Protocol

 

Author

Steven Jansen, Brendan Choat

Overview

This protocol provides basic instructions for the preparation of biological samples for scanning electron microscopy (SEM).

Background

Scanning electron microscopy can produce high resolution three dimensional representation biological samples. The SEM is of great use in examining micromorphological surface features on the leaf, shoot and roots. Samples can be whole mounts or sections through tissue and are usually coated with a very thin coating of metal (gold, gold/palladium alloy, platinum, or carbon). Normally, samples are observed under high vacuum and as such, must be dehydrated before observation. This can lead to the possibility of artefacts developing during preparation, which is a constant cause for concern when the dimensions of an object are important for analysis. To overcome this, samples with delicate structures (e.g., non-lignified cell walls) may be dehydrated using a critical point dryer, which removes the liquid by a process using high pressure liquid carbon dioxide. The liquid CO₂ is heated until its pressure goes beyond the critical point. Alternatively, hydrated (fresh) samples can be observed using an environmental SEM (ESEM), although this technique is much more limited in resolving power. Sample may also be examined while frozen using a cold stage (cryoSEM), a technique which is of great value in observing hydrated biological specimens at high resolution. For more on tissue preparation for cryoSEM, see Cryo-SEM and quantitative cryo-analytical SEM.

Materials/Equipment

• Gloves
• Lab coat
• Ethanol
• Drying oven
• Dissection tools
• Sputter coater

Units, terms, definitions

Scanning electron microscopy (SEM)

Procedure

1. Dissect fixed material that has been stored in 70% ethanol or use fresh material. Make the samples as small as possible. Small samples will dry faster and vacuum pulling (for coating and/or SEM) will be easier than with large ones.
2. Transfer through an ethanol series: 50% (when using fresh material) – 70% – 90% – 100% – 100%. Approximately 1 hour in each solution.
3. Dry material at room temperature or in a warm oven. If material is liable to curve (e.g. leaves), keep it pressed between two clean microscope slides while it is drying.
4. For very delicate material use critical point drier in a well-ventilated room.
5. Mount material onto stubs using double sided sellotape or electron conductive carbon cement. Carbon cement or nail polish is especially helpful for larger samples in which the metal coating does not provide sufficient conductive pathway from the top of the sample.
6. Coat material with gold and/or platinum (Pt) using a sputter coater. The best coating technique should aim to obtain a thin and continuous coating as possible to avoid obstructing details, while giving the specimen stability and avoiding charging. Note that the thickness of the coating layer is about 21 nm after sputter coating for 3 min. using argon at 1×10^-1 mbar with a deposition current of 20 mA and a target to sample distance of 45 mm. Pt is generally considered to be the best choice, because the particle size of Pt is small and the secondary emission efficiency of Pt is very high. The grain size of gold is about 2 nm, and the grain size of gold/palladium is even smaller (in the order of 1.7 nm). Although a smaller grain size may be more ideal, there may not be a recognisable gain in high resolution when using gold/palladium.