Tips and tricks of sample preparation for electron microscopy: illustration by correlative microscopies on plant cells
Claire Boulogne2ѱ Ϯ, Cynthia Gillet2ѱ, Jessica Marionѱ 1, Romain Le Bars1, Béatrice Satiat-Jeunemaitre1.
1 Laboratory Dynamics of CellCompartmentation, Institut de Biologie Intégrative de la Cellule, Université Paris-Saclay, UMR 9198, 91198 Gif sur Yvette France
2 CellBiology pôle Imagerie-Gif, Institut de Biologie Intégrative de la Cellule, Université Paris-Saclay, UMR 9198, 91198 Gif sur Yvette France
ѱ. These authors have equally contributed
Key words: plant cells, electron microscopy, CLEM, EDSX, sample preparation.
Sample preparation is often the bottleneck for the observation of biological specimens by electron microscopy (EM). Transformation of the living cells into fixed and embedding material need a special careto avoid artefacts and preserve cell ultrastructure as close as possible of its native state. The preparation process becomes even more complex when the ultrastructure preservation must be coupled with the preservation of the chemical properties of the sample. This is a daily challenge for scientists working on correlation between EM and other techniques such as chemical spectroscopy or fluorescence microscopy. This talk will deal with 2 examples of advanced EM on plant cells to illustrate the identification of methodological locks and propose protocols to help solving the problems: correlative light and electron microscopy (CLEM) and energy dispersive x-ray spectroscopy (EDXS). Both techniques required a particular attention in the choice of solvents and resin to dehydrate and embed the sample.
CLEM has been performed on root tips of Arabidopsis expressing GFP fused with a well-known autophagy marker, ATG8 (the mammals LC3 homolog in plants). We applied 2 protocols which both succeed to preserve the GFP fluorescence on EM-sections, allowing the recognition of autophagosomes with a fluorescence microscope prior to visualise them at the ultrastructural level with a transmission electron microscope. Advantages of both protocols will be discussed.
EDXS allows the collection of a chemical cartography at the elemental level. This technique is complementary of EELS (energy electron loss spectroscopy) analysis, and can be applied to biological samples if specific precautions are respected. These specific precautions will be discussed and an example of metal cartography in plant tissues will be described.
Le Bars et al., « ATG5 defines a phagophore domain connected to the endoplasmic reticulum during autophagosome formation in plants ».
Mary et al., « Bypassing Iron Storage in Endodermal Vacuoles Rescues the Iron Mobilization Defect in the Natural Resistance Associated-Macrophage protein3natural Resistance Associated-Macrophage protein4 Double Mutant ».