Intravital autofluorescence 2-photon microscopy of murine intestinal mucosa with ultra-broadband femtosecond laser pulse excitation: image quality, photodamage, and inflammation
Intravital autofluorescence 2-photon microscopy of murine intestinal mucosa with ultra-broadband femtosecond laser pulse excitation: image quality, photodamage, and inflammation
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采用超宽带飞秒激光脉冲激发的小鼠肠粘膜活体自发荧光双光子显微镜：图像质量、光损伤和炎症
DOI：
10.1117/1.jbo.20.11.116001
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发表时间：
2015
影响因子：
3.5
通讯作者：
Hüttmann G
中科院分区：
文献类型：
--
作者：
Klinger A;Krapf L;Orzekowsky-Schroeder R;Koop N;Vogel A;Hüttmann G
使用超短脉冲的超宽带激发可以同时激发活体组织中的多个内源荧光团。通过自体荧光 2 光子显微镜对活体肠道粘膜进行成像，将亚 10 fs 钛蓝宝石 (Ti:sapphire) 激光器在 800 nm 中心波长下进行超过 150 nm 的宽激发，并使用基于介电镜的预线性调频，与可调谐 Ti:蓝宝石激光器在 730 和 800 nm 波长下的 220 fs 脉冲进行激发进行比较。评估了激发效率、图像质量和光化学损伤。在相似的激发通量下，两种激光器获得了相同的图像亮度。正如预期的那样，使用超宽带脉冲，同时观察到 NAD(P)H、黄素和脂蛋白的荧光。然而，当激光功率调整到相同的图像亮度时，非线性光损伤会出现得更早，表现为超荧光，并伴有组织的功能和结构改变。仅仅几分钟后，就观察到多形核白细胞迁移到上皮中，并且这些细胞脱粒，这是炎症的迹象。较高的峰值辐照度和/或较长波长下自发荧光的非最佳激发会促进光损伤。我们得出的结论是，对于基于自发荧光的 2 光子显微镜，使用可调谐窄带宽激光器的激发优于超宽带激发，除非可以控制光谱相位以优化激发条件。
Ultra-broadband excitation with ultrashort pulses may enable simultaneous excitation of multiple endogenous fluorophores in vital tissue. Imaging living gut mucosa by autofluorescence 2-photon microscopy with more than 150 nm broad excitation at an 800-nm central wavelength from a sub-10 fs titanium-sapphire (Ti:sapphire) laser with a dielectric mirror based prechirp was compared to the excitation with 220 fs pulses of a tunable Ti:sapphire laser at 730 and 800 nm wavelengths. Excitation efficiency, image quality, and photochemical damage were evaluated. At similar excitation fluxes, the same image brightness was achieved with both lasers. As expected, with ultra-broadband pulses, fluorescence from NAD(P)H, flavines, and lipoproteins was observed simultaneously. However, nonlinear photodamage apparent as hyperfluorescence with functional and structural alterations of the tissue occurred earlier when the laser power was adjusted to the same image brightness. After only a few minutes, the immigration of polymorphonuclear leucocytes into the epithelium and degranulation of these cells, a sign of inflammation, was observed. Photodamage is promoted by the higher peak irradiances and/or by nonoptimal excitation of autofluorescence at the longer wavelength. We conclude that excitation with a tunable narrow bandwidth laser is preferable to ultra-broadband excitation for autofluorescence-based 2-photon microscopy, unless the spectral phase can be controlled to optimize excitation conditions.
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影响因子：
64.8
作者：
通讯作者：
--
影响因子：
2.3
作者：
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影响因子：
3.5
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