I´d like to ask, what can be an origin of the dark (electron-dense) objects (whole first picture) and in the second picture (chloroplast), it is present in the very left side, right side and above chloroplast. These dark objects are appearing during contrasting the sections (uranylacetate and lead citrate). Very often it covers all structures in the cells/tissueas and it is impossible to obtain good photos. We observed also non-contrasted samples and in this case there are no dark objects. I´ve tried also different concentrations od lead citrate (0.2 - 2 %; pH 12-13), but the objects are still present. I use 2% uranylacetate (pH cca 4) (contrasting for 35 min.), then 6x washing by MiliQ water (room temperature, afted boiling, to avoid CO2) and then contrasting by lead citrate for 2.5 min. We are sure these disturbing objects are appearing during contrasting process, but we don´t know how to avoid them. Thanks.
I am not familiar with your specimens, so just generic advice.
Bottom picture looks like one from not sufficiently washed section. More water needed?
First picture - precipitation, but too big crystals, never seen like this before. You can try washing with hot (very hot, almost boiling) water after lead citrate. Some sections can be damaged by hot water, but in most cases it produce good results.
OK... By elimination the other reasons I found out, the origin of these crystals comes from carbon film. We use copper grids covered by carbon film. Pure grids, without contasting process are nicely clear, but after the interaction with uranyl acetate or lead citrate, the crystals are rising up. Thus, the problem originates from the interaction between carbon film and contrasting agent. Now the question is, how to avoid that? Because if I won´t find any solution, I´d need to cut the new samples...
Yes, sir... My colleague recommended the grids with carbon film because of better adhesive properties...
I believe you do not need grids with film. Use standard grids, they are perfect for 95% of embedded specimens. Unneeded use of grids with carbon film decreases contrast, leads to additional artifacts.
Thank you for the recommendation. As I can see, there´s no other possibility, than preparation the new specimens onto pure copper grids. Anyway, thank you!
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