6:01 1085 degrees C. 😅 Not only it does not harm but it is suggested as it stabilizes the formvar residue. Most of the patterned support films are made using an initial formvar template and then are coated with C on top. In the past we used to even wash them with chloroform and heat them on hot plates, even at higher temperatures before use. Nowadays the quality is much better and we do not do that anymore. As a metallurgists who is shifted to biologic stuff, I highly recommend to shift to biological pipettes. A p20 Gilson and D10 tip is the best. Thanks for your great videos. I always forward them to my colleagues.
Thank you for the information about the pipettes, this was certainly something I was obviously not paying a lot of attention to compared to everything else. Also, I tend to avoid support films with formvar; I've had too many issues with instability under the electron beam (though the manufacturers claim over and over that formvar is fine and stable) and plus having to dip grids in chloroform to dissolve the formvar (if needed) can introduce a source of hydrocarbon contamination (though some plasma cleaning may sufficiently address this). Did you ever have hydrocarbon contamination issues when using chloroform to prep grids?
@@NicholasRudawski Hi..it would be great if you introduce us about great variety of TEM grids and their specific applications(I mean when to choose which grid).
@@NicholasRudawski That question brings back a lot of painful memories! Actually, we don’t do that anymore. A chloroform wash can be helpful, but it can also end in a mess. Let me explain the story a bit more. Patterned grids (like quantifoils) are made using a technique called soft lithography. It’s like creating a pattern on a wafer, then making a negative pattern using a soft silicone resin (e.g., Sylgard). This pattern is then used to stamp a polymer solution (like formvar) dissolved in a solvent (probably chloroform) onto a substrate like glass. Afterward, they cover the patterned film with another layer of carbon film on top to make it conductive and stable. For random holey carbon grids, the method is slightly different, but the idea is the same: first, make a patterned formvar film, and then deposit the carbon. However, I’m not sure how lacey grids are made, but I speculate that they’re produced in a similar way. It would be best to consult older EM books. Anyway, the carbon grids are supposed to be pre-washed and free of formvar or polymer backing film from the supplier. But in the past, we often received grids with formvar contamination. In some cases, a chloroform wash wasn’t enough or even made the situation worse. It was like trying to clean a huge mess with a wipe. Also, chloroform sometimes introduced its own contamination, which was really problematic. The contamination could come from mishandled tools or consumables, and in most cases, the source wasn’t clear. It could be from filter papers, mishandled parafilm, tweezers, pipettes, or even contamination in the chloroform bottle. So yes, I did experience contamination after using a chloroform wash. But nowadays, I see far fewer of these issues. Grids are usually clean from the supplier. In case I receive contaminated grids, I pass them on to other EM groups with less strict cleanliness standards. 😂 Finally, back to the original topic: heating the grids helps stabilize many of these microscopic contaminants. However, overdoing it may also make the carbon film fragile. It's also worth mentioning that in cryo-EM, we glow discharge all the grids, which effectively removes loose hydrocarbons. Although in glowdischaging cleanliness is not intended; It’s done to make the grids hydrophilic.
I think deionized water do have some dissolved solids in it...better to use distilled water Correct me if I'm wrong Also tell me ...what kind of twizzer is that?
Both de-ionization and distillation are processes to remove dissolved solids, so either will be a much better option compared to normal tap water. As far as one being "purer" than the other, I'm not entirely sure on that. I've never encountered a problem using our in house DI-water for sample prep, so there really isn't any reason for me to try distilled instead. That being said, it would be an interesting experiment to simply put a drop of DI (or distilled, or tap) water (no particles) on a grid, let it dry, and then image the grid to see what (if anything) still remains. Regarding the tweezers, these are actually correctly described as "self-closing" tweezers (rather than "self-locking"). Here is a link from Ted Pella: www.tedpella.com/twzr-sc_html/twzr-sc.aspx
Such a wonderful description! I picked up so many new ideas. Hope my samples will be better now. Thank you a lot!
