Department of Biomolecular Imaging, Institute of Biomembranes, Utrecht University, Padualaan 8, Utrecht 3584 CH, The Netherlands.
Traffic. 2012 Jul;13(7):926-33. doi: 10.1111/j.1600-0854.2012.01363.x. Epub 2012 Apr 29.
Electron tomography (ET) is an indispensable high-resolution tool for three dimensional (3D) imaging in cell biology. When applied to immuno-labeled cells, ET can provide essential insights in both the cellular architecture and the dynamics. Current protocols for 3D immuno-labeling of intracellular antigens include permeabilization steps that cause random, extensive cell membrane disruption. This permeabilization results in a poor cell ultrastructure, limiting the usefulness of the specimens for high-resolution studies. Here we describe a novel method, based on a well-controlled permeabilization by targeted laser cell perforation, that allows for the 3D immuno-localization of cytoplasmic antigens in cultured cells. The approach is unique since it is applicable to both chemically and cryo-fixed cells and leads to a superior ultrastructural preservation for electron microscopy and tomography.
电子断层扫描（ET）是细胞生物学中三维（3D）成像不可或缺的高分辨率工具。当应用于免疫标记的细胞时，ET 可以提供关于细胞结构和动力学的重要见解。目前用于细胞内抗原的 3D 免疫标记的方案包括通透化步骤，该步骤会导致随机的、广泛的细胞膜破坏。这种通透化导致细胞超微结构较差，限制了标本用于高分辨率研究的用途。在这里，我们描述了一种新的方法，基于靶向激光细胞穿孔的良好控制的通透化，该方法允许在培养细胞中进行细胞质抗原的 3D 免疫定位。该方法是独特的，因为它适用于化学固定和冷冻固定的细胞，并导致电子显微镜和断层扫描的超微结构保存更好。
