利用高压冷冻和连续块面扫描电子显微镜观察植物细胞。

Imaging Plant Cells by High-Pressure Freezing and Serial block-face scanning electron microscopy.

机构信息

Advanced Bioimaging Laboratory, Donald Danforth Plant Science Center, St. Louis, MO, USA.

Department of Ophthalmology and Visual Sciences, University of Wisconsin-Madison, Madison, WI, USA.

出版信息

Methods Mol Biol. 2020;2177:69-81. doi: 10.1007/978-1-0716-0767-1_7.

DOI:10.1007/978-1-0716-0767-1_7

PMID:32632806

Abstract

This chapter describes methods to enhanced contrast of plant material processed by high-pressure freezing and freeze substitution for improved visualization by serial block-face scanning electron microscopy (SBEM). The contrast enhancing steps are based on a protocol involving the sequential incubation of samples in heavy metals and sodium thiocarbohydrazide (OTO staining). We also describe the pipeline for imaging plant tissues in a commercial SBEM system (Gatan 3View) and routines for the image analysis and three-dimensional reconstructions using open-source and commercial software packages.

摘要

本章介绍了用于提高高压冷冻和冷冻替代处理的植物材料对比度的方法，以改善基于连续块面扫描电子显微镜（SBEM）的可视化效果。对比度增强步骤基于一个包含样品在重金属和硫代乙酰胺（OTO 染色）中顺序孵育的方案。我们还描述了在商业 SBEM 系统（Gatan 3View）中成像植物组织的流程，以及使用开源和商业软件包进行图像分析和三维重建的例程。