Thursday, 19 June 2014

Ready for the EM!!!

So after a long week of waiting for me TCV particles to disassemble and re-assemble, carefully changing their buffer every couple of hours making sure they were in the best environment and looking after them, now the time has finally come to see the what's been going on inside my samples.
To image my virus particles I have prepared copper grids that will be used on the Electron Microscope so I will hopefully get some pretty pictures for my virus.

To prepare the samples on the grid, first you have to activate the grids by using the glow discharger. This instrument I have found to be quite temperamental. Maybe it's my inexperience with working with it but it is taking me a while to get it to work. Whilst the instrument is activating the copper disks, it glows a lovely purple colour, which got me thinking, what does the glow discharger actually do, and how does it work? So I did some research and thought it might be interesting if I shared that with you.

The glow discharger works by creating a partial vacuum and when a high voltage is applied between the cathode and anode at each end of the chamber, the electron potential ionizes the gas within the chamber. These negatively charged ions then deposit on the carbon, giving the carbon film an overall hydrophobic surface. The carbon film is on top of the copper grid and is usually hydrophobic (water hating), which would repel your sample so the glow discharger is needed to make this film hydrophilic (water loving) and hence your sample sticks to the film.

After activating the grids and delicately pipetting the sample onto the grid, the samples are then stained with a heavy metal salt that readily absorbs electrons. This is needed as biological molecules contain mainly C, O, N and H atoms which are not very dense, and the amount of electrons they absorb is minimal compared to the intensity of the electron beam of the microscope. This staining process is called "negative staining". This is because you don't see the object itself, you see an area empty of stain surrounded by stain. The area without stain is the object of interest as the sample prevents the stain from depositing onto the carbon layer.

I thought all this was rather interesting, I think it is good to know why you are undertaking a method, rather than just doing it. So now I will be researching electron microscopy, ready for using the microscope tomorrow morning and then hopefully, if all goes to plan, I will have some lovely images of virus capsids to share!

No comments:

Post a Comment