I am preparing some samples (external abdominal walls of lepidopterans) to embed them in resin. There are basically two problems:
To address the first problem, I think that cutting the sample in a specific shape will help me to recognise which part is which. For example, a long rectangle with a sharpened end, or an right triangle.
The second problem seems a bit more difficult. I will be using an Eppendorf tube, about 1 cm high. If the sample is long enough, I will probably be able to pierce it with an entomological pin and leave it like that for a few hours (the long rectangular shape might be better for this). Also, I was thinking about pouring small amounts of resin each time, waiting a few hours for the resin to become a bit thicker before pouring the next amount and so on, thus avoiding sample drift.
I've just found an article that says I can cut the hardened resin, then re-embed the block in more resin in the desired position. Is it possible to do it with all types of resin? (They used JB-4)
Thank you
Dear Richardo,
In my opinion, the best way is to use some type of flat embedding mold. The polymerization of the material in the droplet of embedding resin on the polyethylene film and subsequent cutting and new embedding in the proper orientation is also possible. Every resin is suitable.
Best regards
Pavel Travnik
I agree with Pavel. You can do the first embedding between layers of Aclar plastic film, or even on the surface of a cover slip (dip in liq N2 after polymerization to separate the cured resin and specimen from the glass). Then trim, orient in the bottom of a flat embedding mold, pour in fresh resin. If you draw a picture of the trimmed specimen it is easier to get the orientation right later on when you section.
Thank you very much for your answers. I didn't know you could even embed a sample in a cover slip or on polyethylene sheets! This will help me a lot.
Thank you again.
I agree, the use of flat embedding molds is very helpful. You can then orient your sample in the mold (best under microscope) before the resin is polymerised. Furthermore, when sectioning your sample you should first start with semithin sections of larger areas of the specimen, mount the sections in microscopic glass, color the sections with toluidin blue, observe under light microscope and identify your area of interest, then prepare a smaller pyramid and do ultrathin sections. Semithin sectioning of specimens embedded in gelatine capsules might be useful as well.
The approaches suggested by other respondents are all valid. However, since all are based on flat embedding, the methods are tedious and as you state, after Os treatment you can’t even see tissue features to enable orientation. For this reason, and others, I created a new type of labware that works entirely differently. Warning – my answer is commercial since my company and several EM distributers now sell these labware products.
You can learn about this new way to prepare EM specimens (and grids) from our website www.MicroscopyInnovations.com. In particular, to see the new way specimens are oriented look at the mPrep/s Workstation video, accessed from the website or from YouTube: https://www.youtube.com/watch?v=ZG1d1Me70y0
The video shows several ways to orient tissue. For your tissue the “pinch method” will likely be best. With this method, dissect the tissue so that one end contains the area of interest. Ideally, the specimen should be ~2 to 9 mm long and ~75 um to ~2 mm thick. To orient the specimen, the back end of the tissue (not the region of interest) is clamped in an mPrep/s screen, using the Workstation to open the slit-clamp in the screen. The Workstation is designed for use with a dissecting microscope so you can see what you are doing. Once the tissue is clamped in place it remains oriented during the entire fixation and embedding process. It is then inserted into an mPrep/s capsule (micro-preparation of specimens) that suspends the area of interest in the capsule. The capsules then connect like pipette tips to deliver the reagents to the specimens within. The specimen is even embedded and sectioned inside the capsule.
Tissue can be inserted and removed from mPrep capsules, and oriented at any time before resin infiltration. Some users pre-fix with glut to toughen tissues prior to orienting, and/or stain the tissue to enable seeing the feature of interest during pinch orientation.
If you would like additional information, don’t hesitate to contact me directly. Good luck with your research!
www.MicroscopyInnovations.com
Hello Ricardo,
Many years ago in order to get specimens (megaspores) in the desired position for TEM
we used the following treatment:
We filled gelatine capsules with the resin and let it polymerize without a specimen.
Then with dental drills we centrally drilled a hole into the upper horizontal surface
of the polymerized block, a little bit larger than the specimen.
With a drop of resin the specimen was then oriented within that hole.
Finally, the remaining open space was filled with resin for polymerization.
In this way we got oriented specimens in the center of the finally prepared pyramid
for ultra-thin sectioning.
Finally, I would also try to study a specimen without osmium treatment.
Thank you, that was very ingenious. I will try to do something similar, your answers have been really useful
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