Three-dimensional imaging modalities for optically dense connective tissues such as tendons are limited and typically have a single imaging methodological endpoint. Here, we have developed a bimodal procedure utilising fluorescence-based confocal microscopy and x-ray micro-computed tomography for the imaging of adult tendons to visualise and analyse extracellular sub-structure and cellular composition in small and large animal species. Using fluorescent immunolabelling and optical clearing, we visualised the expression of the novel cross-species marker of tendon basement membrane, laminin-α4 in 3D throughout whole rat Achilles tendons and equine superficial digital flexor tendon 5 mm segments. This revealed a complex network of laminin-α4 within the tendon core that predominantly localises to the interfascicular matrix compartment. Furthermore, we implemented a chemical drying process capable of creating contrast densities enabling visualisation and quantification of both fascicular and interfascicular matrix volume and thickness by x-ray micro-computed tomography. We also demonstrated that both modalities can be combined using reverse clarification of fluorescently labelled tissues prior to chemical drying to enable bimodal imaging of a single sample. Whole-mount imaging of tendon allowed us to identify the presence of an extensive network of laminin-α4 within tendon, the complexity of which cannot be appreciated using traditional 2D imaging techniques. Creating contrast for x-ray micro-computed tomography imaging of tendon using chemical drying is not only simple and rapid, but also markedly improves on previously published methods. Combining these methods provides the ability to gain spatio-temporal information and quantify tendon substructures to elucidate the relationship between morphology and function.

中文翻译：

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使用共聚焦显微镜和 X 射线微计算机断层扫描的肌腱双峰整体成像。

用于光致密结缔组织（例如肌腱）的三维成像方式是有限的，并且通常具有单一的成像方法终点。在这里，我们开发了一种双峰程序，利用基于荧光的共聚焦显微镜和 X 射线微计算机断层扫描对成人肌腱进行成像，以可视化和分析小型和大型动物物种的细胞外亚结构和细胞组成。使用荧光免疫标记和光学清除，我们在整个大鼠跟腱和马浅表指屈肌腱 5 mm 节段中以 3D 形式显示了腱基底膜的新型跨物种标记层粘连蛋白-α4 的表达。这揭示了肌腱核心内的复杂层粘连蛋白-α4 网络，主要定位于束间基质隔室。此外，我们实施了一种化学干燥过程，能够产生对比密度，从而通过 X 射线微计算机断层扫描对束状和束间基质的体积和厚度进行可视化和量化。我们还证明了这两种方式可以在化学干燥之前使用荧光标记组织的反向澄清来组合，以实现单个样本的双峰成像。肌腱的整体成像使我们能够识别肌腱内广泛的层粘连蛋白-α4 网络的存在，其复杂性无法使用传统的 2D 成像技术来理解。使用化学干燥为肌腱的 X 射线微计算机断层扫描成像创建对比度不仅简单快速，而且显着改进了以前发表的方法。