Determination of collagen fibril size via absolute measurements of second-harmonic generation signals
Determination of collagen fibril size via absolute measurements of second-harmonic generation signals
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DOI：
10.1038/ncomms5920
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发表时间：
2014-09-01
影响因子：
16.6
通讯作者：
Schanne-Klein, Marie-Claire
中科院分区：
文献类型：
--
作者：
Bancelin, Stephane;Aime, Carole;Schanne-Klein, Marie-Claire
胶原原纤维大小的定量是研究与细胞外基质结构缺陷相关的病理障碍的一个主要问题。二次谐波产生显微镜是表征未染色生物组织中胶原的大分子结构的一种强有力的技术。然而，由于这种非线性光信号具有复杂的相干结构，到目前为止还没有被用来测量纤维直径。在这里，我们报告了通过相关的二次谐波电子显微镜的实现，从孤立的纤维到直径30 nm的二次谐波信号的绝对测量。此外，通过分析和数值计算，我们证明了这种技术的高灵敏度源于胶原蛋白三螺旋在纤维内的平行排列以及随后的二次谐波辐射的建设性干扰。最后，我们使用这些绝对测量值作为体外定量糖尿病大鼠角膜后弹力膜中纤维直径的校准。
The quantification of collagen fibril size is a major issue for the investigation of pathological disorders associated with structural defects of the extracellular matrix. Second-harmonic generation microscopy is a powerful technique to characterize the macromolecular organization of collagen in unstained biological tissues. Nevertheless, due to the complex coherent building of this nonlinear optical signal, it has never been used to measure fibril diameter so far. Here we report absolute measurements of second-harmonic signals from isolated fibrils down to 30 nm diameter, via implementation of correlative second-harmonic-electron microscopy. Moreover, using analytical and numerical calculations, we demonstrate that the high sensitivity of this technique originates from the parallel alignment of collagen triple helices within fibrils and the subsequent constructive interferences of second-harmonic radiations. Finally, we use these absolute measurements as a calibration for ex vivo quantification of fibril diameter in the Descemet's membrane of a diabetic rat cornea.