Bone healing involves the interplay of immune cells, mesenchymal cells, and vasculature over the time course of regeneration. Approaches to quantify the spatiotemporal aspects of bone healing at cellular resolution during long bone healing do not yet exist. Here, a novel technique termed Limbostomy is presented, which combines intravital microendoscopy with an osteotomy. This design allows a modular combination of an internal fixator plate with a gradient refractive index (GRIN) lens at various depths in the bone marrow and can be combined with a surgical osteotomy procedure. The field of view (FOV) covers a significant area of the fracture gap and allows monitoring cellular processes in vivo. The GRIN lens causes intrinsic optical aberrations which have to be corrected. The optical system was characterized and a postprocessing algorithm was developed. It corrects for wave front aberration-induced image plane deformation and for background and noise signals, enabling us to observe subcellular processes. Exemplarily, we quantitatively and qualitatively analyze angiogenesis in bone regeneration. We make use of a transgenic reporter mouse strain with nucleargreen fluorescent protein and membrane-bound tdTomato under the Cadherin-5 promoter. We observe two phases of vascularization. First, rapid vessel sprouting pervades the FOV within 3-4 days after osteotomy. Second, the vessel network continues to be dynamically remodeled until the end of our observation time, 14 days after surgery. Limbostomy opens a unique set of opportunities and allows further insight on spatiotemporal aspects of bone marrow biology, for example, hematopoiesis, analysis of cellular niches, immunological memory, and vascularization in the bone marrow during health and disease. © 2020 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

中文翻译：

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肢体造口术：小鼠截骨术中的纵向活体显微内窥镜检查。

骨愈合涉及免疫细胞、间充质细胞和脉管系统在再生过程中的相互作用。尚不存在在长骨愈合期间以细胞分辨率量化骨愈合的时空方面的方法。在这里，介绍了一种称为 Limbostomy 的新技术，它将活体显微内窥镜检查与截骨术相结合。这种设计允许内固定器板与骨髓中不同深度的梯度折射率 (GRIN) 透镜的模块化组合，并且可以与外科截骨术程序相结合。视野 (FOV) 覆盖了骨折间隙的一个重要区域，并允许在体内监测细胞过程。GRIN 镜头会导致必须校正的固有光学像差。对光学系统进行了表征，并开发了后处理算法。它校正波前像差引起的图像平面变形以及背景和噪声信号，使我们能够观察亚细胞过程。例如，我们定量和定性地分析了骨再生中的血管生成。我们利用具有核绿色荧光蛋白和膜结合 tdTomato 在 Cadherin-5 启动子下的转基因报告小鼠品系。我们观察到血管化的两个阶段。首先，在截骨术后 3-4 天内，快速的血管萌芽遍布 FOV。其次，血管网络继续动态重塑，直到我们的观察时间结束，即手术后 14 天。造口术开辟了一系列独特的机会，并允许进一步了解骨髓生物学的时空方面，例如，造血、细胞壁龛分析、免疫记忆以及健康和疾病期间骨髓中的血管形成。© 2020 作者。Cytometry Part A 由 Wiley Periodicals, Inc. 代表 International Society for Advancement of Cytometry 出版。