Compared to 2PE (two‐photon excitation) microscopy, 3PE microscopy has superior spatial resolution, deeper tissue penetration, and less defocused interference. The design of suitable agents with a large Stokes shift, good three‐photon absorption (3PA), subcellular targeting, and fluorescence lifetime imaging (FLIM) properties, is challenging. Now, two Ir^III complexes (3PAIr1 and 3PAIr2) were developed as efficient three‐photon phosphorescence (3PP) agents. Calculations reveal that the introduction of a new group to the molecular scaffold confers a quadruple promotion in three‐photon transition probability. Confocal and lifetime imaging of mitochondria using Ir^III complexes as 3PP agents is shown. The complexes exhibit low working concentration (50 nm), fast uptake (5 min), and low threshold for three‐photon excitation power (0.5 mW at 980 nm). The impressive tissue penetration depth (ca. 450 μm) allowed the 3D imaging and reconstruction of brain vasculature from a living specimen.

中文翻译：

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三光子磷光生物成像的环金属化铱（III）配合物的合理设计

与2PE（双光子激发）显微镜相比，3PE显微镜具有更高的空间分辨率，更深的组织穿透力和更少的散焦干扰。具有大斯托克斯位移，良好的三光子吸收（3PA），亚细胞靶向和荧光寿命成像（FLIM）特性的合适试剂的设计具有挑战性。现在，开发了两种Ir ^III复合物（3PAIr1和3PAIr2）作为有效的三光子磷光（3PP）试剂。计算表明，向分子支架中引入新的基团使三光子跃迁几率提高了四倍。显示了使用Ir ^III复合物作为3PP试剂对线粒体进行共聚焦和终生成像。复合物的工作浓度低（50 n m），快速吸收（5分钟）和低阈值的三光子激发功率（在980 nm处为0.5 mW）。令人印象深刻的组织穿透深度（约450μm）允许从活体标本进行3D成像和脑血管重建。