Scanning electron microscopy is being used by the SSRC to determine species of dinoflagellates in the Bellingham Bay and Nooksack River.
Images below were taken at Western Washington University (WWU) with the assistance of Dr. Michael Kraft. Due to COVID-19 restrictions, all sessions are virtual. Thankfully, we can run the instrument from the SSRC. Although, I do miss touching the instrument.
These images were taken with a Tescan VEGA3 microscope at 10.0 kV.
The above SEM image is of Alexadrium fundyense cells that have been fixed using 2% glutaraldehyde and 1% osmium tetroxide (OsO4), and freeze dried for 5 minutes on glass slides.
The above SEM image is of a Prorocentrum cell cultured in L1 broth and collected from the Bellingham Bay Buoy (Se'lhaem) in October 2020. The sample has been fixed using Lugol's, 2% glutaraldehyde and 100% hexamethyldisilazane (HMDS), and airdried for 5 minutes on glass slides.
The use of OsO4 is very toxic and hard to handle. Ideally, we would use a Critical Point Drying (CPD) apparatus or machine to achieve the temperature/pressure combination to completely dehydrate a small specimen for SEM work. However, due to COVID-19 restrictions, we are unable to access this kind of equipment at WWU. HMDS (hexamethyldisilazane) may be used to replace the CPD step in sample preparation of cells with "armor" (for instance the Alexandrium are “armored dinoflagellates” meaning it has thecal plates made of cellulose surrounding the cell like armor) for SEM imaging. All HMDS steps need to be carried out in the fume hood wearing the necessary personal protection gear as it is also highly toxic.
I hope to acquire more quality images with better preservation in the future. For instance, I would have liked to have seen flagella in the image of the Prorocentrum cell. In future experiments, I would like to try filtering the environmental samples, instead of using a centrifuge. I think Lugol's, 2% glutaraldehyde and 100% hexamethyldisilazane (HMDS) steps can easily be done with a filter tower in the fume hood. I cannot imagine a safe way to handle the OsO4 in the fume hood and with a filter tower, at this time.