Optimizing CLEM protocols for plants cells: GMA embedding and cryosections as alternatives for preservation of GFP fluorescence in Arabidopsis roots.

Abstract

Recently, a number of diverse correlative light and electron microscopy (CLEM) protocols have been developed for several model organisms. However, these CLEM methods have largely bypassed plant cell research, with most protocols having little application to plants. Using autophagosome identification as a biological background, we propose and compare two CLEM protocols that can be performed in most plant research laboratories, providing a good compromise that preserves fluorescent signals as well as ultrastructural features. These protocols are based on either the adaptation of a high pressure fixation/GMA acrylic resin embedding method, or on the Tokuyasu approach. Both protocols suitably preserved GFP fluorescence while allowing the observation of cell ultrastructure in plants. Finally, the advantages and disadvantages of these protocols are discussed in the context of multiscale imaging of plant cells.