Monday, 23 June 2014

The first battle with the EM microscope....

Today I am heading into my third week working in the Stockley lab at the University of Leeds and I can't believe how fast the time is going! 8 weeks will have flown by in no time!!

Last Friday, I was introduced to the electron microscope. Let me tell you, using that thing is pretty hard and it takes a lot of skill and patience, which I'm finding is something that is needed in science as there is a lot of waiting for things to take their course or run. Taking this into account, I think that my first attempt at obtaining images of my TCV sample was quite successful.
I had trouble getting the images in focus and so they aren't of the best resolution. We think this is mainly because of my sample preparation technique, so I have prepared more samples this morning in order to test different staining methods to see which works best for me as each method I have read seems to use a different method.

These are a few of the images I took with the electron microscope on my first trial, although they are not of great resolution, they do confirm that my experiments these last two weeks have been successful, which is great news!!

These first two images are of my TCV sample that I purified and concentrated in my first week. The dark marks are of the expected size of around 30 nm and the edge of the virus VLP can almost be seen.

This image is from the sample of dissociated virus. There are no clear structures seen in the image; this is excepted as the TCV VLP's have come apart and are now just capsid proteins and RNA. In the left corner of the image there is a area of high concentration of salt, this may be because of the buffer the virus was dissociated in as it was high in salt and contained Tris, which isn't always ideal for imaging with the electron microscope.

These last two images that I took were from the same copper grid, but from different areas of the grid. They are both from the sample of re-assembled virus. The top image shows that the re-assembly of the TCV VLP's was a success as small circular shapes have reformed, although they are not as uniform as the previous sample of TCV VLP's but this was expected. The second image looks a little different, the shapes seen are not as circular and are most likely to be the salt stable complex that has not re-assembled with the RNA to form VLP's.

As mentioned before the images are the best quality and the contrast between the background and the sample does not allow for a lot of detail to be seen of the virus structures. Hopefully by investigating different staining techniques I will be able to obtain a better quality image. Stay tuned for the results.....

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