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

Summary

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.

Abstract

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.

Introduction

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,

Protocol

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. Ideally, immerse the material after 48 h in FAA (steps 1.1.1-1.1.3) to avoid excessive dehydration in the ethanol. If a fume cupboard is accessible to the ....

Results

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.......

Discussion

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, .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Acetic acid	No specific supplier		Skin irritation, eye irritation
aluminium stubs	Ted Pella, Inc.	16221	www.tedpella.com
Centrifuge tubes	No specific supplier
Critical Point Dryer	Polaron Quatum Technologies	CPD7501
D-(+)-Glucose	Merck	1,083,421,000
Double sided sellotape	No specific supplier
Ethanol absolute	No specific supplier		Flammable
European bacteriological agar	Conda	1800.00	www.condalab.com
Filter paper	No specific supplier
Forceps	No specific supplier
Formalin 4%	No specific supplier		Harmful, acute toxicity, skin sensitisation, carcinogenicity. Flammable
Glass cover slips	No specific supplier
Glass hermetic container	No specific supplier
Glutaraldehyde 25% DC 253857.1611  (L)	Dismadel S.L.	3336	www.dismadel.com
Mycological peptone	Conda	1922.00	www.condalab.com
needles	No specific supplier
Petri dishes	No specific supplier
Plastic containers	No specific supplier
Sample holder with lid  for the critical point dryer	Ted Pella, Inc.	4591	www.tedpella.com
scalpels	No specific supplier
Scanning Electron Microscope	Hitachi	S3000N
Software for SEM
Solution A: NaH2PO4
Solution B: Na2HPO4
Specimen holders	No specific supplier
Sputter coater	Balzers	SCD 004
Stereomicroscope	No specific supplier
Transmission Electron Microscope (TEM) grids	Electron Microscopy Sciences	G200 (Square Mesh)	www.emsdiassum.com
Tweezers	No specific supplier