Hi Denis,

I understand your predicament and I'm not sure how feasible this would be for your situation, but one way around this would be if you could take the HPF to the animal. Our EMPACT is very moveable but I have never moved it between buildings.

None, you should organize isopentane cooling or any other cooling.

Thanks. Sorry if my additional question looks idiotic (this area is pretty new for me), but which design of experiment do you mean? The main idea of HPF is prevention of ice crystals formation, if the sample is frozen with a conventional way (isopentane, etc) it yet contains ice crystals and HPF won't have any sense.

Hi Denis,

You're right - the less time spent between dissection and HPF, the better. I work with plant tissues, but the same basic principles apply. If your samples air dry, you'll see plasmolysis/damage that even the best freezing, fixation and processing can't fix. If you can't dissect tissues of interest right before freezing, can you at least submerge them in cryoprotectant (commonly hexadecene or sucrose solutions in plants)?

I also recommend running ;)

Hi Teagen,

Thanks. Yep, I was running with a tube between buildings :-)

All manipulations with animals are strictly regulated with law and we cannot just take a mouse and kill it near the HPF... I think, it is not a big concern for the mouse - where it will be killed, but we cannot break the rules.

I am not sure about cryoprotectant - the function of extracellular cryoprotectant is just prevention of ice crystals formation around the sample. How can it prevent post-mortem tissue degradation?

There should be an "acceptable time" which can vary between tissues. Degradation has a fixed rate - not immediately. I was looking for literature about a self-evident methodological experiment: freeze directly after dissection, in 10 min, in 20 min... etc, then compare the results pointing the differences. Nothing. Definitely, someone conducted such studies, I don't want to reinvent the wheel :-)

A little example. Once I didn't synchronize the schedule with a colleague who dissected a mouse and the sample (testis) was stored in PBS (+ 4 C, in the fridge) at least 1 h and about 20 min for HPF starting. I didn't discard that sample (just from curiosity) , frozen it (EM PACT2), then conducted AFS, embedding and slicing. A few TEM pictures is attached. Degeneration of ultrastructure looks not so dramatic. of course, it is not an excuse for long time tissue storage before freezing, but it points that the post-mortem degradation is not super-fast.

Cheers,

Denis

Denis, also I am not aware of any study comparing different storing conditions after excision prior to HPF. You've shown the problem clearly...It might be a challenge to document the timeline of any specimen preparation you might have in the future just to do like we had to do in describing exactly the procedere of our animal specimen preparations for inclusion into our thesis / theses....(e. g. right after excision..... primary fixation of.... was applied immediately / within sec's mins .... or: 'specimens were pooled and at least after a period of maximum 1 hr at RT, 4°C, ' etc....

I am sure you know what I mean...(:-)).

In my former 'professional' life it was a crucial point to get (documented!) at least the initial timepoint of excision AND primary fixation for diagnostic samples from the surgical theatre sent to the EM-Lab....so I knew at least a bit about the possibility of autolysis-inetrnal degeneration (physiological degradation as well as e. g. temperature- or humidity-related tissular alterations. Usually I always was present with my fixatives in a room situated before the operating room.... so I always saw how the surgical specimen was i) treated, ii) transferred in my hands and iii) how the spec was treated, cut and finally, when it was placed into the fixative proper.

But I know the difference between chemical and physical fixation procedures well... and therefore knowing too, it might be very difficult to end up with real time "freezing" and to be able to display moments of physiological processes reliably and reproducible, as long as you can't culture your cells (or organ systems) in the HPF-object- /specimen holder... [having seen right now the specification of the HPM 010 High Pressure Freezer (RMC Boeckeler) ....:

(quote) "Well frozen specimens of 200µm thickness and 2 mm diameter without use of cyroprotectant!) (end of quote).

