Development of a real-time flexible multiphoton microendoscope for label-free imaging in a live animal.
Development of a real-time flexible multiphoton microendoscope for label-free imaging in a live animal.
复制标题
在活动物中开发用于无标记成像的实时柔性多光子微镜。
DOI：
10.1038/srep18303
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发表时间：
2015-12-17
影响因子：
4.6
通讯作者：
Louradour F
中科院分区：
文献类型：
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作者：
Ducourthial G;Leclerc P;Mansuryan T;Fabert M;Brevier J;Habert R;Braud F;Batrin R;Vever-Bizet C;Bourg-Heckly G;Thiberville L;Druilhe A;Kudlinski A;Louradour F
我们提出了一种双光子显微内窥镜能够在体内无标记的深层组织高分辨率快速成像通过一个很长的光纤。首先，先进的光脉冲光谱时间整形装置最佳地预补偿内窥镜光纤内传播期间发生的线性和非线性失真。这使得能够在5米光纤的输出处递送亚40 fs持续时间的红外激发脉冲。其次，内窥镜光纤是一种定制的双包层保偏光子晶体光纤，专门设计用于优化成像分辨率和本征发光后向收集。第三，2.2 mm外径的小型化光纤扫描仪允许以每秒8帧的速度同时进行二次谐波产生（SHG）和双光子激发自发荧光（TPEF）成像。该显微内窥镜的横向和轴向分辨率分别为0.8 μm和12 μm，视场达450 μm。该显微内窥镜的前所未有的能力在无标记成像过程中得到了验证，在各种固定的人体组织样本上进行了离体成像，并在麻醉小鼠肾脏上进行了体内成像，证明了器官表面以下的成像穿透深度大于300 μm。本文报道的结果证实，非线性显微内窥镜可以成为一种有价值的临床工具，用于实时原位评估病理状态。
We present a two-photon microendoscope capable of in vivo label-free deep-tissue high-resolution fast imaging through a very long optical fiber. First, an advanced light-pulse spectro-temporal shaping device optimally precompensates for linear and nonlinear distortions occurring during propagation within the endoscopic fiber. This enables the delivery of sub-40-fs duration infrared excitation pulses at the output of 5 meters of fiber. Second, the endoscopic fiber is a custom-made double-clad polarization-maintaining photonic crystal fiber specifically designed to optimize the imaging resolution and the intrinsic luminescence backward collection. Third, a miniaturized fiber-scanner of 2.2 mm outer diameter allows simultaneous second harmonic generation (SHG) and two-photon excited autofluorescence (TPEF) imaging at 8 frames per second. This microendoscope’s transverse and axial resolutions amount respectively to 0.8 μm and 12 μm, with a field-of-view as large as 450 μm. This microendoscope’s unprecedented capabilities are validated during label-free imaging, ex vivo on various fixed human tissue samples, and in vivo on an anesthetized mouse kidney demonstrating an imaging penetration depth greater than 300 μm below the surface of the organ. The results reported in this manuscript confirm that nonlinear microendoscopy can become a valuable clinical tool for real-time in situ assessment of pathological states.