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Two-Photon Near Infrared Fluorescent Turn-On Probe Toward Cysteine and Its Imaging Applications
面向半胱氨酸的双光子近红外荧光导通探针及其成像应用

Junfeng Wang, † † ^(†){ }^{\dagger} Bin Li, † † ^(†){ }^{\dagger} Weiyu Zhao, † † ^(†){ }^{\dagger} Xinfu Zhang, † † ^(†){ }^{\dagger} Xiao Luo, † † ^(†){ }^{\dagger} Mark E. Corkins, ‡ ‡ ^(‡){ }^{\ddagger} Sara L. Cole, § § ^(§){ }^{\S}§ Chao Wang, |I Yi Xiao, ‖ ‖ ^(||){ }^{\|}Xiaoman Bi, ⊥ ⊥ ^(_|_){ }^{\perp} Yi Pang, ⊥ ⊥ ^(_|_){ }^{\perp} Craig A. McElroy, † † ^(†){ }^{\dagger} Amanda J. Bird, ‡ ‡ ^(‡){ }^{\ddagger} and Yizhou Dong*, † † ^(†){ }^{\dagger}
王俊峰， † † ^(†){ }^{\dagger} 李斌， † † ^(†){ }^{\dagger} 赵伟宇， † † ^(†){ }^{\dagger} 张新福， † † ^(†){ }^{\dagger} 罗晓， † † ^(†){ }^{\dagger} Mark E. Corkins， ‡ ‡ ^(‡){ }^{\ddagger} Sara L. Cole， § § ^(§){ }^{\S}§ 王超， |I Yi Xiao、 ‖ ‖ ^(||){ }^{\|} Xiaoman Bi、 ⊥ ⊥ ^(_|_){ }^{\perp} Yi Pang、 ⊥ ⊥ ^(_|_){ }^{\perp} Craig A. McElroy、 † † ^(†){ }^{\dagger} Amanda J. Bird ‡ ‡ ^(‡){ }^{\ddagger} 和 Yizhou Dong*， † † ^(†){ }^{\dagger} † † ^(†){ }^{\dagger} Division of Pharmaceutics & Pharmaceutical Chemistry, College of Pharmacy, † † ^(†){ }^{\dagger} Department of Molecular Genetics, and § § ^(§){ }^{\S}§ Campus Microscopy and Imaging Facility, The Ohio State University, Columbus, Ohio 43210, United States
† † ^(†){ }^{\dagger} 俄亥俄州立大学药剂学与药物化学系， † † ^(†){ }^{\dagger} 分子遗传学系， § § ^(§){ }^{\S}§ 以及校园显微镜和成像设施，哥伦布，俄亥俄州43210，美国 "State Key Laboratory of Fine Chemicals, Dalian University of Technology, Dalian 116024, P. R. China "State Key Laboratory of Fine Chemicals, Dalian University of Technology, Dalian 116024, P. R. China  ^(""State Key Laboratory of Fine Chemicals, Dalian University of Technology, Dalian 116024, P. R. China "){ }^{\text {"State Key Laboratory of Fine Chemicals, Dalian University of Technology, Dalian 116024, P. R. China }} ⊥ ⊥ ^(_|_){ }^{\perp} Department of Chemistry & Maurice Morton Institute of Polymer Science, The University of Akron, Akron, Ohio 44325, United States
⊥ ⊥ ^(_|_){ }^{\perp} 阿克伦大学化学系和莫里斯·莫顿高分子科学研究所，阿克伦，俄亥俄州44325，美国

S Supporting Information S 支持信息

Abstract 抽象

Two-photon excitable ( 850 nm ) NIR fluorescent turn-on ( 702 nm ) probe TP-NIR was synthesized for selective detection of cysteine (Cys). The probe itself shows is turned on by reaction with Cys in aqueous buffer. In addition, the probe displays greater selectivity for Cys over other thiols, including glutathione (GSH) and homocysteine (Hcy). Moreover, the large Stokes shift, NIR excitation, and NIR emission make this probe suitable for biological imaging.
合成双光子可激发 （ 850 nm ） NIR 荧光开启 （ 702 nm ） 探针 TP-NIR 用于选择性检测半胱氨酸 （Cys）。探针本身显示通过与水性缓冲液中的 Cys 反应而打开。此外，该探针对 Cys 的选择性高于其他硫醇，包括谷胱甘肽 （GSH） 和同型半胱氨酸 （Hcy）。此外，较大的斯托克斯位移、NIR 激发和 NIR 发射使该探针适用于生物成像。

KEYWORDS: two-photon, near-infrared, probe, cysteine, dicyanomethylene-4H-pyran, Stokes shift, imaging
关键词：双光子， 近红外， 探针， 半胱氨酸， 双氰亚甲基-4H-吡喃， 斯托克斯位移， 成像
Biological tissues strongly scatter visible light, which makes it incredibly difficult to conduct high-resolution and deep tissue imaging. 1 1 ^(1){ }^{1} Fluorophores with long emissions at the nearinfrared (NIR) region from 700 to 1000 nm are promising to address the issues for in vivo imaging, 2 − 4 2 − 4 ^(2-4){ }^{2-4} as NIR emission exhibits appealing features including deep tissue penetration, low tissue photodamage, and little autofluorescence interference. 5 − 10 5 − 10 ^(5-10){ }^{5-10} A number of cyanine-based dyes reported in the literature possessed strong NIR absorption and emission properties, but their small Stokes shifts (typically 20-50 nm) greatly hampered their application. 11 − 16 11 − 16 ^(11-16){ }^{11-16} Two-photon (TP) probe-based fluorescent imaging, an emerging technique, has great potential for deep-tissue imaging with prolonged observation time; however, the majority of the existing TP probes have short emission wavelengths ranging from 380 to 550 nm . 17 550 nm . 17 550nm.^(17)550 \mathrm{~nm} .{ }^{17} To overcome the current problems with deep tissue imaging, an ideal fluorescent probe should have both excitation and emission in the NIR range (NIR-NIR). 18 , 19 18 , 19 ^(18,19){ }^{18,19} Meanwhile a large Stokes shift can highly improve the sensitivity of fluorescence microscopy wherein emission photons can be detected against the background from excitation photons. 11 11 ^(11){ }^{11} Consequently, new probes with the above properties are in great demand.
