High-pressure freezing and low-temperature processing of plant tissue samples for electron microscopy

Use of electron tomography methods improves image resolution of transmission electron microscopy especially in the z-direction, enabling determination of complicated 3D structures of organelles and cytoskeleton arrays. The increase in resolution necessitates preservation of cellular structures close to the native states with minimum artifacts. High-pressure freezing (HPF) that immobilizes molecules in the cell instantaneously has been used to avoid damages caused by convention chemical fixation. Despite the advantages of HPF, cells could still be damaged during dissection prior to HPF. Therefore, it is critical to isolate cells/tissues of interest quickly and carefully. The samples frozen by HPF are often processed by freeze substitution (FS), and FS should be carried out under appropriate conditions. Here we describe dissection, HPF, and FS methods that we have utilized to prepare plant samples for electron tomography/immuno-electron microscopy.