Denis,

your main problem is that there is moderate freezing damage. You're not going to be able to squeeze more out of those samples with all the FS methods. You have two options: HPF in 100 um carriers (my choice) or adding a cryoprotectant (in this case I would suggest 20% BSA in PBS).

HPF users should be reminded that the ability to freeze a 200 um slice of tissue without crystal formation remains the exception, it only works if the tissue contains little water, contains endogenous cryoprotectants (like sugars) or if cryoprotectants are added before HPF. Daniel Studer in Bern, Switzerland has done a lot of work on cooling simulations and HPF of brain (which is probably similar to testis from a prep standpoint). See Article Electron microscopy of high pressure frozen samples: Bridgin...

I also highly encourage you to read Jacques Dubochet's work on the vitrification of biological samples. People thought he was crazy at the time, he deserved the Nobel prize many times over...

Hi Denis,

in general I would advise to first try freezing it without cryoprotectants in a shallow carrier (100 um) and polymerize in the carrier. As long as your washes during FS and infiltration are gentle enough you shouldn't displace the tissue. Your best bet is probably going to be a methacrylate resin because of the lower viscosity.

If you have to go with a deeper carrier things get tricky. I would guess that 20% BSA (or a BSA-gelatin mix) is going to give you the best compromise between cryoprotection, cohesion and low infiltration barrier. Hexadecene or sugars won't come out well during FS and won't help with keeping your tissue in place. I'm happy to provide more detail if needed.

Chris

Hi Chris,

Thank you so much. I used 200 um carriers (Leica EM PACT2 and Leica ICE freezers) without any cryoprotectants and with hexadecene (carriers coated before tissue placing). I used only Epon resin, going to use HM20 now. The sample is mouse testis. Have tried a few AFS protocols from "Leica EM AFS recipe book":

http://otolic.stanford.edu/documents/Recipe_book.pdf

The staining was a problem - very bad contrast. The first acceptable results were reached with high (up to 4%) concentration of OsO4 in acetone and UA extra staining. I tried to use polymerization in the carriers. I am preparing the samples for FIB-SEM and it is pretty easy to use them right with the carriers - just cut in diameter with a thin blade. But it didn't have any advantages in comparison with the conventional way (remove tissue before polymerizing) and I have skipped it.

The current results (conventional TEM, Epon) for mouse testis are attached. Do you have any ideas - what is wrong with them?

Cheers,

Denis

Hi Denis,

I concur with Chris, try to use 100um carriers if possible. On some images it looks as if there is a little freeze damage, others look ok (maybe provide a higher resolution image?). Thus, I would focus on the FS. Out of the top of my head a couple of things you can try to increase contrast:

1) Try additional staining agents. For instance tannic acid can be used as a mordant for OsO4 (eg. start the FS at -90C in 1% tannic acid in acetone over night, then wash thoroughly with acetone @ -90C and continue with OsO4 protocol as usual) or after FS, eg. Article Tannic acid-mediated osmium impregnation after freeze-substi...

2) Include up to 5% water in your FS mix to increase membrane contrast: Article Freeze-substitution: The addition of water to polar solvents...

3) Warming the FS mix after the AFS run. A lot of FS protocols end at 4C or even 0C, but letting the sample sit at room temperature for half an hour before rinsing in acetone can make a huge difference. Even more so if you heat the sample to 40C or 60C for a short period of time, but the drawback is that there is usually some sort of degradation.

4) Addition of up to 3% glutaraldehyde together with OsO4 and UA has been reported to increase membrane contrast. I have not made that observation in my personal experience, but it could be worth a try.

5) Experiment with different resins/resin mixes. Not sure how flexible you are with the FIB SEM though.

Good luck!

Martin

Hi Martin,

Thank you very much.

1. I was trying 1% TA in acetone without osmication. The results were not good:

https://drive.google.com/open?id=1EobihjA4GUe8shrp0ezhpw03gsYNWEnu

Combination with osmication (what about additional UA?) looks promising. Thanks. I will try it.

2. Thanks. I have read about that effect of water. But I am still little confused - a lot of protocols suggest to use analytical grade "dry acetone". In general, the question itself is interesting. Leica AFS "revolvers" are not sealed and it looks possible that some water (1% or more?) can be just absorbed from the atmosphere during substitution. But it is just a guess.

3. Yep. The first good stained samples were prepared with warming up to 0 C with OsO4, p. 47:

http://otolic.stanford.edu/documents/Recipe_book.pdf

But with room temperature OsO4 in acetone chemically destroys the tissue:

https://drive.google.com/open?id=1qigEEaRSKWydgN0rtv06UmQsKGtbitzJ

4. Interesting point. Have never heard about this way. What about thin structure preservation? GA can bind the proteins and induce some artifacts.