生物组织会强烈散射可见光，这使得进行高分辨率和深层组织成像变得非常困难。 1 1 ^(1){ }^{1} 在 700 至 1000 nm 的近红外 （NIR） 区域具有长发射的荧光团有望解决体内成像的问题， 2 − 4 2 − 4 ^(2-4){ }^{2-4} 因为 NIR 发射表现出吸引人的特性，包括深层组织穿透、低组织光损伤和很少的自发荧光干扰。 5 − 10 5 − 10 ^(5-10){ }^{5-10} 文献中报道的许多基于花青素的染料具有很强的 NIR 吸收和发射特性，但它们的小斯托克斯位移（通常为 20-50 nm）极大地阻碍了它们的应用。 11 − 16 11 − 16 ^(11-16){ }^{11-16} 基于双光子 （TP） 探针的荧光成像是一种新兴技术，在观察时间长的情况下具有巨大的深组织成像潜力;然而，大多数现有的 TP 探针具有较短的发射波长，范围从 380 到 550 nm . 17 550 nm . 17 550nm.^(17)550 \mathrm{~nm} .{ }^{17} 为了克服当前深层组织成像的问题，理想的荧光探针应同时具有 NIR 范围内的激发和发射 （NIR-NIR）。 18 , 19 18 , 19 ^(18,19){ }^{18,19} 同时，较大的斯托克斯位移可以大大提高荧光显微镜的灵敏度，其中可以在激发光子的背景下检测到发射光子。 11 11 ^(11){ }^{11} 因此，具有上述特性的新探针需求量很大。
Sensing of biological thiols has aroused tremendous interest due to their essential roles in human physiology. 20 − 24 20 − 24 ^(20-24){ }^{20-24} For instance, a deficiency of cysteine (Cys) causes various health problems, such as retarded growth, hair depigmentation, lethargy, liver damage, muscle and fat loss, and skin lesions. 25 25 ^(25){ }^{25} A number of fluorescent probes toward biothiols have been
由于生物硫醇在人体生理学中的重要作用，对生物硫醇的感知引起了极大的兴趣。 20 − 24 20 − 24 ^(20-24){ }^{20-24} 例如，半胱氨酸 （Cys） 缺乏会导致各种健康问题，例如生长迟缓、头发色素脱失、嗜睡、肝损伤、肌肉和脂肪流失以及皮肤损伤。 25 25 ^(25){ }^{25} 许多针对生物硫醇的荧光探针已被
developed, most of which exhibit emission or absorption within the ultraviolet or visible range. 26 − 61 26 − 61 ^(26-61){ }^{26-61} Although a few NIR fluorescent probes toward biothiols (Cys, GSH, and Hcy) have been developed, 62 − 69 62 − 69 ^(62-69){ }^{62-69} the probes suffer from either a small Stokes shift or short excitation wavelength. Recently, Yu et al. reported a biothiols-selective near-infrared fluorescent probe with a large Stoke shift. This probe showed great promise for biological imaging. However, the excitation wavelength is 560 nm , which is not in the NIR range and limit its broader applications. 70 70 ^(70){ }^{70} Herein, we elucidated the features of this probe (named TP-NIR, Scheme 1) using two-photon-excited fluorescence (TPEF), investigated the Cys-triggered process using an in situ 1 H 1 H ^(1)H{ }^{1} \mathrm{H} NMR study, and applied TP-NIR to visualize cells using a multiphoton laser scanning confocal microscope.
发达，其中大多数在紫外或可见光范围内表现出发射或吸收。 26 − 61 26 − 61 ^(26-61){ }^{26-61} 尽管已经开发出一些针对生物硫醇（Cys、GSH 和 Hcy）的 NIR 荧光探针，但 62 − 69 62 − 69 ^(62-69){ }^{62-69} 这些探针具有较小的斯托克斯位移或短激发波长。最近，Yu 等人报道了一种具有大 Stoke 位移的生物硫醇选择性近红外荧光探针。该探针显示出生物成像的巨大前景。然而，激发波长为 560 nm ，这不在 NIR 范围内，限制了其更广泛的应用。 70 70 ^(70){ }^{70} 在此，我们使用双光子激发荧光 （TPEF） 阐明了该探针（命名为 TP-NIR，方案 1）的特征，使用原位 1 H 1 H ^(1)H{ }^{1} \mathrm{H} NMR 研究研究了 Cys 触发的过程，并应用 TP-NIR 使用多光子激光扫描共聚焦显微镜观察细胞。

EXPERIMENTAL SECTION 实验部分

Materials. All solvents for fluorescence experiments were analytical grade and purchased from Fisher Scientific and used without further purification. Probe TP-NIR was dissolved in DMSO ( 10 mM ) as a stock solution and 100 mM biologically relevant analytes (Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, Cys, Hcy, GSH) were dissolved in distilled water. 10 mM PBS solution ( pH = 7.4 pH = 7.4 pH=7.4\mathrm{pH}=7.4 ) was prepared as the buffer solution. UV-vis spectra were acquired on a molecular devices Spectramax M5
材料。用于荧光实验的所有溶剂均为分析级溶剂，购自 Fisher Scientific，无需进一步纯化即可使用。将探针 TP-NIR 作为储备液溶于 DMSO （10 mM） 中，将 100 mM 生物相关分析物（Ala、Arg、Asn、Asp、Gln、Glu、Gly、His、Ile、Leu、Lys、Met、Phe、Pro、Ser、Thr、Trp、Tyr、Val、Cys、Hcy、GSH）溶于蒸馏水中。制备 10 mM PBS 溶液 （ pH = 7.4 pH = 7.4 pH=7.4\mathrm{pH}=7.4 ） 作为缓冲溶液。紫外-可见光谱是在分子器件 Spectramax M5 上采集的
Scheme 1. Synthesis of Probe TP-NIR and the Proposed Response Mechanism to Cys
方案 1.探针 TP-NIR 的合成及对 Cys 的响应机制

spectrometer. Fluorescence spectra were measured by RF-5301PC and Spectramax M5 spectrometer.
