High-Pressure Freezing and Freeze Substitution for Transmission Electron Microscopy Imaging and Immunogold-Labeling.

Electron microscopy enables the unbiased imaging of organelles and cellular structures at nano-meter scale resolution. The combination of cryofixation/freeze-substitution methods with other imaging techniques such as correlative light and electron microscopy (CLEM), electron tomography (ET), and immunogold-labeling provides unique opportunities to understand structural changes associated with cellular processes. This chapter presents the main steps in the preparation of Arabidopsis thaliana roots, cotyledons, anthers, and developing seeds by high-pressure freezing and freeze-substitution for structural analysis and immunogold-labeling using transmission electron microscopy.