Thank you, glad to hear you found my video helpful; do let me know how your samples turn out for you.
6:01 1085 degrees C. 😅
Not only it does not harm but it is suggested as it stabilizes the formvar residue. Most of the patterned support films are made using an initial formvar template and then are coated with C on top. In the past we used to even wash them with chloroform and heat them on hot plates, even at higher temperatures before use. Nowadays the quality is much better and we do not do that anymore.
As a metallurgists who is shifted to biologic stuff, I highly recommend to shift to biological pipettes. A p20 Gilson and D10 tip is the best.
Thanks for your great videos. I always forward them to my colleagues.
Thank you for the information about the pipettes, this was certainly something I was obviously not paying a lot of attention to compared to everything else. Also, I tend to avoid support films with formvar; I've had too many issues with instability under the electron beam (though the manufacturers claim over and over that formvar is fine and stable) and plus having to dip grids in chloroform to dissolve the formvar (if needed) can introduce a source of hydrocarbon contamination (though some plasma cleaning may sufficiently address this). Did you ever have hydrocarbon contamination issues when using chloroform to prep grids?
@@NicholasRudawski Hi..it would be great if you introduce us about great variety of TEM grids and their specific applications(I mean when to choose which grid).
@@NicholasRudawski
That question brings back a lot of painful memories! Actually, we don’t do that anymore. A chloroform wash can be helpful, but it can also end in a mess. Let me explain the story a bit more.
Patterned grids (like quantifoils) are made using a technique called soft lithography. It’s like creating a pattern on a wafer, then making a negative pattern using a soft silicone resin (e.g., Sylgard). This pattern is then used to stamp a polymer solution (like formvar) dissolved in a solvent (probably chloroform) onto a substrate like glass. Afterward, they cover the patterned film with another layer of carbon film on top to make it conductive and stable.
For random holey carbon grids, the method is slightly different, but the idea is the same: first, make a patterned formvar film, and then deposit the carbon. However, I’m not sure how lacey grids are made, but I speculate that they’re produced in a similar way. It would be best to consult older EM books.
Anyway, the carbon grids are supposed to be pre-washed and free of formvar or polymer backing film from the supplier. But in the past, we often received grids with formvar contamination. In some cases, a chloroform wash wasn’t enough or even made the situation worse. It was like trying to clean a huge mess with a wipe. Also, chloroform sometimes introduced its own contamination, which was really problematic. The contamination could come from mishandled tools or consumables, and in most cases, the source wasn’t clear. It could be from filter papers, mishandled parafilm, tweezers, pipettes, or even contamination in the chloroform bottle. So yes, I did experience contamination after using a chloroform wash.
But nowadays, I see far fewer of these issues. Grids are usually clean from the supplier. In case I receive contaminated grids, I pass them on to other EM groups with less strict cleanliness standards. 😂
Finally, back to the original topic: heating the grids helps stabilize many of these microscopic contaminants. However, overdoing it may also make the carbon film fragile. It's also worth mentioning that in cryo-EM, we glow discharge all the grids, which effectively removes loose hydrocarbons. Although in glowdischaging cleanliness is not intended; It’s done to make the grids hydrophilic.
Nice video!
Thank you for your support; I hope you found the video useful.
I think deionized water do have some dissolved solids in it...better to use distilled water
Correct me if I'm wrong
Also tell me ...what kind of twizzer is that?
Both de-ionization and distillation are processes to remove dissolved solids, so either will be a much better option compared to normal tap water. As far as one being "purer" than the other, I'm not entirely sure on that. I've never encountered a problem using our in house DI-water for sample prep, so there really isn't any reason for me to try distilled instead. That being said, it would be an interesting experiment to simply put a drop of DI (or distilled, or tap) water (no particles) on a grid, let it dry, and then image the grid to see what (if anything) still remains. Regarding the tweezers, these are actually correctly described as "self-closing" tweezers (rather than "self-locking"). Here is a link from Ted Pella: www.tedpella.com/twzr-sc_html/twzr-sc.aspx