光谱仪。荧光光谱通过 RF-5301PC 和 Spectramax M5 光谱仪测量。
Synthesis of Probe TP-NIR. Compound 1 (100 mg) was dissolved in DCM ( 5 mL ) followed by addition of TEA ( 90 mg ), and then acryloyl chloride ( 117 mg ) was added slowly. 70 70 ^(70){ }^{70} The resulting mixture was stirred at room temperature for 10 min . Probe TP-NIR was purified by silica column chromatography ( DCM :Hexanes = 7 : 3 = 7 : 3 =7:3=7: 3 ) as yellow solid in 92 % 92 % 92%92 \% yield. 1 H 1 H ^(1)H{ }^{1} \mathrm{H} NMR ( 400 MHz , CDCl 3 400 MHz , CDCl 3 400MHz,CDCl_(3)400 \mathrm{MHz}, \mathrm{CDCl}_{3} ): δ 8.94 δ 8.94 delta8.94\delta 8.94 ( 1 H , dd , J = 1.2 Hz , J = 8.4 Hz ) , 7.77 ( 1 H , dt , J = 1.2 Hz , J = 7.2 Hz ) ( 1 H , dd , J = 1.2 Hz , J = 8.4 Hz ) , 7.77 ( 1 H , dt , J = 1.2 Hz , J = 7.2 Hz ) (1H,dd,J=1.2Hz,J=8.4Hz),7.77(1H,dt,J=1.2Hz,J=7.2Hz)(1 \mathrm{H}, \mathrm{dd}, J=1.2 \mathrm{~Hz}, J=8.4 \mathrm{~Hz}), 7.77(1 \mathrm{H}, \mathrm{dt}, J=1.2 \mathrm{~Hz}, J=7.2 \mathrm{~Hz}), 7.66 − 7.62 ( 3 H 7.66 − 7.62 ( 3 H 7.66-7.62(3H7.66-7.62(3 \mathrm{H}, overlapping), 7.59 ( 1 H , dd , J = 1.2 Hz , J = 8.4 Hz ) 7.59 ( 1 H , dd , J = 1.2 Hz , J = 8.4 Hz ) 7.59(1H,dd,J=1.2Hz,J=8.4Hz)7.59(1 \mathrm{H}, \mathrm{dd}, J=1.2 \mathrm{~Hz}, J=8.4 \mathrm{~Hz}), 7.48 ( 1 H , dt , J = 1.2 Hz , J = 7.2 Hz ) , 7.66 − 7.62 ( 2 H 7.48 ( 1 H , dt , J = 1.2 Hz , J = 7.2 Hz ) , 7.66 − 7.62 ( 2 H 7.48(1H,dt,J=1.2Hz,J=7.2Hz),7.66-7.62(2H7.48(1 \mathrm{H}, \mathrm{dt}, J=1.2 \mathrm{~Hz}, J=7.2 \mathrm{~Hz}), 7.66-7.62(2 \mathrm{H}, overlapping), 6.90 ( 1 H , s ) , 6.81 ( 1 H , d , J = 16.0 Hz ) , 6.67 ( 1 H , dd , J = 1.2 Hz , J = 6.90 ( 1 H , s ) , 6.81 ( 1 H , d , J = 16.0 Hz ) , 6.67 ( 1 H , dd , J = 1.2 Hz , J = 6.90(1H,s),6.81(1H,d,J=16.0Hz),6.67(1H,dd,J=1.2Hz,J=6.90(1 \mathrm{H}, \mathrm{s}), 6.81(1 \mathrm{H}, \mathrm{d}, J=16.0 \mathrm{~Hz}), 6.67(1 \mathrm{H}, \mathrm{dd}, J=1.2 \mathrm{~Hz}, J= 17.2 Hz ) , 6.36 ( 1 H , q , J = 8.4 Hz ) , 6.08 ( 1 H , dd , J = 1.2 Hz , J = 8.4 17.2 Hz ) , 6.36 ( 1 H , q , J = 8.4 Hz ) , 6.08 ( 1 H , dd , J = 1.2 Hz , J = 8.4 17.2Hz),6.36(1H,q,J=8.4Hz),6.08(1H,dd,J=1.2Hz,J=8.417.2 \mathrm{~Hz}), 6.36(1 \mathrm{H}, \mathrm{q}, J=8.4 \mathrm{~Hz}), 6.08(1 \mathrm{H}, \mathrm{dd}, J=1.2 \mathrm{~Hz}, J=8.4 Hz ). 13 C 13 C ^(13)C{ }^{13} \mathrm{C} NMR ( 100 MHz , CDCl 3 100 MHz , CDCl 3 100MHz,CDCl_(3)100 \mathrm{MHz}, \mathrm{CDCl}_{3} ): δ 164.2 , 157.2 , 152.8 , 152.3 δ 164.2 , 157.2 , 152.8 , 152.3 delta164.2,157.2,152.8,152.3\delta 164.2,157.2,152.8,152.3, 152.2 , 137.7, 134.7, 133.2, 132.4, 129.1, 127.6, 126.0, 125.9, 122.4, 118.9, 118.6, 117.8, 116.7, 115.6, 107.0. MS-ESI + ( m / z ) : [ M + H ] + + ( m / z ) : [ M + H ] + ^(+)(m//z):[M+H]^(+){ }^{+}(\mathrm{m} / \mathrm{z}):[\mathrm{M}+\mathrm{H}]^{+} calcd for C 23 H 15 N 2 O 3 , 367.11 C 23 H 15 N 2 O 3 , 367.11 C_(23)H_(15)N_(2)O_(3),367.11\mathrm{C}_{23} \mathrm{H}_{15} \mathrm{~N}_{2} \mathrm{O}_{3}, 367.11; found, 367.10. 1 H 1 H ^(1)H{ }^{1} \mathrm{H} NMR and 13 C 13 C ^(13)C{ }^{13} \mathrm{C} NMR spectra were obtained using a Bruker AVIII-HD 400 MHz NMR spectrometer. Electrospray ionization (ESI) mass spectra were acquired with an amaZon ETD mass spectrometer.
探针 TP-NIR 的合成。将化合物 1 （100 mg） 溶于 DCM （ 5 mL ） 中，然后加入 TEA （ 90 mg ），然后缓慢加入丙烯酰氯 （ 117 mg ）。 70 70 ^(70){ }^{70} 将所得混合物在室温下搅拌 10 分钟。探针 TP-NIR 通过硅胶柱色谱法（DCM：己烷 = 7 : 3 = 7 : 3 =7:3=7: 3 ）纯化为黄色固体 92 % 92 % 92%92 \% ，收率为黄色固体。 