Department of Medicine Huddinge, Center for Infectious Medicine, ANA Futura, Karolinska Institutet, Karolinska University Hospital Huddinge, Stockholm, Sweden.
Methods Mol Biol. 2020;2164:55-63. doi: 10.1007/978-1-0716-0704-6_7.
Histology, or the study of tissue microanatomy, is essential to understanding the in situ function of varying cell types within an organ. How cells are distributed throughout organs provides an indication of how they interact with other cells and structures within the organ microanatomy. The tortuous shape and large size of liver macrophages limits the value of standard tissue thickness of 5-10 μm. As a result, imaging of specimens ideally thicker than 100 μm is necessary to investigate the liver microanatomy and the how macrophages are distributed within this. Modern methods of microscopy, such as confocal and light sheet microscopy, allow for the analysis of tissue specimens of a thickness well beyond 100 μm in the z-dimension. Liver tissue is an opaque tissue, and as a result, different techniques are needed to ameliorate light diffraction within the tissue. These techniques, in conjunction with antibody staining and refractive index matching of the tissue, have allowed researchers to image liver tissue specimens of more than 100 μm thickness. Two of these techniques are modified versions of the clearing methods known as clearing-enhanced 3D (Ce3D) and fructose, urea, and glycerol for imaging (FUnGI). Here, we discuss the steps involved in preparing tissue specimens for optically clearing tissue using Ce3D and FUnGI for subsequent analysis of the distribution of macrophages in three dimensions using a confocal microscope.
组织学，即组织微观解剖学的研究，对于理解器官内不同细胞类型的原位功能至关重要。细胞在器官中的分布方式表明它们如何与器官微观解剖结构中的其他细胞和结构相互作用。肝巨噬细胞的曲折形状和较大尺寸限制了标准组织厚度为 5-10μm 的应用价值。因此，为了研究肝微观解剖结构以及巨噬细胞在其中的分布情况，最好对厚度超过 100μm 的标本进行成像。现代显微镜方法，如共聚焦和光片显微镜，允许在 z 方向上分析厚度超过 100μm 的组织标本。肝组织是一种不透明的组织，因此需要不同的技术来改善组织内的光衍射。这些技术与抗体染色和组织的折射率匹配相结合，使研究人员能够对超过 100μm 厚的肝组织标本进行成像。其中两种技术是称为增强型 3D 清除（Ce3D）和果糖、尿素和甘油成像（FUnGI）的清除方法的改良版本。在这里，我们讨论了使用 Ce3D 和 FUnGI 为光学清除组织准备组织标本的步骤，以便随后使用共聚焦显微镜分析巨噬细胞在三维空间中的分布。
