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Electron Microscopy (EM) Grid and Carbon film Preparation for Cryo-Electron Microscopy. Cryo-EM tutorial July25~29, 2005 Zongli Li. What I will talk about……. Some basic topics about EM grid Home-made grid preparation Grid cleaning
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What is EM grid?
What we need to do with EM grid before we can use it?
How to chose right grid-
some preliminary considerations
Home-made grid Vs. commercially available grid
*: Some pictures and materials are borrowed from web site
http://bilbo.bio.purdue.edu/~baker/documentation/sample_and_prep/b2.htm for this section
3) Clean a glass slide with water
5) Remove Formvar film from slide and add grids.
3) Clean a glass slide with water and detergent.
5) Preparing holey grid.
Ethylene dichloride, chloroform are carcinogenic, ethyl acetate and methanol are toxic. Care should be taken to avoid contact with these solvents or inhaling their vapors. Although acetone is less toxic, breathing moderate- to-high levels of acetone for short periods of time, can also cause nose, throat, lung, and eye irritation and other health problems.
Home-made holey grid (1)
Home-made holey grid (2)
In the final stage of the production process of QUANTIFOIL® holey film, carbon is evaporated onto a plastic holey film, and the plastic is dissolved. The grids are washed with chloroform, ethyl acetate and acetone. However, not all plastic can be removed. Therefore, the final thickness of the foil is about 20 nm, although only 10 nm of carbon is evaporated onto the plastic.
R1.2/1.3 quantifoil grid
Diagram for cleaning grids-
carbon film preparation
The evaporator has a small vacuum chamber and is very sensitive to oil contamination. To keep it oil-free the following rules are necessary:
Cleaning the Bell Jar:1) Carefully remove the bell jar by pulling it straight up and past the implosion shield. Be extremely careful when handling the bell jar because it is expensive. Remove both gaskets.2) Soak the bell jar with soapy water for a couple of minutes then gently rub it with paper towels. When the jar is clean, rinse it with deionized water and wipe it dry with Kimwipes.3) Apply a thin coating of Bell Bright to the inner surface of the jar. There should not be any liquid droplets on the surface. Let it dry for a couple of minutes and then replace the gaskets. Do not forget to use Bell Bright or the carbon will be nearly impossible to remove the next time it is cleaned.
To get holey carbon grids or grids with continuous carbon film.
There are different reasons for putting more carbon on holey carbon grid