1 H 1 H ^(1)H{ }^{1} \mathrm{H} 核磁共振 （ 400 MHz , CDCl 3 400 MHz , CDCl 3 400MHz,CDCl_(3)400 \mathrm{MHz}, \mathrm{CDCl}_{3} ）： δ 8.94 δ 8.94 delta8.94\delta 8.94 ( 1 H , dd , J = 1.2 Hz , J = 8.4 Hz ) , 7.77 ( 1 H , dt , J = 1.2 Hz , J = 7.2 Hz ) ( 1 H , dd , J = 1.2 Hz , J = 8.4 Hz ) , 7.77 ( 1 H , dt , J = 1.2 Hz , J = 7.2 Hz ) (1H,dd,J=1.2Hz,J=8.4Hz),7.77(1H,dt,J=1.2Hz,J=7.2Hz)(1 \mathrm{H}, \mathrm{dd}, J=1.2 \mathrm{~Hz}, J=8.4 \mathrm{~Hz}), 7.77(1 \mathrm{H}, \mathrm{dt}, J=1.2 \mathrm{~Hz}, J=7.2 \mathrm{~Hz}) ， 7.66 − 7.62 ( 3 H 7.66 − 7.62 ( 3 H 7.66-7.62(3H7.66-7.62(3 \mathrm{H} ， 重叠）， 7.59 ( 1 H , dd , J = 1.2 Hz , J = 8.4 Hz ) 7.59 ( 1 H , dd , J = 1.2 Hz , J = 8.4 Hz ) 7.59(1H,dd,J=1.2Hz,J=8.4Hz)7.59(1 \mathrm{H}, \mathrm{dd}, J=1.2 \mathrm{~Hz}, J=8.4 \mathrm{~Hz}) ， 7.48 ( 1 H , dt , J = 1.2 Hz , J = 7.2 Hz ) , 7.66 − 7.62 ( 2 H 7.48 ( 1 H , dt , J = 1.2 Hz , J = 7.2 Hz ) , 7.66 − 7.62 ( 2 H 7.48(1H,dt,J=1.2Hz,J=7.2Hz),7.66-7.62(2H7.48(1 \mathrm{H}, \mathrm{dt}, J=1.2 \mathrm{~Hz}, J=7.2 \mathrm{~Hz}), 7.66-7.62(2 \mathrm{H} ， 重叠）， 6.90 ( 1 H , s ) , 6.81 ( 1 H , d , J = 16.0 Hz ) , 6.67 ( 1 H , dd , J = 1.2 Hz , J = 6.90 ( 1 H , s ) , 6.81 ( 1 H , d , J = 16.0 Hz ) , 6.67 ( 1 H , dd , J = 1.2 Hz , J = 6.90(1H,s),6.81(1H,d,J=16.0Hz),6.67(1H,dd,J=1.2Hz,J=6.90(1 \mathrm{H}, \mathrm{s}), 6.81(1 \mathrm{H}, \mathrm{d}, J=16.0 \mathrm{~Hz}), 6.67(1 \mathrm{H}, \mathrm{dd}, J=1.2 \mathrm{~Hz}, J= 17.2 Hz ) , 6.36 ( 1 H , q , J = 8.4 Hz ) , 6.08 ( 1 H , dd , J = 1.2 Hz , J = 8.4 17.2 Hz ) , 6.36 ( 1 H , q , J = 8.4 Hz ) , 6.08 ( 1 H , dd , J = 1.2 Hz , J = 8.4 17.2Hz),6.36(1H,q,J=8.4Hz),6.08(1H,dd,J=1.2Hz,J=8.417.2 \mathrm{~Hz}), 6.36(1 \mathrm{H}, \mathrm{q}, J=8.4 \mathrm{~Hz}), 6.08(1 \mathrm{H}, \mathrm{dd}, J=1.2 \mathrm{~Hz}, J=8.4 Hz ）。 13 C 13 C ^(13)C{ }^{13} \mathrm{C} 核磁共振 （ 100 MHz , CDCl 3 100 MHz , CDCl 3 100MHz,CDCl_(3)100 \mathrm{MHz}, \mathrm{CDCl}_{3} ）： δ 164.2 , 157.2 , 152.8 , 152.3 δ 164.2 , 157.2 , 152.8 , 152.3 delta164.2,157.2,152.8,152.3\delta 164.2,157.2,152.8,152.3 ， 152.2 ， 137.7， 134.7， 133.2， 132.4， 129.1， 127.6， 126.0， 125.9， 122.4， 118.9， 118.6， 117.8， 116.7， 115.6， 107.0.MS-ESI + ( m / z ) : [ M + H ] + + ( m / z ) : [ M + H ] + ^(+)(m//z):[M+H]^(+){ }^{+}(\mathrm{m} / \mathrm{z}):[\mathrm{M}+\mathrm{H}]^{+} calcd 为 C 23 H 15 N 2 O 3 , 367.11 C 23 H 15 N 2 O 3 , 367.11 C_(23)H_(15)N_(2)O_(3),367.11\mathrm{C}_{23} \mathrm{H}_{15} \mathrm{~N}_{2} \mathrm{O}_{3}, 367.11 ;找到，367.10。 1 H 1 H ^(1)H{ }^{1} \mathrm{H} NMR 和 13 C 13 C ^(13)C{ }^{13} \mathrm{C} NMR 波谱是使用 Bruker AVIII-HD 400 MHz NMR 波谱仪获得的。使用 amaZon ETD 质谱仪采集电喷雾电离 （ESI） 质谱。
Spectroscopic Measurement. The UV-vis and fluorescence experiments were performed using 10 μ M 10 μ M 10 muM10 \mu \mathrm{M} of TP-NIR in PBS buffer ( pH = 7.4 , 10 mM ) pH = 7.4 , 10 mM ) pH=7.4,10mM)\mathrm{pH}=7.4,10 \mathrm{mM}) containing 50 % 50 % 50%50 \% DMSO at room temperature. 100 μ M μ M muM\mu \mathrm{M} amino acids ( 100 μ M 100 μ M 100 muM100 \mu \mathrm{M} or 10 mM of GSH) were added and the fluorescence data were recorded after 30 min . To test if GSH affects the reaction between TP-NIR and cysteine, TP-NIR ( 10 μ M ) 10 μ M ) 10 muM)10 \mu \mathrm{M}) was incubated in three different mixtures of cysteine and GSH ( 100 μ M ( 100 μ M (100 muM(100 \mu \mathrm{M} cysteine and 1 mM GSH; 100 μ M 100 μ M 100 muM100 \mu \mathrm{M} cysteine and 5 mM GSH; and 100 μ M μ M muM\mu \mathrm{M} cysteine and 10 mM GSH) and 100 μ M 100 μ M 100 muM100 \mu \mathrm{M} cysteine solution only, then emission spectra (excitation 557 nm ) were scanned every 1 min to monitor the kinetics until the fluorescent signal did not change. Two-photon absorption spectrum and cross section of compound 1 ( 100 μ M ) ( 100 μ M ) (100 muM)(100 \mu \mathrm{M}) and the product from the reaction of TP-NIR ( 100 μ M ) ( 100 μ M ) (100 muM)(100 \mu \mathrm{M}) with Cys ( 500 μ M ( 500 μ M (500 muM(500 \mu \mathrm{M}, incubated for 30 min at RT) in PBS buffer ( pH = ( pH = (pH=(\mathrm{pH}= 7.4) containing 50 % 50 % 50%50 \% of DMSO were measured with the two-photonexcited fluorescence (TPEF) method using the femtosecond laser pulses. Experiments were conducted on a confocal microscopy system (FV1000, Olympus) combined with femtosecond-pulsed wavelength switchable laser source (Mai Tai Deepsee, 80 MHz , > 2.1 W, Opticalphysics). Laser beam was