Ducourthial Guillaume, Leclerc Pierre, Mansuryan Tigran, Fabert Marc, Brevier Julien, Habert Rémi, Braud Flavie, Batrin Renaud, Vever-Bizet Christine, Bourg-Heckly Geneviève, Thiberville Luc, Druilhe Anne, Kudlinski Alexandre, Louradour Frédéric
XLIM, UMR-CNRS 7252, Université de Limoges, France.
PhLAM, UMR-CNRS 8523, Université Lille I, Villeneuve d'Ascq, France.
Sci Rep. 2015 Dec 17;5:18303. doi: 10.1038/srep18303.
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.
我们展示了一种双光子微型内窥镜,它能够通过一根非常长的光纤进行体内无标记的深层组织高分辨率快速成像。首先,一种先进的光脉冲光谱时间整形装置可对在内窥镜光纤内传播过程中出现的线性和非线性失真进行最佳预补偿。这使得在5米光纤的输出端能够传输持续时间低于40飞秒的红外激发脉冲。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的反向收集。第三,一个外径为2.2毫米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生成(SHG)和双光子激发自发荧光(TPEF)成像。这种微型内窥镜的横向分辨率和轴向分辨率分别为0.8微米和12微米,视野高达450微米。这种微型内窥镜前所未有的能力在无标记成像过程中得到了验证,在体外对各种固定的人体组织样本进行了验证,并在体内对一只麻醉小鼠的肾脏进行了验证,证明成像穿透深度大于器官表面以下300微米。本手稿中报告的结果证实,非线性微型内窥镜可以成为一种有价值的临床工具,用于对病理状态进行实时原位评估。