Scanning electron microscopy (SEM) protocols for problematic plant, oomycete, and fungal samples

Common problems in the processing of biological samples for observations with the scanning electron microscope (SEM) include cell collapse, treatment of samples from wet microenvironments and cell destruction. Using young floral tissues, oomycete cysts, and fungi spores (Agaricalesas examples, specific protocols to process delicate samples are described here that overcome some of the main challenges in sample treatment for image capture under the SEM. Floral meristems fixed with FAA (Formalin-Acetic-Alcohol) and processed with the Critical Point Dryer (CPD) did not display collapsed cellular walls or distorted organs. These results are crucial for the reconstruction of floral development. A similar CPD-based treatment of samples from wet microenvironments, such as the glutaraldehyde-fixed oomycete cysts, is optimal to test the differential growth of diagnostic characteristics (e.g., the cyst spines) on different types of substrates. Destruction of nurse cells attached to fungi spores was avoided after rehydration, dehydration, and the CPD treatment, an important step for further functional studies of these cells. The protocols detailed here represent low-cost and rapid alternatives for the acquisition of good-quality images to reconstruct growth processes and to study diagnostic characteristics.This project has received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement No. 634429. This publication reflects the views only of the author, and the European Commission cannot be held responsible for any use which may be made of the information contained therein. We also acknowledge the financial contribution made by the Real Jardín Botánico, CSIC. SR is grateful to the European Union [ITN-SAPRO-238550] for the support of her research in Saprolegnia. We also want to thank Francisco Calonge for kindly provide the Phellorinia herculanea images and B. Pueyo for processing samples (Figure 5). All images were taken by the SEM service at the Real Jardín Botánico-CSIC in Madrid