm, λem = 430–470 nm) and PI (5 μM, λex = 561 nm, λem = 570–670 nm). The fluorescence intensity of live cells incubated with SF was defined as 1.0. Scale bar: 10 μm. (F) Confocal fluorescence images of live cells incubated with squaraine derivatives and with Hoechst 33342 (5 μM, λex = 405 nm, λem = 430–470 nm) and DCFH-DA (5 μM, λex = 488 nm, λem = 500–600 nm). The fluorescence intensity of live cells incubated with SF was defined as 1.0. Scale bar: 10 μm. Error bars were calculated from three repeated measurements.
Fig.6 (A) Molecular structure of Mito-SF–F-S. (B) UV–vis-NIR absorption spectrum and fluorescence emission spectrum of Mito-SF–F-S. (C) Comparison between SIM and wide-field images of live cells incubated with Mito-SF–F-S (10 μM). Scale bar: 10 μm. (D) SIM images of live cells incubated with mitochondrial tracker (5 μM) and Mito-SF–F-S (10 μM), and the overlay of the two fluorescence images. Scale bar: 10 μm. (E) SIM images of live cells incubated with mitochondrial tracker (5 μM) and Mito-SF–F-S (10 μM) at different time points (50 W/cm2). Scale bars: 10 μm. (F) Change of relative fluorescence intensity of the mitochondrial tracker and Mito-SF–F-S shown in (E). Error bars were calculated from three repeated measurements. (G, I) SIM images of live cells incubated with lysosomal tracker (5 μM) and Mito-SF–F-S (10 μM) and then stimulated with CCCP (10 μM) at different time points. Scale bars: 0.5 μm. (H) Shape changes of mitochondria shown in (G). (J) Trajectories of lysosomes in cells shown in (I). Mito-SF–F-S: λex = 640 nm, λem = 663–738 nm; Mitochondrial tracker: λex = 488 nm, λem = 500–545 nm.
Fig.7 SIM images of live cells incubated Mito-SF–F-S (15 μM, λex = 640 nm, λem = 663–738 nm) in the (A) absence and (B) presence of CCCP (10 μM) at different time points (0, 2, 4, 6, 8, 10, 12, and 14 min) (50 W/cm2). Scale bars: 10 μm. The ROI area: 5 × 5 μm. (C) The relative mean length and (D) area of the mitochondrial perimeter under different conditions shown in (A) and (B). Error bars were calculated from three repeated measurements.
创新与提升
本文核心创新在于突破传统方酸菁的对称骨架设计，利用不对称分子天线打破对称性与巯基取代的协同效应，从分子层面抑制了系间窜越与光氧化通路。实验数据显示，所开发的探针不仅实现了近红外发射和长荧光寿命（2.78 ns），更将光漂白耐受时间提升数倍，单线态氧产生量与光毒性较传统染料大幅降低，有力回应了SIM成像因持续光照易导致荧光淬灭与细胞损伤的痛点。该研究为开发兼具高亮度、超长耐光性和低光毒性的超分辨探针提供了新范式；论文同时指出，当前的溶酶体动态轨迹基于离散时间点图像重构，未来需结合超快连续时间序列成像进一步深化细胞器动力学表征。
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