Here is one picture of dark spots. I prepared 1.5% formvar in chloroform and diluted it into 0.5%. After o/n incubation I performed carbon-coating.
Could it be the result of insuficient drying or there is another problem with formvar solution?
Thank you for help.
Lenka
Did you cast your films on water? My issues with dirty formvar always came down to dirty water, either when casting them or in the rinse water during staining.
John-Erich Haight , I cast the film into fresh dH2O and didnt perform any rising. I will try new beaker and water but dont think it results from this. I also experienced water dirt but it looked differently.
My experience in casting films was the everything had to be absolutely clean. I acid cleaned the glassware and slides, and used double distilled water. Maybe try a different water source and a new box of slides.
Dear Lenka Koptašiková , unfortunately the image you enclosed is low power mag ( and - unfortunately - out of focus ). So it is impossible to infer from the "morphology" of "precipitates?/dark spots that they are either impurities of "your" dH2O / water (it would be interesting which source your dH2O is and if you could try the same again with ddH2O or even Ultrapure water, if available), or dust, or eventually 'carbon sparks' from uneven carbon coating..... Except for the 'holes' in seen in the image of one Cu-Grid mesh at least the thickness of your formvar film seems to be homogenous over the whole area.... congrats....(:-))
PS:
I second John-Erich Haight 's opinion on scrupulously clean(ed) glassware and tools used in the process of making the film. This includes also tough / thorough cleaning of the grids, especially if you apply the film to grids taken out from the 'suppliers box' (grids always need to be cleaned in an - at least - two step cleaning process, including defatting / degreasing and getting rid of any remnants of organic / inorganic material from their production and 'shielding' for storage.... most recommended 1-2x acetone, ethanol or mixture of both (3-5 mins) , + 3 washings in petroleum benzine, boiling range: 40-60 °C, or even -140-180°C)using an ultrasonic ) Other sources could be the formvar-powder itself (deteriorated by - evt.lly also long-term - storage in unfavourable conditions (deterioration, kind of molding due to air moisture, etc.).
Best wishes and regards, W.H.M.
I've seen this before and it had nothing to do with spotlessly clean glassware or the water used in the process.
Formvar takes a couple of days to fully dissolve in chloroform. It may look dissolved soon after mixing, but small crystals remain undissolved for a couple of days. If you make films with this, the result is these dark spots in the film = undissolved formvar crystals.
When I make formvar, I mix it and then let it sit for a week (in a brown glass bottle in a dark chemical cabinet) before making films.
Wolfgang H. Muss and Brent E Gowen , thank you for your responses, I will try to implement your advices. Especially the water purity and also I will try longer formvar dissolving.
Best,
Lenka
Dear Lenka,
I have seen a pretty similar effect, but the spots were dozen times smaller - metal (electron density) particles about 10-20 nm. Surprisingly, it was an artifact of carbon coating (old manual system, not well-sharpened electrodes, overheating of the holder), was fixed with good electrode sharpening and cleaning of holder - the evaporator had been used for Ni and Au coating too and there was a thin metal layer on the heated part of the electrodes holder. Try to visualize a grid without carbon coating.
Why did you use a SEM for a TEM grid visualization? :)
Did you make the formvar film with a glass slide? The best formvar films which I have ever seen were made with a droplet of 0.2% formvar in dichloroethane on the water surface as described in this forgotten article:
Article Ultrathin Formvar Support Films for Transmission Electron Microscopy
Good luck!
Denis
Dear Denis,
In our facility we have just SEM so far, so for TEM I need to commute. I needed to check the grids quickly and this was the only way. I am not using SEM that often...
We have quite new high-vaccuum coater so I think it is not in carbon coating (?).
As a method I cast cover glass with formvar (in chloroform) into the water.
Thank you for the article recommendation!
Best,
Lenka
Dear all,
I finally got clean formvar grids. It is hard to say where exactly the problem was. I cleaned all glasses properly, used ddH2O, prepared new 0.5% formvar in chloroform.
I found out that our carbon coating is making us some troubles.
You can find attached SEM pictures of TEM grids with formvar only or sections without formvar with carbon coating.
Best,
Lenka
Dear Lenka Koptašiková , your informative reply greatly is appreciated!
Best regards, WHM.
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