I used TEM equipped with tungsten gun to obtain attached image (several other too, like shown).
Side blotting technique followed by negative staining (1% PTA for 30 sec) was used. I cannot get the more clear background and there's also problem of resolution.
Please suggest how can I improve sample preparation? or any other suggestion to get better images.
The sample is polyelectrolyte complex in water.
In negative staining the resolution of the microscope is not a problem - any normal TEM is Ok, if it is aligned properly. The really important factors:
1. Thickness and stability of the support film;
2. Sample concentration and purification (critically important!);
3. Staining. Empirically, for different viruses, phages, VLP, and similar objects, the best results (from my personal experience) can be reached using 1% water solution of uranyl acetate (works like negative and positive stain simultaneously) with pretty fast (from 10 to 30 sec) staining without washing (just remove the droplet with filter paper).
As the first step, I'd recommend to purify and slightly (1:10) dilute the sample. Uranyl acetate can be useful too.
Good luck!
Article Ultrastructural Aspects of Photodynamic Inactivation of High...
In negative staining the resolution of the microscope is not a problem - any normal TEM is Ok, if it is aligned properly. The really important factors:
1. Thickness and stability of the support film;
2. Sample concentration and purification (critically important!);
3. Staining. Empirically, for different viruses, phages, VLP, and similar objects, the best results (from my personal experience) can be reached using 1% water solution of uranyl acetate (works like negative and positive stain simultaneously) with pretty fast (from 10 to 30 sec) staining without washing (just remove the droplet with filter paper).
As the first step, I'd recommend to purify and slightly (1:10) dilute the sample. Uranyl acetate can be useful too.
Good luck!
Article Ultrastructural Aspects of Photodynamic Inactivation of High...
Everything in terms of the preparation looks good to me; you just need to underfocus a bit more. You can clearly see areas with no stain; just blank support film. So, I think everything is good except your focus. Don't use the wobbler. Focus to what looks like the best contrast using your eyes.
Denis Korneev Thanks for the response,
My lab mates had tried Uranyl acetate for staining in the past, and they all recommended me not to use it. I don't exactly know the reason why? but probably due to some sort of interaction with the acetate. I'll give it a try.
Brent E Gowen Thanks for your response.
I am actually not authorized to operate TEM myself, but I'll convey your suggestion to the operator.
Is it ok if i'll quickly wash the grid after staining?, just may be few quick drops of water. Will it make my background more brighter, or on the contrary wipe out the salt of contrast?
Thanks again.
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