Naked Science Forum
Non Life Sciences => Technology => Topic started by: joe.r on 03/06/2008 01:01:30
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When performing TEM on tissue samples, our group prepares a thin (1-2 microns) platinum replica to examine (2 mm in diameter). In this process, after spraying carbon for structural support, we dissolve the tissue from the replica by inserting it into bleach. When we remove the replica from the bleach and place it in DI water for cleansing, the surface tension of the water fractures it. Our group would greatly appreciate any advice to add avoid this fracturing.
One idea we have is to use a mesh screening to add structural support.
Another idea is to slowing drain the bleach from the petree dish until the replica sits on a soft, structurally strong surface. Then, add a dissolvable medium (maybe a gel) to add structural support upon entering the DI water. Lastly, drain the DI water slowly.
Any thoughts are greatly appreciated.
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I am unlikely to have an answer for you but am interested in the process you are using. It is not clear to me how you get the platinum replica to start with. Could you explain step by step?
You could check that it is really the surface tension causing the platinum to fracture by adding a little detergent to the water; this should reduce the surface tension. If the replica still fractures it may be a different mechanism; is the water and the platinum at the same temperature for example?
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Great point. I appreciate your comment. I have to look into that.
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As for the step by step, I'll get back to you. In short, at an extremely low temperature the platinum is sprayed on top of the sample and then a small amount of carbon is sprayed. I will try to get the exact quantities for you and a better explanation.
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well first what do you mean by TEM? plz when doing any post avoid the short forms.
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Short forms like plz?
Anyway, since someone has replied I think his post is clear enough. My personal guess is the difference in surface tension between water and bleach. Swelling of whatever he's imaging is another possibility, but that would only make sense if the bleach hasn't had time to do its job.
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TEM = Transmission Electron Microscopy. Hence needing a thin sample.
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A quick explanation of the sample preparation from wikipedia.... The fresh tissue or cell suspension is frozen rapidly (cryofixed), then fractured by simply breaking or by using a microtome while maintained at liquid nitrogen temperature. The cold fractured surface (sometimes "etched" by increasing the temperature to about -100°C for several minutes to let some ice sublime) is then shadowed with evaporated platinum or gold at an average angle of 45° in a high vacuum evaporator. A second coat of carbon, evaporated perpendicular to the average surface plane is often performed to improve stability of the replica coating. The specimen is returned to room temperature and pressure, then the extremely fragile "pre-shadowed" metal replica of the fracture surface is released from the underlying biological material by careful chemical digestion with acids, hypochlorite solution or SDS detergent. The still-floating replica is thoroughly washed from residual chemicals, carefully fished up on EM grids, dried then viewed in the TEM.
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Can anyone think of a liquid to use in place of DI water that would have a lower surface tension?
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I said in my first reply, add a little detergent to the water. A drop or two of Fairy liquid will do.
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we used a surfacant, but no luck... thanks tho... we may have a swelling issue
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OK. Well at least that eliminates the surface tension as the cause of the fracturing. If the temperatures of the sample and the water are matched I can only think that there is some residual organic material that is absorbing the water and swelling up. The carbon and platinum should not be unduly affected by the water.