directed into the scan-module of the microscopy system, and focused onto the sample sealed in a cell culture dish. The fluorescence emission was collected by the same objective, projected onto PMT, and the fluorescence emission curve and intensity were then plotted. Fluorescence emission excited by different laser wavelength, from 750 to 900 nm ( 10 nm per step), was collected. The intensities of the two-photon induced fluorescence spectra of the reference and sample emitted at the same excitation wavelength were recorded. All parameters remain constant during the data collection at different wavelengths. The TPA cross section ( δ ) ( δ ) (delta)(\delta) of samples in DMSO/PBS ( pH = 11 , 100 μ M pH = 11 , 100 μ M pH=11,100 muM\mathrm{pH}=11,100 \mu \mathrm{M} ) were measured using
光谱测量。使用 10 μ M 10 μ M 10 muM10 \mu \mathrm{M} PBS 缓冲液（ pH = 7.4 , 10 mM ) pH = 7.4 , 10 mM ) pH=7.4,10mM)\mathrm{pH}=7.4,10 \mathrm{mM}) 在室温下含有 50 % 50 % 50%50 \% DMSO。加入 100 μ M μ M muM\mu \mathrm{M} 个氨基酸（ 100 μ M 100 μ M 100 muM100 \mu \mathrm{M} 或 10 mM GSH）中的 TP-NIR 进行紫外-可见分光光度计和荧光实验，并在 30 分钟后记录荧光数据。为了测试 GSH 是否影响 TP-NIR 和半胱氨酸之间的反应， 10 μ M ) 10 μ M ) 10 muM)10 \mu \mathrm{M}) TP-NIR （ 在半胱氨酸和 GSH ( 100 μ M ( 100 μ M (100 muM(100 \mu \mathrm{M} 半胱氨酸以及 1 mM GSH 的三种不同混合物中孵育; 100 μ M 100 μ M 100 muM100 \mu \mathrm{M} 半胱氨酸和 5 mM GSH;和 100 个 μ M μ M muM\mu \mathrm{M} 半胱氨酸和 10 mM GSH）和 100 μ M 100 μ M 100 muM100 \mu \mathrm{M} 半胱氨酸溶液，然后每 1 分钟扫描一次发射光谱（激发 557 nm）以监测动力学，直到荧光信号没有变化。使用双光子激发荧光 （TPEF） 方法测量化合物 1 ( 100 μ M ) ( 100 μ M ) (100 muM)(100 \mu \mathrm{M}) 的双光子吸收光谱和横截面以及 TP-NIR ( 100 μ M ) ( 100 μ M ) (100 muM)(100 \mu \mathrm{M}) 与 Cys ( 500 μ M ( 500 μ M (500 muM(500 \mu \mathrm{M} 反应的产物，在 RT） 中孵育 30 分钟）在含有 50 % 50 % 50%50 \% DMSO 的 PBS 缓冲液 ( pH = ( pH = (pH=(\mathrm{pH}= 7.4） 中使用飞秒激光脉冲。在共聚焦显微镜系统（FV1000，奥林巴斯）与飞秒脉冲波长可切换激光源（Mai Tai Deepsee，80 MHz，> 2.1 W，光学物理学）相结合的共聚焦显微镜系统上进行实验。激光束被引导到显微镜系统的扫描模块中，并聚焦在密封在细胞培养皿中的样品上。用同一物镜收集荧光发射，投影到 PMT 上，然后绘制荧光发射曲线和强度。收集 750 至 900 nm （每步 10 nm） 不同激光波长激发的荧光发射。 记录了在相同激发波长下发射的参比和样品的双光子诱导荧光光谱的强度。在收集不同波长的数据集期间，所有参数都保持不变。DMSO/PBS 中样品 ( δ ) ( δ ) (delta)(\delta) 的 TPA 横截面 （ pH = 11 , 100 μ M pH = 11 , 100 μ M pH=11,100 muM\mathrm{pH}=11,100 \mu \mathrm{M} ） 使用
fluorescein in water ( pH = 11 , 10 μ M ) ( pH = 11 , 10 μ M ) (pH=11,10 muM)(\mathrm{pH}=11,10 \mu \mathrm{M}) as the reference. 71 , 72 71 , 72 ^(71,72){ }^{71,72} The serum stability of the probe TP-NIR was measured by incubating TPNIR in fetal bovine serum dialyzed with a 3 K membrane and subsequently recording the fluorescence spectra at different time points.
水中 ( pH = 11 , 10 μ M ) ( pH = 11 , 10 μ M ) (pH=11,10 muM)(\mathrm{pH}=11,10 \mu \mathrm{M}) 荧光素作为参考。 71 , 72 71 , 72 ^(71,72){ }^{71,72} 通过在用 3 K 膜透析的胎牛血清中孵育 TPNIR 来测量探针 TP-NIR 的血清稳定性，随后记录不同时间点的荧光光谱。
Flow Cytometer and Cell Imaging Analysis. For flow cytometer analysis, Hep 3B cells were cultured in cysteine free medium, regular medium ( 0.03 g / L 0.03 g / L 0.03g//L0.03 \mathrm{~g} / \mathrm{L} cysteine) or regular medium supplemented with cysteine ( 0.3 g / L 0.3 g / L 0.3g//L0.3 \mathrm{~g} / \mathrm{L} cysteine) prior to seeding to 6 well plate. After 2 h treatment, cells were analyzed by flow cytometer (BD LSR II). For florescence imaging, Hep 3B cells were incubated with 10 μ M 10 μ M 10 muM10 \mu \mathrm{M} of TP-NIR ( 2 μ L 2 μ L 2muL2 \mu \mathrm{~L} of the stock solution was dissolved in 2 mL of PBS) in regular medium ( 0.03 g / L 0.03 g / L 0.03g//L0.03 \mathrm{~g} / \mathrm{L} cysteine), cysteine free medium or regular medium supplemented with cysteine ( 0.3 g / L 0.3 g / L 0.3g//L0.3 \mathrm{~g} / \mathrm{L} cysteine) for 30 min and imaged under a Nikon ECLIPSE Ti inverted fluorescence microscopy (collected with a Cy 5 filter) or an Olympus FV1000 MPE Multiphoton Laser Scanning Confocal microscope (excited at 850 nm and the NIR emission signal was selected from 575 to 630 nm ).