5. Yep. AFS for HM20 is running right now. At least it will be possible to compare with Epon and understand - do we have any problems with infiltration/embedding or not.

Cheers,

Denis

FIB-SEM preparation for the sample polymerized in the carrier. But it doesn't have any advantages and the carriers, which can be used a few times, are not so cheap. I don't use this method now.

I think the TA staining does not look that bad. Could it be that the sample is not in the best state before you freeze it? Maybe some sperm cells are already headed towards self destruction? Also, sometimes it's simply unavoidable to chemically fix a sample before HPF/FS. How do you harvest the sample?

Since you mentioned decay when you warmed the FS to rt, you might want to use additional fixatives in your FS mix. Osmium tetroxide is not great at fixing proteins, in fact at higher temperatures (above 0C if I remember correctly) it acts as a protease. Thus, I always include some UA (0.1% or 0.2% from 5% methanolic stock solution), and you can also include glutaraldehdye in the mix. For example: 1% OsO4, 0.5% GA, 0.2% UA, and 2% water in acetone. I usually take the sample out as soon as it reaches 0C and incubate at room temperature for a while .

The effect of water is an interesting one. Obviously there is ALWAYS going to be water in your FS mix, namely the water that will come out of your sample! (besides the atmospheric contamination esp. in areas with high humidity). Why does the addition of water cause a better contrast? I don't know, I can only refer you back to Chris' publication. I'm usually including 2-5% water in my FS mix (for animal cells at least, it doesn't work for plant samples).

One more thing that might make trouble shooting easier. With most samples you can get away with very short FS times. I usually do quick FS in a chilled metal block on a shaker for 2h, see literature from McDonald/Rick Webb. When using the AFS I mostly run programs overnight (ca. 18h), most of the time at temperatures below -70C (below temperatures where OsO4 will start to react) to allow for good substitution, then you can warm relatively quickly to final temperature.

Denis,

your main problem is that there is moderate freezing damage. You're not going to be able to squeeze more out of those samples with all the FS methods. You have two options: HPF in 100 um carriers (my choice) or adding a cryoprotectant (in this case I would suggest 20% BSA in PBS).

HPF users should be reminded that the ability to freeze a 200 um slice of tissue without crystal formation remains the exception, it only works if the tissue contains little water, contains endogenous cryoprotectants (like sugars) or if cryoprotectants are added before HPF. Daniel Studer in Bern, Switzerland has done a lot of work on cooling simulations and HPF of brain (which is probably similar to testis from a prep standpoint). See Article Electron microscopy of high pressure frozen samples: Bridgin...

I also highly encourage you to read Jacques Dubochet's work on the vitrification of biological samples. People thought he was crazy at the time, he deserved the Nobel prize many times over...

@Martin, thanks a lot. I will try to add GA in the FS mix. Which effect does it have? I think, with pretty low concentration (less than 1%) it could prevent some degradation of protein based structures like microtubules at temperature above 0C after substitution.

i used pretty simple preparation: the mouse testis was put to PBS buffer (this organ has a capsule and buffer shouldn't be a big deal) and delivered to HPF room, then it was cut in a Petri dish with a sharp blade under PBS and a piece of the seminiferous tubule (diameter is about 150 um) were put into hexadecene coated 200 um carrier with a sharp tweezer.The carrier contained a small droplet of PBS which was looking as a little meniscus (as described in HPF Leica ICE manual).

@Chris, thank you very much. I will read the papers. The diameter of the seminiferous tubules is about 150 um. Is it Ok to use 100 um carriers for such samples?

Wild et al. studied the effect of Os/GA regarding membrane staining in FS Article Enhanced resolution of membranes in cultured cells by cryoim...

I have no experience with this tissue, however, do not underestimate the influence that buffers and general handling can have on a tissue.

To mitigate the freezing damage, try to use 100um cups or 150um cups, or try slot cups (e.g. 200um x 200um x 2mm). Use 20% BSA/PBS instead of PBS only for loading.

@Martin, thank you so much. We have only 200 um carriers now, but I will order some 100 um and 150 um (if they exist for EM PACT2).

Cheers

The problem was completely solved with the new freezer. Wohlwend is the best! :-)

Wohlwend makes the best HPF hands down. I'm happy for you!

Indeed. It is a really great machine. Old good engineering without plastic and "intellectual interface" - like a gun shutter :-)

Denis Korneev I did not see this in time, but I feel that it is still worth mentioning for anyone with an EMPACT2: If its soft enough to be deformable like a thick liquid (or even biopsied with a needle punch), the "tube carrier" system is 100% the way to go. When it comes to thermal conductance, I'd choose a copper tube over a flat disc any day. If it fits in the tube, it works.