Transmission electron microscopy (TEM) to study histology of pollen and pollen tubes

Abstract

Here, we describe methods of transmission electron microscopy (TEM) based on conventional chemical fixation and high-pressure freezing (HPF) and freeze-substitution (FS) to examine the ultrastructure of Arabidopsis pollen grains and pollen tubes. Compared to other plant samples, such as root or leaf, pollen grains have thick pollen coat and cell wall which limit the permeation of fixative. Thus, it is difficult to obtain high-quality ultrastructural images of pollen. Moreover, pollen tube is very soft and the traditional procedure is too harsh to get an intact pollen tube sample. Up to now, there is no available mature protocol for TEM sample preparation of Arabidopsis pollen tube. Here, we describe the details and step-by-step procedures of chemical fixation, HPF, FS, and resin-embedding protocols for Arabidopsis pollen and pollen tube. In addition, we also provide a method on how to get longitudinal sections of pollen tubes.