流式细胞仪和细胞成像分析。对于流式细胞仪分析，在接种到 6 孔板之前，将 Hep 3B 细胞在无半胱氨酸培养基、普通培养基（ 0.03 g / L 0.03 g / L 0.03g//L0.03 \mathrm{~g} / \mathrm{L} 半胱氨酸）或补充有半胱氨酸（ 0.3 g / L 0.3 g / L 0.3g//L0.3 \mathrm{~g} / \mathrm{L} 半胱氨酸）的常规培养基中培养。处理 2 小时后，通过流式细胞仪 （BD LSR II） 分析细胞。对于荧光成像，将 Hep 3B 细胞与 10 μ M 10 μ M 10 muM10 \mu \mathrm{M} TP-NIR（ 2 μ L 2 μ L 2muL2 \mu \mathrm{~L} 储备溶液溶解在 2 mL PBS 中）在常规培养基（ 0.03 g / L 0.03 g / L 0.03g//L0.03 \mathrm{~g} / \mathrm{L} 半胱氨酸）、无半胱氨酸培养基或补充有半胱氨酸（ 0.3 g / L 0.3 g / L 0.3g//L0.3 \mathrm{~g} / \mathrm{L} 半胱氨酸）的普通培养基中孵育 30 分钟，并在尼康 ECLIPSE Ti 倒置荧光显微镜（用 Cy 5 滤光片收集）或奥林巴斯 FV1000 MPE 多光子激光扫描共聚焦显微镜（在 850 nm 和 NIR 发射下激发）下成像信号选择在 575 至 630 nm 之间）。

RESULTS AND DISCUSSION 结果与讨论

In order to generate an NIR-NIR fluorescent probe for biological imaging, TP-NIR was first designed and synthesized through acylation of dicyanomethylene-4H-pyran derivative 1 with acryloyl chloride in 92% yield (Figure S1). Structures of products were confirmed by 1 H 1 H ^(1)H{ }^{1} \mathrm{H} NMR spectroscopy and mass spectrometry (Supporting Information, and Figure S9 and S12). This design was based on the following rationale: (1) emission at long wavelength of chromophore dicyanomethy-lene-4H-pyran derivatives; 73 73 ^(73){ }^{73} (2) two-photon absorption of dicyanomethylene-4H-pyran derivatives; 17 17 ^(17){ }^{17} (3) photostability; (4) acrylate as a reliable Cys selective trigger (Scheme 1). 74 74 ^(74){ }^{74}
为了生成用于生物成像的 NIR-NIR 荧光探针，首先通过二氰亚甲基-4H-吡喃衍生物 1 与丙烯酰氯的酰化以 92% 的产率设计并合成 TP-NIR（图 S1）。通过 1 H 1 H ^(1)H{ }^{1} \mathrm{H} NMR 波谱和质谱证实产品结构（支持信息，以及图 S9 和 S12）。该设计基于以下基本原理：（1） 发色团二氰基-lene-4H-吡喃衍生物在长波长处发射; 73 73 ^(73){ }^{73} （2） 双氰亚甲基-4H-吡喃衍生物的双光子吸收; 17 17 ^(17){ }^{17} （3） 光稳定性;（4） 丙烯酸酯作为可靠的 Cys 选择性触发器（方案 1）。 74 74 ^(74){ }^{74}
The Cys response of TP-NIR was examined in 10 mM PBS buffer ( pH = 7.4 pH = 7.4 pH=7.4\mathrm{pH}=7.4 ) containing 50% DMSO. TP-NIR exhibited a maximum absorption at 381 nm , and a large UV-vis spectral red-shift ( λ max = 557 nm ) λ max = 557 nm (lambda_(max)=557(nm))\left(\lambda_{\max }=557 \mathrm{~nm}\right) was observed upon addition of Cys (Figure 1a), which revealed the deacylation of the probe. The response of TP-NIR toward Cys was very fast (within 2 min ) with significant enhancement of fluorescence intensity (up to 35 -fold increase in 30 min , Figure 1 b and Figure S2). The twophoton properties of probe TP-NIR were then studied, and the two-photon active absorption cross section of TP-NIR was calculated by using the following formula: δ = δ = δ r × ( S s × Φ r δ = δ = δ r × S s × Φ r delta=delta=delta_(r)xx(S_(s)xxPhi_(r):}\delta=\delta=\delta_{\mathrm{r}} \times\left(S_{\mathrm{s}} \times \Phi_{\mathrm{r}}\right. × ϕ r × c r ) / ( S r × Φ s × ϕ s × c s ) × ϕ r × c r / S r × Φ s × ϕ s × c s {: xxphi_(r)xxc_(r))//(S_(r)xxPhi_(s)xxphi_(s)xxc_(s))\left.\times \phi_{\mathrm{r}} \times c_{\mathrm{r}}\right) /\left(S_{\mathrm{r}} \times \Phi_{\mathrm{s}} \times \phi_{\mathrm{s}} \times c_{\mathrm{s}}\right), where fluorescein (aqueous, pH = 11 = 11 =11=11 ) was used as a reference probe. 75 , 76 75 , 76 ^(75,76){ }^{75,76} In the presence of Cys, the probe was calculated to have a two-photon absorption cross section of δ max = 16 GM δ max  = 16 GM delta_("max ")=16GM\delta_{\text {max }}=16 \mathrm{GM} at 830 nm , which was consistent with that of compound 1 1 1\mathbf{1} (Figure S3). These results demonstrate that the excitation and emission of TP-NIR are both in NIR range with a large Stokes shift ( ∼ 150 nm ∼ 150 nm ∼150nm\sim 150 \mathrm{~nm} ), which makes it promising for in vivo imaging.
在含有 50% DMSO 的 10 mM PBS 缓冲液 （ pH = 7.4 pH = 7.4 pH=7.4\mathrm{pH}=7.4 ） 中检查 TP-NIR 的 Cys 反应。TP-NIR 在 381 nm 处表现出最大吸收，并且在添加 Cys 时观察到较大的 UV-vis 光谱红移 ( λ max = 557 nm ) λ max = 557 nm (lambda_(max)=557(nm))\left(\lambda_{\max }=557 \mathrm{~nm}\right) （图 1a），这揭示了探针的脱酰基化。TP-NIR 对 Cys 的响应非常快（在 2 分钟内），荧光强度显着增强（在 30 分钟内增加高达 35 倍，图 1 b 和图 S2）。然后研究探针 TP-NIR 的双光子特性，并使用以下公式计算 TP-NIR 的双光子主动吸收截面： δ = δ = δ r × ( S s × Φ r δ = δ = δ r × S s × Φ r delta=delta=delta_(r)xx(S_(s)xxPhi_(r):}\delta=\delta=\delta_{\mathrm{r}} \times\left(S_{\mathrm{s}} \times \Phi_{\mathrm{r}}\right. × ϕ r × c r ) / ( S r × Φ s × ϕ s × c s ) × ϕ r × c r / S r × Φ s × ϕ s × c s {: xxphi_(r)xxc_(r))//(S_(r)xxPhi_(s)xxphi_(s)xxc_(s))\left.\times \phi_{\mathrm{r}} \times c_{\mathrm{r}}\right) /\left(S_{\mathrm{r}} \times \Phi_{\mathrm{s}} \times \phi_{\mathrm{s}} \times c_{\mathrm{s}}\right) ，其中荧光素（水，pH = 11 = 11 =11=11 ）用作参考探针。 75 , 76 75 , 76 ^(75,76){ }^{75,76} 在 Cys 存在下，计算探针 δ max = 16 GM δ max  = 16 GM delta_("max ")=16GM\delta_{\text {max }}=16 \mathrm{GM} 在 830 nm 处具有双光子吸收截面，这与化合物 1 1 1\mathbf{1} 的吸收截面一致（图 S3）。这些结果表明，TP-NIR 的激发和发射都在 NIR 范围内，具有较大的斯托克斯位移 （ ∼ 150 nm ∼ 150 nm ∼150nm\sim 150 \mathrm{~nm} ），这使得它有希望用于体内成像。
The response of probe TP-NIR toward other physiological amino acids was further investigated (Figure 1b). Compared with Cys, no apparent changes in fluorescent spectra were observed upon addition of other relevant analysts (Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val). Fluorescence experiments with other biological thiol-containing molecules such as Hcy and GSH at the same condition, resulted in very little conversion of TP-NIR to compound 1 (Figure 1b). Moreover, considering the high concentration of GSH in cells, we measured the fluorescent intensity at 10 mM of GSH. Similarly, no dramatic signal was detected (Figure S4). In addition, the spectra profiles of TPNIR treated with Cys were consistent with that of compound 1 1 1\mathbf{1}, indicating the proposed reaction mechanism (Figure S5).
