The wound healing assay provides essential information about collective cell migration and cell-to-cell interactions. It is a simple, effective, and widely used tool for observing the effect of numerous chemical treatments on wound healing speed. To perform and analyze a wound healing assay, various imaging techniques have been utilized. However, image acquisition and analysis are often limited in two-dimensional space or require the use of exogenous labeling agents. Here, we present a method for imaging large-scale wound healing assays in a label-free and volumetric manner using optical diffraction tomography (ODT). We performed quantitative high-resolution three-dimensional (3D) analysis of cell migration over a long period without difficulties such as photobleaching or phototoxicity. ODT enables the reconstruction of the refractive index (RI) tomogram of unlabeled cells, which provides both structural and biochemical information about the individual cell at subcellular resolution. Stitching multiple RI tomograms enables long-term (24 h) and large field-of-view imaging (> 800 × 400 μm²) with a lateral resolution of 110 nm. We demonstrated the thickness changes of leading cells and studied the effects of cytochalasin D. The 3D RI tomogram also revealed increased RI values in leading cells compared to lagging cells, suggesting the formation of a highly concentrated subcellular structure.

中文翻译：

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伤口愈合过程中迁移细胞的三维无标记成像和定量

伤口愈合分析提供了有关集体细胞迁移和细胞间相互作用的基本信息。它是一种简单，有效且广泛使用的工具，可用于观察多种化学治疗对伤口愈合速度的影响。为了执行和分析伤口愈合测定法，已经利用了各种成像技术。但是，图像获取和分析通常限于二维空间，或者需要使用外源标记剂。在这里，我们提出了一种使用光学衍射断层扫描（ODT）以无标记和体积方式成像大规模伤口愈合试验的方法。我们对细胞迁移进行了长时间的定量高分辨率三维（3D）分析，而没有诸如光漂白或光毒性之类的困难。ODT能够重建未标记细胞的折射率（RI）断层图，从而以亚细胞分辨率提供有关单个细胞的结构和生化信息。拼接多个RI断层图可实现长期（24 h）和大视野成像（> 800×400μm²）具有110nm的横向分辨率。我们证明了前导细胞的厚度变化并研究了细胞松弛素D的作用。3DRI断层图也显示与落后的细胞相比，前导细胞的RI值增加，表明形成了高度浓缩的亚细胞结构。