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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