Methods used for scanning electron microscopy

There are a number of ways to prepare plant samples for SEM. Our plan was to use fresh (not fixed) material so there would be minimal change in the structure of the plant parts. This is challenging as each part of the plant must be very small – no bigger than 1 cm2 and the pieces must be completely dry. Since plants contain a lot of water, this can be challenging. Also, the plant pieces must be kept absolutely clean as even a speck of dust will be detected.

It ended up that the three methods were needed to get accurate pictures of the plant parts. The first method was air-drying over drierite in a sealed glass jar for 72 hours. The second method was air-drying by pulling a vacuum in a sealed container for 72 hours. The third method was freeze-drying the samples for 72 hours. To prepare the samples, a corner of the lab was cleaned, the preparer wore a lab coat, mask and gloves and no one else was in the room. Plant samples were cut on a clean cutting board using a sharp razor blade. Then samples were put in a new plastic petri dish and placed in one of the above containers/machines.

Once dried, the samples had to be sputter coated with an ultra-thin layer of an electrically conducting metal (like silver, gold etc.). When the sample is bombarded with electrons an image is produced. Below are pictures of bean plant parts on the stubs – before and after sputter-coating. Once coated the stubs are put into the SEM*for viewing.  

Fig 7. Pieces of red kidney bean plant before sputter coating. Credit: You (Joe) Zhou

Fig. 8. Pieces of red kidney bean plant after sputter coating. Credit: You (Joe) Zhou

*The University of Nebraska-Lincoln Nebraska Center for Biotechnology – Microscopy Center (https://biotech.unl.edu/microscopy) has many high magnification microscopes for plant, animal and human research. These include the Hitachi S4700 Field-Emission SEM (scanning electron microscope) which was used to capture these bean pictures.