进一步研究了探针 TP-NIR 对其他生理氨基酸的响应 （图 1b）。与 Cys 相比，在添加其他相关分析人员（Ala、Arg、Asn、Asp、Gln、Glu、Gly、His、Ile、Leu、Lys、Met、Phe、Pro、Ser、Thr、Trp、Tyr、Val）后，未观察到荧光光谱的明显变化。在相同条件下使用其他含生物巯基分子（如 Hcy 和 GSH）进行荧光实验，导致 TP-NIR 向化合物 1 的转化非常少（图 1b）。此外，考虑到细胞中 GSH 的高浓度，我们测量了 10 mM GSH 时的荧光强度。同样，没有检测到显着信号（图 S4）。此外，Cys 处理的 TPNIR 的光谱曲线与化合物 1 1 1\mathbf{1} 的光谱曲线一致，表明了所提出的反应机制（图 S5）。
(a) （一）

(b) （二）
Figure 1. UV-vis (a) and fluorescent spectra (b) of TP-NIR ( 10 μ M 10 μ M 10 muM10 \mu \mathrm{M} ) in PBS buffer ( pH = 7.4 pH = 7.4 pH=7.4\mathrm{pH}=7.4 ) containing 50 % 50 % 50%50 \% of DMSO upon addition of 100 μ M 100 μ M 100 muM100 \mu \mathrm{M} of thios and amino acids (Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, Cys, Hcy, GSH) for 30 min at RT. Excitation wavelength, 557 nm .
图 1.在含有 50 % 50 % 50%50 \% DMSO 的 PBS 缓冲液 （ pH = 7.4 pH = 7.4 pH=7.4\mathrm{pH}=7.4 ） 中加入 100 μ M 100 μ M 100 muM100 \mu \mathrm{M} 硫和氨基酸（Ala、Arg、Asn、Asp、Gln、Glu、Gly、His、Ile、Leu、Lys、Met、Phe、Pro、Ser、Thr、Trp、Tyr、Val、Cys、Hcy、GSH）在室温下放置 30 分钟后，TP-NIR （ 10 μ M 10 μ M 10 muM10 \mu \mathrm{M} ） 的紫外-可见光 （a） 和荧光光谱 （b） 。激发波长，557 nm。
Besides, the detection limit of TP-NIR toward Cys was as low as 0.2 μ M 0.2 μ M 0.2 muM0.2 \mu \mathrm{M} calculated from the calibration curve (Figure S6). Since the intracellular concentration of GSH is higher than that of cysteine, we next evaluated the effect of GSH concentration on the reaction between TP-NIR and cysteine. We observed a concentration-dependent effect (Figure S7). High GSH concentration intensely interfered with the reaction of TPNIR and Cys.
此外，TP-NIR 对 Cys 的检测限与根据校准曲线 0.2 μ M 0.2 μ M 0.2 muM0.2 \mu \mathrm{M} 计算的检测限一样低（图 S6）。由于 GSH 的细胞内浓度高于半胱氨酸，我们接下来评估了 GSH 浓度对 TP-NIR 和半胱氨酸反应的影响。我们观察到浓度依赖性效应（图 S7）。高 GSH 浓度强烈干扰 TPNIR 和 Cys 的反应。
To elucidate the reaction process, an in situ 1 H 1 H ^(1)H{ }^{1} \mathrm{H} NMR study of the reaction between TP-NIR and Cys/GSH was carried out. When TP-NIR was incubated with Cys, fast conversion ( 2 min ) to compound 1 1 1\mathbf{1} was clearly seen by the newly formed doublet at 6.86 ( 1 H 1 H ^(1)H{ }^{1} \mathrm{H} NMR) and the singlet at 6.93 ( 1 H 1 H ^(1)H{ }^{1} \mathrm{H} NMR) (Figure 2), which is consistent with the fluorescent response. Prolonged reaction time ( 70 min ) resulted in complete deacylation from
为了阐明反应过程，对 TP-NIR 和 Cys/GSH 之间的反应进行了原位 1 H 1 H ^(1)H{ }^{1} \mathrm{H} NMR 研究。当 TP-NIR 与 Cys 一起孵育时，新形成的双峰在 6.86 （ 1 H 1 H ^(1)H{ }^{1} \mathrm{H} NMR） 和新形成的单峰在 6.93 （ 1 H 1 H ^(1)H{ }^{1} \mathrm{H} NMR） 处清楚地看到快速转化（2 分钟）到化合物 1 1 1\mathbf{1} （图 2），这与荧光反应一致。延长的反应时间 （ 70 分钟 ） 导致完全脱酰基
Figure 2. In situ 1 H 1 H ^(1)H{ }^{1} \mathrm{H} NMR spectra of TP-NIR upon addition of 10 equiv Cys over time in d 6 − DMSO : D 2 O = 9 : 1 d 6 − DMSO : D 2 O = 9 : 1 d_(6)-DMSO:D_(2)O=9:1d_{6}-\mathrm{DMSO}: \mathrm{D}_{2} \mathrm{O}=9: 1.
图 2.TP-NIR 在 中随时间添加 10 个当量 Cys 时的原位 1 H 1 H ^(1)H{ }^{1} \mathrm{H} NMR 谱图 d 6 − DMSO : D 2 O = 9 : 1 d 6 − DMSO : D 2 O = 9 : 1 d_(6)-DMSO:D_(2)O=9:1d_{6}-\mathrm{DMSO}: \mathrm{D}_{2} \mathrm{O}=9: 1 。
the intermediate 2 2 2\mathbf{2} to yield compound 1 1 1\mathbf{1} (Figure S9a and S9b). Similarly, the reaction of GSH to TP-NIR was monitored by in situ 1 H 1 H ^(1)H{ }^{1} \mathrm{H} NMR. TP-NIR underwent the Michael addition reaction with GSH, as evidenced by the disappearance of characteristic alkenyl protons H 2 , H b H 2 , H b H_(2),H_(b)\mathrm{H}_{2}, \mathrm{H}_{\mathrm{b}}, and H c H c H_(c)\mathrm{H}_{\mathrm{c}} (Figure S10, 30 min ). The reaction product TP-NIR-GSH was also identified by mass spectrometry at m / z 674.20 m / z 674.20 m//z 674.20m / z 674.20 (Figure S9c, calculated for [ M + H ] + C 33 H 32 N 5 O 9 S : [ M + H ] + C 33 H 32 N 5 O 9 S : [M+H]^(+)C_(33)H_(32)N_(5)O_(9)S:[\mathrm{M}+\mathrm{H}]^{+} \mathrm{C}_{33} \mathrm{H}_{32} \mathrm{~N}_{5} \mathrm{O}_{9} \mathrm{~S}: 674.19). However, during this reaction, very low levels of compound 1 were detected after 70 min of well-mixing. These results further validated the specific response of TP-NIR to Cys in comparison to other biological thiols. To further evaluate the stability of probe TPNIR, we incubated TP-NIR with fetal bovine serum (FBS) and dialyzed FBS. TP-NIR in dialyzed FBS displayed much less signal (Figure S11), suggesting that TP-NIR was stable in the dialyzed FBS for at least 48 h .
