Department of Pathology and Cancer Research Institute, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA.
Ludwig Center at Harvard Medical School, Boston, MA, USA.
Nat Protoc. 2020 May;15(5):1649-1672. doi: 10.1038/s41596-020-0300-1. Epub 2020 Apr 1.
In pathology, microscopy is an important tool for the analysis of human tissues, both for the scientific study of disease states and for diagnosis. However, the microscopes commonly used in pathology are limited in resolution by diffraction. Recently, we discovered that it was possible, through a chemical process, to isotropically expand preserved cells and tissues by 4-5× in linear dimension. We call this process expansion microscopy (ExM). ExM enables nanoscale resolution imaging on conventional microscopes. Here we describe protocols for the simple and effective physical expansion of a variety of human tissues and clinical specimens, including paraffin-embedded, fresh frozen and chemically stained human tissues. These protocols require only inexpensive, commercially available reagents and hardware commonly found in a routine pathology laboratory. Our protocols are written for researchers and pathologists experienced in conventional fluorescence microscopy. The conventional protocol, expansion pathology, can be completed in ~1 d with immunostained tissue sections and 2 d with unstained specimens. We also include a new, fast variant, rapid expansion pathology, that can be performed on <5-µm-thick tissue sections, taking <4 h with immunostained tissue sections and <8 h with unstained specimens.
在病理学中，显微镜是分析人体组织的重要工具，无论是用于疾病状态的科学研究还是用于诊断。然而，病理学中常用的显微镜受到衍射的分辨率限制。最近，我们发现通过化学过程，可以使保存的细胞和组织在尺寸上各向同性地膨胀 4-5 倍。我们称这个过程为扩展显微镜（ExM）。ExM 可以在传统显微镜上实现纳米级分辨率成像。在这里，我们描述了各种人体组织和临床标本的简单有效的物理扩展方案，包括石蜡包埋、新鲜冷冻和化学染色的人体组织。这些方案仅需要廉价的、市售的试剂和在常规病理实验室中常见的硬件。我们的方案是为有经验的常规荧光显微镜研究人员和病理学家编写的。传统方案（扩展病理学）可以在 1 天内完成免疫染色的组织切片，2 天内完成未染色的标本。我们还包括一种新的快速变体，快速扩展病理学，可在<5-µm 厚的组织切片上进行，免疫染色的组织切片<4 小时，未染色的标本<8 小时。
