Cryo-TEM: Biological sample preparation technique

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• By Jahnavi Zalki M

Transmission electron microscopy (TEM) is used to look into the details at nanoscale levels. Electrons generated for imaging purposes are allowed to pass through the sample for imaging purposes. Under these conditions, the sample is required to be dry. Therefore, it gets difficult to analyse soft materials and biological samples that are contained in the liquid. Cryo-TEM overcomes this drawback by freezing the samples to maintain their shape and size. Sample preparation is a very important part of electron microscopy. The sample preparation procedure for Cryo-TEM imaging is different and goes something like this. Epoxy resins are used to hold the sample. Soft materials that do not contain water are directly placed into the epoxy resin; whereas, the biological samples that do contain water are dehydrated in series to obtain a minimal amount of water required to image the sample. This involves the freezing of the sample that still has water in it. Freezing gives a better resolution. Biological samples are fixed with formaldehyde to arrest dehydration. Dehydration of biological samples can cause them to lose their size, shape and regular morphology. Heavy metals are used to stain the biological samples to develop a contrast for imaging. To remove the extra water from the sample, it is given an ethanol wash in series that involves treatment of the sample with 0%, 25%, 50%, 75% and 100% ethanol to remove the additional water. Once the water is removed, the sample is put into epoxy resin and the resin is hardened in the oven. Once the resin hardens, thin slices of resin are made using a microtome, which has a diamond knife to finely chop off thin sections of our sample along with the resin. These thin sections are taken on a TEM grid and are suspended in a drop of water. This drop of water containing the resin and sample is taken for plunge freezing that involves liquid ethane or propane, which gives ice form to the suspended drop of water. The ice obtained here is vitreous ice, which is amorphous and does not have any crystal structure. This makes it easy to visualise through the TEM. The obtained sample is now ready to be imaged through Cryo-TEM.

Keywords: Cryo-TEM, Transmission electron microscopy, Plunge freeze, Microtome, Epoxy resin, Vitreous ice

Image source:
Liquid helium cryogenic transmission electron microscope by EMSL, available under a Creative Commons Attribution Licence 2.0 at https://www.flickr.com/photos/emsl/15543986365/in/photolist-tXJAZw-t25aGj-tXJBr3-t25dwY-tFv4rS-2k1ra2U-dYBEXW-dgLBRo-Nicasg-pFDfP9-SrM74N-9Um8om-2jbfhi6-Nrtu1C-dgLPFx-dgXW4K-oJHoR5-EFDi9r-dgLEcy-dgLzg9-dgLMxv-pp5nT8-qxkgh6-pp5CCD-ppaqAJ-dZUKr7-nuJiPb-xg81sN-pp5pAr-pFDoKQ-oJLust-pp8MNz-pFDqxh-pFz1w2-pFyTBF-oJHpLG-pDtxJh-pp5tw2-ppaCNj-pFz4ip-pFyZAV-fbFDXu-dgLmfa-dgLwfY-9zWKzy-dgLGVk-dgLtty-dgLKqu-2g8mkmX-drmYyj

Citation:
Jahnavi Zalki M. Cryo-TEM: Biological sample preparation technique. The Torch. 2020. 1 (2).
Available from: https://www.styvalley.com/pub/magazines/torch/read/cryo-tem-biological-sample-preparation-technique/.

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