中间 2 2 2\mathbf{2} 产率化合物 1 1 1\mathbf{1} （图 S9a 和 S9b）。同样，通过原位 1 H 1 H ^(1)H{ }^{1} \mathrm{H} NMR 监测 GSH 对 TP-NIR 的反应。TP-NIR 与 GSH 发生 Michael 加成反应，特征烯基质子 H 2 , H b H 2 , H b H_(2),H_(b)\mathrm{H}_{2}, \mathrm{H}_{\mathrm{b}} 的消失证明了这一点，并且 H c H c H_(c)\mathrm{H}_{\mathrm{c}} （图 S10,30 分钟）。反应产物 TP-NIR-GSH 也通过质谱鉴定 m / z 674.20 m / z 674.20 m//z 674.20m / z 674.20 （图 S9c，计算为 [ M + H ] + C 33 H 32 N 5 O 9 S : [ M + H ] + C 33 H 32 N 5 O 9 S : [M+H]^(+)C_(33)H_(32)N_(5)O_(9)S:[\mathrm{M}+\mathrm{H}]^{+} \mathrm{C}_{33} \mathrm{H}_{32} \mathrm{~N}_{5} \mathrm{O}_{9} \mathrm{~S}: 674.19）。然而，在该反应过程中，充分混合 70 分钟后检测到非常低水平的化合物 1。这些结果进一步验证了与其他生物硫醇相比，TP-NIR 对 Cys 的特异性反应。为了进一步评估探针 TPNIR 的稳定性，我们将 TP-NIR 与胎牛血清 （FBS） 和透析的 FBS 一起孵育。透析的 FBS 中的 TP-NIR 显示的信号要少得多（图 S11），表明 TP-NIR 在透析的 FBS 中稳定至少 48 小时。
In order to study the potential applications of TP-NIR for biomedical imaging, we treated cells with TP-NIR. First, we studied the effect of Cys concentrations ( 0 , 0.03 , 0.3 g / L 0 , 0.03 , 0.3 g / L 0,0.03,0.3g//L0,0.03,0.3 \mathrm{~g} / \mathrm{L}; Cys in regular medium = 0.03 g / L = 0.03 g / L =0.03g//L=0.03 \mathrm{~g} / \mathrm{L} ) on TP-NIR in living cells. According to the florescence images (Figure S8a), TP-NIR showed dramatic fluorescence signal in Cys regular ( 0.03 g / L 0.03 g / L 0.03g//L0.03 \mathrm{~g} / \mathrm{L} ) and Cys high medium ( 0.3 g / L 0.3 g / L 0.3g//L0.3 \mathrm{~g} / \mathrm{L} ), while no evident signal was observed in Cys free medium. Moreover, flow cytometry analysis displayed significantly higher signal in Cys high medium compared to that in Cys regular medium (Figure S8b). These results indicated that Cys concentration played an important role in TP-NIR imaging. Lastly, we investigated twophoton imaging of TP-NIR in cells. Compared to untreated cells, dramatic fluorescent signal ( 575 − 630 nm 575 − 630 nm 575-630nm575-630 \mathrm{~nm} ) was observed in the TP-NIR treated cells after excitation at 850 nm via an Olympus FV1000 MPE multiphoton laser scanning confocal microscope (Figure 3). Taken together, these results demonstrated that TP-NIR is a compatible probe for biological imaging applications.
为了研究 TP-NIR 在生物医学成像中的潜在应用，我们用 TP-NIR 处理细胞。首先，我们研究了 Cys 浓度 （ 0 , 0.03 , 0.3 g / L 0 , 0.03 , 0.3 g / L 0,0.03,0.3g//L0,0.03,0.3 \mathrm{~g} / \mathrm{L} ;常规培养基 = 0.03 g / L = 0.03 g / L =0.03g//L=0.03 \mathrm{~g} / \mathrm{L} 中的 Cys ） 对活细胞中的 TP-NIR 的影响。根据荧光图像（图 S8a），TP-NIR 在 Cys 常规 （ 0.03 g / L 0.03 g / L 0.03g//L0.03 \mathrm{~g} / \mathrm{L} ） 和 Cys 高培养基 （ ） 中显示出明显的荧光信号 0.3 g / L 0.3 g / L 0.3g//L0.3 \mathrm{~g} / \mathrm{L} ，而在 Cys 游离培养基中没有观察到明显的信号。此外，流式细胞术分析显示，与 Cys 常规培养基相比，Cys 高培养基中的信号显着更高（图 S8b）。这些结果表明 Cys 浓度在 TP-NIR 成像中起重要作用。最后，我们研究了细胞中 TP-NIR 的双光子成像。与未处理的细胞相比，通过奥林巴斯 FV1000 MPE 多光子激光扫描共聚焦显微镜在 850 nm 激发后，在 TP-NIR 处理的细胞中观察到显着的荧光信号 （ 575 − 630 nm 575 − 630 nm 575-630nm575-630 \mathrm{~nm} ）（图 3）。综上所述，这些结果表明 TP-NIR 是一种适用于生物成像应用的探针。

CONCLUSION 结论

In summary, we characterized TP-NIR via two-photon-excited fluorescence and applied it to selectively detect Cys in vitro. The probe showed a fast response (within 2 min ) and specific selectivity toward Cys over Hcy, GSH, and other amino acids.
综上所述，我们通过双光子激发的荧光表征了 TP-NIR，并将其应用于体外选择性检测 Cys。探针显示出快速响应（2 分钟内）和对 Cys 的特异性选择性，而不是 Hcy、GSH 和其他氨基酸。
Figure 3. Confocal fluorescence images of Hep 3B cells on an Olympus FV1000 MPE multiphoton laser scanning confocal microscope. Hep 3B cells were imaged after 30 min incubation with 10 μ M 10 μ M 10 muM10 \mu \mathrm{M} of TP-NIR (excited at 850 nm , collected from 575 to 630 nm ; Scale bars: 20 μ m 20 μ m 20 mum20 \mu \mathrm{~m} ).
图 3.奥林巴斯 FV1000 MPE 多光子激光扫描共聚焦显微镜上 Hep 3B 细胞的共聚焦荧光图像。Hep 3B 细胞与 10 μ M 10 μ M 10 muM10 \mu \mathrm{M} TP-NIR（在 850 nm 处激发，从 575 至 630 nm 收集;比例尺： 20 μ m 20 μ m 20 mum20 \mu \mathrm{~m} ）。
  1. Received: December 4, 2015
    收稿日期： 2015-12-04
    Accepted: June 1, 2016 录用日期： 2016-06-01
    Published: June 1, 2016 发布时间：2016 年 6 月 1 日