Good day to everyone!
The situation - I have a plunge-frozen sample on TEM grid and I need to perform cryo-ultramicrotomy of the sample together with the grid (that is I need to cut perpendicular to the grid plane). Theoretically, if to forget that I will destroy my knife by cutting the ice together with copper, I could put the grid in the holder. But the grid is a way too flexible and most likely going to behave like a flat spring making the ultramicrotomy almost impossible. That's why I need to provide a thick layer of something around my grid to provide enough of rigidity, thus I could trim the "ice" block and then cut.
Now there is a question - what I could use to drop on the grid at let say -100 °C, that will solidify upon touching the grid and will not cause devitrification of the sample and formation of cubic ice? Any ideas? There are plenty organic compounds that have meting point at around -95 °C, like hexane, heptane, toluene, ethylbenzene, even ethanol and aceton - but what will happen to my frozen sample if I drop on it chilled to -100 °C ethanol? Did anybody ever try to cut something like frozen ethanol? And then to put it on another TEM grid and make cryo-TEM?
Tell me, there is a simple solution of such situation... Please.
Thanks for the inputs.
Yaroslav
Hi Yaroslav,
anything with a temperature higher than -138C will devitrify your sample.
what you are looking for is the cryo-glue first described by Karsten Richter
(https://doi.org/10.1111/j.1365-2818.1994.tb03436.x)
which consists of ethanol (100%) and 2‐propanol in a 2:3 ratio.
you apply this into a holder or cryo-microtome pin (the slotted type) using a wire loop at -140 to -145. At this temperature the glue is still viscous and you can slide the grid in using pre-cooled tweezers. next you lower the temperature of the ultramicrotome to -160 to -165, now the glue will be rigid allowing you to trim and section. be careful to remove most of the glue when trimming because it will have different sectioning properties than the rest of the sample. Also I would recommend sectioning with the grid in a vertical position (same sectioning properties in during the entire stroke) and not to make a to high blockface (this will induce curling of the sections).
The copper of the grid bar will not be that much of an issue (if you section thinner than 50nm) i've had decent results with sectioning gold plated copper grids after HPF, though if you can do the whole thing using gold grids it will section a lot easier.
best of luck!
Rob
Dear Rob Mesman, thanks a lot for the advise! Will definitely try out.
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