How to embed and cut Diatoms algae for transmission electron microscopy?

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Harry Kenn Tolentino Dela Rosa

Embedding and cutting diatoms for transmission electron microscopy (TEM) is a multi-step process that involves preserving the samples, embedding them in a resin, sectioning the embedded samples, and preparing the samples for TEM analysis. Here is a general outline of the procedure:

Sample preservation: Fresh diatom samples need to be fixed and preserved to maintain their structural integrity during the embedding and sectioning process. A common fixative for diatoms is 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.4.

Embedding: After fixation, the samples are embedded in a resin that will provide structural support and allow for sectioning. A commonly used resin for TEM embedding is epoxy resin, which can be polymerized by heating the samples at 60-70°C for several hours.

Sectioning: The embedded samples are sectioned using a microtome or ultramicrotome. The sections need to be thin enough to be electron transparent, typically around 50-70 nm.

Staining: The sections are typically stained with an electron dense stain, such as uranyl acetate, to enhance the contrast between different structures in the sample.

Mounting: The stained sections are mounted on a TEM grid and are ready for TEM analysis.

Note that this is a general outline of the procedure and some specific steps or modifications may be required depending on the sample and the TEM instrument being used. It is also important to carefully control the conditions and reagents used in each step to minimize artifacts and ensure the preservation of the sample's structures.

Francesco Boldrin

Many thanks Harry, I knew the procedure, They are 12 years that I am an electron microscopist. My question was concerning about some critical tips about these microalgae that because of their silica skeleton are difficult to cut as the diamond blade will be heavily damaged from the silica

Thanks

Patrick Kociolek

Hi Francesco: This was not my primary research area, but I did know folks who would expose the diatoms to HF to break down the glass cell wall before sectioning. And while the silica can degrade the diamond knife, we would get our diamond knife re-sharpened. Not cheap, but much cheaper than a new knife.

Thanks!