Three-dimensional cell cultures are able to better mimic the physiology and cellular environments found in tissues in vivo compared to cells grown in two dimensions. In order to study the structure and function of cells in 3-D cultures, light microscopy is frequently used. The preparation of 3-D cell cultures for light microscopy is often destructive, including physical sectioning of the samples, which can result in the loss of 3-D information. In order to probe the structure of 3-D cell cultures at high resolution, we have explored the use of expansion microscopy and compared it to a simple immersion clearing protocol. We provide a practical method for the study of spheroids, organoids and tumor-infiltrating immune cells at high resolution without the loss of spatial organization. Expanded samples are highly transparent, enabling high-resolution imaging over extended volumes by significantly reducing light scatter and absorption. In addition, the hydrogel-like nature of expanded samples enables homogenous antibody labeling of dense epitopes throughout the sample volume. The improved labeling and image quality achieved in expanded samples revealed details in the center of the organoid which were previously only observable following serial sectioning. In comparison to chemically cleared spheroids, the improved signal-to-background ratio of expanded samples greatly improved subsequent methods for image segmentation and analysis.

三维细胞培养能够更好地模仿组织中发现的生理和细胞环境 体内与二维生长的细胞相比。为了研究3-D培养物中细胞的结构和功能，经常使用光学显微镜。用于光学显微镜的3-D细胞培养物的制备通常具有破坏性，包括样品的物理切片，这可能会导致3-D信息丢失。为了以高分辨率探测3-D细胞培养物的结构，我们探索了扩展显微镜的使用，并将其与简单的浸入清除协议进行了比较。我们为研究球体，类器官和浸润肿瘤的免疫细胞提供了一种实用的方法，可以实现高分辨率而又不损失空间组织。扩展的样品高度透明，可通过显着减少光的散射和吸收，实现扩展体积的高分辨率成像。此外，扩展样品的水凝胶样性质使得在整个样品体积中均能对致密表位进行均匀的抗体标记。在扩展样品中获得的改进的标记和图像质量揭示了类器官中心的细节，这些细节以前只能在连续切片后才能观察到。与化学清除的球体相比，扩展样本的改善的信噪比大大改善了后续的图像分割和分析方法。