I will be cryo-fixing a variety of plant species (woody eudicots) for ultrastructure studies via transmission electron microscopy. Previously, I have used standard chemical fixation techniques, which have not provided optimal morphology preservation. Given that cryo-fixation may be better suited for preservation and eliminating artifacts, I am interested in maximizing leaf sucrose content (sucrose serves as a cryo-protectant) in order to minimize crystal formation.
In the past, my lab has left plants in our dark room overnight (16 hours) to allow for starch degradation. My thought is that degradation of starch for chemical fixation is ideal, as starch decreases fixation penetration efficiency and time. In the context of cryo-fixing, I would guess that maximizing starch content is ideal as this will allow for faster freezing.
Any thoughts or suggestions on minimizing artifacts with cryo-fixing plant tissues would be greatly appreciated!
Approximately 1 M to 1.2 M sucrose shall be fine, you may try using ethylene glycol or propylene glycol those are more permeating cryoprotectants.
It's difficult to give a clear answer since it is unclear which chemical fixatives you are using, and which cryopreservation technique you want to employ. I do want to put out a reminder though, that fixation has 2 different roles: to kill quickly and to preserve structure.
I raise this point, cryo actually does these 2 things separately (Freeze to kill, then freeze substitute to preserve), whereas Glutaraldehyde does both at the same time (which can be a problem, since it permeates slowly through plant tissue).
All kinds of biological things can happen while you're waiting for a fixative to kill your sample. It doesn't matter how good a fixative is at preserving morphology if that morphology was already disrupted by slow death of the cell. Sometimes, its better to kill very fast with just enough preservation, to buy you enough time to use a better preservation technique.
Here's why i'm mentioning all this: Paraformaldehyde (PFA) isn't fantastic for preserving morphology on its own, but it kills fast and penetrates far more rapidly than glutaraldehyde. It also slightly permeabilizes the sample, which speeds up the penetration of any subsequent fixatives you use. You can also combine this with cryotechniques. I've actually tried this; fix with PFA to kill the sample, then high pressure freeze to allow for freeze substitution. The results can be remarkable, so dont be afraid to combine your techniques.
Hope that helps
Maybe you want to share more details about the methods you've tried so far, and the kind of artifacts you observed?
In the best case you have access to a high pressure freezer to cryofix the samples. If you sucrose infiltrate plant tissue without prior fixation it might result in ultrastructural changes, too. If you sucrose infiltrate fixed tissue, you might want to take a look at protocols describing the Tokuyasu technique of cryosectioning (for plants e.g. Ripper et al. 2008), where most commonly 2.3M sucrose is used as cryoprotectant. That being said, in his original 1974 paper Tokyasu used sucrose concentrations of 0.6M to 2.3M.
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