Scanning Electron Microscopy (SEM) Protocols for Problematic Plant, Oomycete, and Fungal Samples

Problems in the processing of biological samples for scanning electron microscopy observation include cell collapse, treatment of samples from wet microenvironments and cell destruction. Low-cost and relatively rapid protocols suited for preparing challenging samples such as floral meristems, oomycete cysts, and fungi (Agaricales) are compiled and detailed here.

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 (Agaricales) as 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.

In biology, the use of scanning electron microscopy (SEM) has been extended to studies of structural evolution, comparative morphology, organ development, and characterization of populations or species¹. With its two-dimensional view of microscopic structures, areas such as micromorphology and systematics profited from SEM technique advances since the second half of the 20^th century. For example, the introduction of the sputter coating methodology in the 1970s made possible observations of delicate materials such as shoot apices and flowers enhancing the imaging of non-conductive tissues^2,

NOTE: This protocol includes six main sections, three devoted to specific organisms (sections 1-3), and three describing the procedures common to all (4-6). Asterisks (*) indicate steps modified by the experimenters.

1. Studies of Developing and Fully Formed Plant Structures

1. Collection and fixation
   1. If the plant material is collected in a place with no access to a fume cupboard, introduce and immerse the material in 70% ethanol in centrifuge tubes. Ideal.......

Floral Development and Fixation of Developing and Fully Formed Plant Structures

Using the FAA-CPD protocol described here, young and mature plant tissues are optimally fixed and dehydrated for SEM imaging. Processes such as floral development can be reconstructed because the topography and shape of the buds is not distorted by cell shrinking (Figures 1b, 1d, 4a-f). Structures with complex shapes can be successfull.......

With respect to standard SEM protocols, the procedures presented here include relatively rapid, easy to follow, and low-cost methodologies. Depending on the amount of samples and on the ease of processing, it takes four to five days to acquire good quality images. Including adequate safety precautions for the CPD and SEM operation, the procedures are easy to handle. Particular caution should be taken with formalin and the glutaraldehyde (see steps 1.1.1 to 1.1.3 and 2.1.5 of the protocol). There are certain steps where, .......

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 (Fig....