SEM of Biological Samples without Coating
Utilizing Bulk Conductivity for Increased Versatility
- Fig. 1: Leaf of a stinging nettle (Urtica dioica), fresh hydrated, without metal coating. Overlay of SE and BSE signal. The inset shows compositional contrast of the mineralized hairs using the BSE detector.
- Fig. 2: Superhydrophobic leaf of Xanthosoma robustum. The fresh hydrated sample (a, b) shrinks rapidly in the SEM, whereas the glycerol-infiltrated sample (c) remains stable for hours.
- Fig. 3: Frozen Xanthosoma leaf samples without metal coating. (a) Charging occurs at -120°C due to insufficient conductivity, but disappears at -80°C (b). (c) Cryo-fracture imaged at -70°C.
- Fig. 4: HCl vapour-treated Pinus pollen.
Biological SEM samples usually require a metal coating to avoid charging . However, a good knowledge of the properties and behavior of the samples offers ways to utilize internal electrical conductivity of biological samples for Scanning Electron MIcroscopy (SEM) examination without the need for metal coating. This not only simplifies the preparation, viewing and imaging. It also opens up new ways to examine natural, uncoated surfaces, for example compositional contrast imaging or for the study of adhesion effects.
In conventional (high vacuum) SEMs, biological and other non-conductive samples need a conductive coating, usually gold, to obtain a good image quality. Alternatively, special techniques such as imaging with very low beam acceleration voltages or the use of low-vacuum (variable-pressure-) SEMs are used to image non-conductive specimens. Here we demonstrate the utilization of internal conductivity on the basis of several botanical samples in four different SEM techniques, using a conventional SEM.
1. Fresh, Hydrated Samples 
Using fresh hydrated plant samples is the easiest and fastest preparation method. Most plant leaves are covered with a cuticle with low water permeability. Many of them dry so slowly even under high vacuum that they can be examined for several minutes up to more than an hour, before they collapse and become non-conductive. As long as the samples contain moisture, they are sufficiently conductive and can be examined without metal coating (fig. 1).
2. Glycerol Substitution 
Samples, which dry out too rapidly (fig. 2 a, b), can be examined in the SEM after replacing the internal water by glycerol, which has a very low vapour pressure (fig. 2 c). This is particularly useful for leaves with hydrophobic surfaces; infiltration from the underside leaves the hydrophobic surface dry. Remarkably, the glycerol-infiltrated samples also have sufficient conductivity to avoid charging. (However, an additional metal coating is advantageous to obtain improved contrast of fine structures.)
3. Cryo-SEM 
This is the most reliable preparation technique to avoid drying artifacts. Electrical conductivity of frozen hydrated samples depends on temperature. Above ca -100°C charging disappears and the image quality increases dramatically, so that imaging without metal coating is successful (fig. 3 a, b). Even slow-freezing of the samples (with ice crystals growing in the tissue) can be tolerated, when only the surfaces have to be examined, since these are usually not affected by internal restructuring (fig. 3 c). Without the requirements for metal coating and shock freezing, the simplified cryo-SEM method can be executed with self-made, low-cost equipment.
4. Chemical Treatment of Dry Material
A simple vapour treatment induces protonic conductivity in various biological materials. The reaction of HCl vapour with dry tissue can generate sufficient conductivity to avoid charging. For seeds, pollen, or paper, 5 to 20 minutes of vapour treatment are sufficient (fig. 4). Samples with low permeability require longer reaction times. Safety precautions should be used to avoid insertion of HCl vapour into the SEM and for the handling of concentrated hydrochloric acid.
The four methods presented, all utilizing the internal electrical conductivity, simplify sample preparation. Omitting metal coating also improves the scope of material contrast imaging. Additionally, these techniques leave the sample surfaces completely unaltered since they do away with immersing the samples in liquid of any kind. They extend the range of quick and easy SEM preparation techniques for biological materials and are useful in a range of fields such as the study of superhydrophobic surfaces, biomineralization, contamination, plant pathogens etc.
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 Ensikat, H.J. et al.: Microscopy: Science, Technology, Applications and Education. Méndez-Vilas, A.; Alvarez, J. D., (Eds.); Vol. 1 (13); Formatex Research Center, Bajados, Spain, 248-255, (2010)
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Hans-Jürgen Ensikat (corresponding author via eail request button below)
Prof. Dr. Maximilian Weigend
University of Bonn
Nees Institute for Biodiversity of Plants