Hi Rob,

I'm in the US and purchase acetone and other EM grade chemicals for freeze substitution from EMS

https://www.emsdiasum.com/microscopy/products/chemicals/acetate.aspx

In the Netherlands their products are distributed through Aurion, see

https://aurion.nl/products/products-of-ems/

Good luck!

Martin

Hi Rob,

Sorry for the idiotic question (HPF/FS is pretty new area for me), but how important is the solvent purity?

For c.elegans worms (AFS from -90, acetone, 0.1% UA) we have not seen any differences between technical (just from a wash bottle) acetone and analytical grade from fresh opened bottle. In addition, some papers suggest to add a few per cent of water that can slightly increase membrane staining. In addition, if AFS is running in open vessels like Leica "revolvers" it absorbs some water from the atmosphere.

Did you compere solvents with different degree of purity?

Cheers,

Denis

@Martin; Thanks for the suggestion, I hadn't considered EMS yet. Though on their site they do not state the percentage of residual water in the glass distilled acetone. I'll check with them if they can send me more data on that.

@Dennis; in my experience solvent quality has quite some effect on the freeze substitution. This is mostly visible if you use osmium in the substitution mix (which tends to react with several of the trace compounds found in technical grade solvents).

The water content also has an effect in the solubility of uranyl acetate in the acetone (unless you work from a 20% UA in methanol stock, though this will offset the % of water you need to enhance membrane contrast since methanol can contain more water at -90℃ than acetone). If the acetone contains more than 0.5 water the UA will precipitate once you cool the solvent down to -90℃.

During FS the uptake of water from the atmosphere can be limited even with open containers if you properly set the TF (nitogen gas flow). On both the AFS 1 and 2 you can use this to fill up the chamber with dry nitrogen gas before you fully close the lid.

Cheers,

Rob

Rob, water content in the glass distilled and reagent grade acetone is stated as 0.2% on the label.

thanks for the info Martin!,

The VWR anhydrous acetone contains 0.01% water (the bottle just came in), I still have tot test whether it is suitable for use with Osmium. I'll post an update as soon as I have the results.

Hi Rob,

Did you get some results?

Cheers,

Denis

I finally had the time to test a few things;

First of all, the VWR acetone bottle is sealed with a septum covered with a non-removable plastic cap with 6 positions to pierce with a needle. The septum doesn't re-seal very well though so I resorted to removing the top of the cap with the septum so I can access the bottle with a 5ml pipet, and flush it with dry nitrogen gas before closing the bottle.

The Osmium crystals (4% w/v, as a test) dissolved well, and judging by the nice faint yellow color there are no reactive contaminants in the acetone.

Also the Uranyl acetate (0.2% w/v) dissolved without issues.

I'm currently running a freeze-substitution with the 0.2% UA for low-temperature infiltration with Lowicryl HM20, so I'm curious how that will turn out.

Rob Mesman, thanks for sharing the information. I am slightly confused - are you sure that it is so important to have super-dry solvent? Do you have some illustrations of the low water concentration effects (I don't know, perhaps some self-evident artifacts on the pictures, etc.)?

How do you transfer the freezing carriers from LN2 to substitution cocktail?

@ Dennis;

I don't think you'll be able to see much difference between samples substituted with solvent containing 0.1 or 0.2% water, especially not is you do the infiltration with resin above -30C. For me the (for now) preference for the dryer solvent is a matter of personal choice, coming from a solvent containing only 0.075% water the step to 0.1% is a 33% increase, going to 0.2% would be a 166% increase. Since I have to compare the samples I make now to those I've made in the past few years i'd like to keep the change as small as possible.

For low temperature infiltration with non-polar resins, there might actually be an effect. Lowicryl HM-20 for instance only tolerates up to 0.1% water in the sample for polymerization, and historically has been reported not to be very soluble in acetone. I've never had issues with the solubility of HM20 in the acetone I used, but it is quite likely that the original reported problems were due to to high water concentrations in the solvent.

One other thing is that minor additions of a polar solvent (in this case water) to a less polar solvent (acetone) van have quite influence on the properties of the mixture (which is often non-linear compared to the ratio of the 2 solvents).

For instance if you look at the addition of water in the freeze-substitution mix

as described by Walther & ZieglerArticle Freeze substitution of high-pressure frozen samples: The vis...

and Buser & Walther Article Freeze-substitution: The addition of water to polar solvents...

In my hands the addition of 1% of water already gave me the enhanced membrane contrast shown there for 3%. Additionally I could not observe differences between 3% and 5% water while in both articles that is only observed for 5% and higher.

The only difference in the approach it that in my case the uranly acetate is directly dissolved in the acetone (this doesn't work for all brands of uranyl acetate) and in the articles mentioned a stock solution of 10 to 20% of uranyl acetate in methanol is used. Thus the end concentration of 0.5 - 1% of methanol in the substitution mix increases the amount of water required for membrane contrast quite dramatically.

Regarding the transfer of the carriers; I have my substitution mix cooled down to -90C and transfer my samples from liquid nitrogen into the tubes containing the substitution mix using Leica flow-trough capsules (# 16702738) inside the AFS.

As soon as the substitution fluid melts again, I check whether the carrier is facing the right way up (I marked the other side of the carrier using a pencil before freezing) and adjust it if needed using an insect needle to manipulate the carrier.

Best,

Rob

Hi Rob,

Thank you very much for the detailed description. I used the same transfer of the carriers and Leica capsules (geometry is stupid, why don't they make a notch for a needle or sharp tweezers? it is not so easy to flip the carrier in the capsule).

There are some protocols when the carriers are put on the surface of frozen substitution cocktail and it is melted to -90, but I have never used them.

Cheers,

Denis