High-Pressure Freezing and Freeze Substitution of In Vivo and In Vitro Cultured Plant Samples

Abstract

The progress of plant biology, as well as many other biological subdisciplines, has been based on the microscopical observation of tissues, cells, and subcellular elements. For conventional electron microscopy, the specimens have to be fixed and processed, before they can be imaged. The preservation of subcellular structures during different processing steps has been a major concern of electron microscopists. For decades, structural preservation has relied on the use of aldehyde-based chemical fixatives. They are relatively inexpensive, easy to use, and very efficient for the preservation of certain subcellular elements. However, they present important limitations, including the induction of damages and artifacts that may alter significantly the cellular ultrastructure. To overcome this, one of the best options is to process specimens by high-pressure freezing (HPF) followed by freeze substitution (FS) procedures. Here I describe combined HPF and FS methods currently used in our laboratory to process different plant cells and tissues, grown in vivo and in vitro, for ultrastructural analyses and in situ localization studies.