On the other hand one could assume that some minutes of delay - say 5 -7 mins (provided the use of a perhaps more sophisticated cooling- [down to 4-6°C] and moisty chamber device as [eventually rapid] transfer unit and that initial specimen size is 5-10 times bigger than the finally again trimmed to be HP-frozen specimen) - and if such a consistent delay routinely could be maintained during every performed preparation -

COULD be acceptable in terms of "nearly in vivo conditions" prior to HP-freezing itself... but naturally it depends on the task you have to analyze in the examination of the achieved cryosections in your TEM then....

To be honest: in vivo (as you want to show it) not always does mean in vivo....(taken from: https://www.leica-microsystems.com/fileadmin/academy/2013/WF_HighPressureFreezing_07_13.pdf):

(quote) " The Leica EM PACT2 high pressure freezer serves

the needs of molecular and cell biologists and all researchers who want an “in vivo” impression of their cellular structures and functions in question

– without the artefacts of chemical fixation but with the high resolution information of EM immunocytochemistry, frozen hydrated sections and

freeze fracturing.

The Rapid Transfer System EM RTS allows correlative LM/EM experiments, taking a live specimen from a light microscope (e. g. a confocal microscope) to freezing in less than 5 seconds. In the same way, time resolved experiments are

possible. Safety and reproducibility for the specimen are increased while operator mistakes are reduced." (end of quote).

So - finally - it might be advisable to get an extra permission for running your HPF at the site of 'dissection' (or at least in a room nearby....)

With my respect and best wishes for your research work...cordially yours, W.H.M.

Disclaimer: not affiliated with or financially interested in any of the mentioned companies.

Dear Wolfgang,

Thank you so much for the answer and upvoting :)

I am really surprised, why there are not any publications about ultrastructure degradation kinetics? Theoretically, HPF provides a possibility to show even very little changes...

Cheers,

Denis

Dear Denis, you're right....so - after having machines at the highest standards ever and deep insight into the physical processes of "freezing" (conventional, rapid, or HPF) it is now up to young and ambitious researchers like you to analyze and provide evidence of time-related tissular ultratsructure degeneration/degradation kinetics...(:-))

Dear Wolfgang, thank you. Sometimes I am trying to "reinvent the wheel" (as not so young but pretty ambitious researcher :)) and spend time for things which were well-known a lot years ago, HPF is not so young method and I thought there are some basic methodological studies about tissue preparation.

Hi Denis,

I understand your predicament and I'm not sure how feasible this would be for your situation, but one way around this would be if you could take the HPF to the animal. Our EMPACT is very moveable but I have never moved it between buildings.

Hi Christine,

Thanks. But it doesn't work.

I am a guest and the distance between the buildings is about 1.5 km. They are very kind and enthusiastic people, but not so much :)

Hi Denis,

there hasn't been much systematic study on this because a) there are too many variables (buffers, fixatives, freezing quality) and b) the effects vary between different sample types. So it's a lot of work and doesn't translate easily to other biological systems.

Keep the time short and your samples on ice. Dumping a tissue into buffer can cause damage too... If you really want to know, compare:

Lots of work!

Chris

Hi Chris,

Thanks. Indeed, it looks like a huge amount of work... Nevertheless, some basic regularities for most common tissues (brain, muscles, kidney, etc.) could be empirically estimated.

I am not sure about ice (the cooling can induce some morphological changes itself) and paraformaldehyde (this pretty strong chemical can reduce the main idea of HPF - fast fixation of intact tissue). But buffer should have an influence, yes.

Cheers,

Denis

I fully agree on ice and formaldehyde, you will have to compromise somewhere. The formaldehyde has some effect but after that the samples are stable and do not deteriorate further in storage. Because of that it would be a good reference point. I have been on the receiving end of this advice and reacted the same way as you (defies the purpose of HPF) but I have come to see the benefits. I now routinely keep an aliquot of my HPF samples in 4% formaldehyde in PBS as backup.

Chris

@Chris, thank you for the useful idea. It is a good way to have a backup. At least, it will be possible to compare the results HPF samples with conventional chemical fixation and understand where is the problem: in the sample itself or in HPF/AFS preparation.

Cheers,

Denis