Hage C H, Leclerc P, Brevier J, Fabert M, Le Nézet C, Kudlinski A, Héliot L, Louradour F
Université de Limoges, XLIM, UMR CNRS 7252, 123 Avenue A. Thomas, 87060 Limoges, France.
Univ. Lille, CNRS, UMR 8523 - PhLAM - Physique des Lasers, Atomes et Molécules, F-59000 Lille, France.
Biomed Opt Express. 2017 Dec 8;9(1):142-156. doi: 10.1364/BOE.9.000142. eCollection 2018 Jan 1.
fluorescence lifetime imaging microscopy (FLIM) in an endoscopic configuration of the endogenous biomarker nicotinamide adenine dinucleotide (NADH) has a great potential for malignant tissue diagnosis. Moreover, two-photon nonlinear excitation provides intrinsic optical sectioning along with enhanced imaging depth. We demonstrate, for the first time to our knowledge, nonlinear endogenous FLIM in a fibered microscope with proximal detection, applied to NADH in cultured cells, as a first step to a nonlinear endomicroscope, using a double-clad microstructured fiber with convenient fiber length (> 3 m) and excitation pulse duration (≈50 fs). Fluorescence photons are collected by the fiber inner cladding and we show that its contribution to the impulse response function (IRF), which originates from its intermodal and chromatic dispersions, is small (< 600 ps) and stable for lengths up to 8 m and allows for short lifetime measurements. We use the phasor representation as a quick visualization tool adapted to the endoscopy speed requirements.
在内窥镜配置下，利用内源性生物标志物烟酰胺腺嘌呤二核苷酸（NADH）进行荧光寿命成像显微镜（FLIM）在恶性组织诊断方面具有巨大潜力。此外，双光子非线性激发提供了固有的光学切片以及增强的成像深度。据我们所知，我们首次展示了在具有近端检测功能的光纤显微镜中进行非线性内源性FLIM，将其应用于培养细胞中的NADH，这是迈向非线性内窥镜显微镜的第一步，使用了具有便利光纤长度（> 3米）和激发脉冲持续时间（≈50飞秒）的双包层微结构光纤。荧光光子由光纤内包层收集，并且我们表明其对源于其模式间和色散的脉冲响应函数（IRF）的贡献很小（< 600皮秒），并且对于长达8米的长度是稳定的，并且允许进行短寿命测量。我们使用相量表示作为一种适应内窥镜速度要求的快速可视化工具。
