Microtome Carrot / Styrofoam sandwich issues

[Michael Cytrynowicz]

Dear microscopists,

I have been trying to use the sandwich method with carrot (pith, styrofoam) that our experts so successfully used (that's you Fahrenheit :-)  and I am finding it hard to avoid an interesting issue - see the picture attached.  When I tighten the carrot sandwich with the sample inside (say a leaf section, shown in green in the drawing) - the Jung Hand Cylinder microtome grabs th bottom of the sandwich, and even though the clamps move slightly inwards (in the drawing I just show them parallel), still the pressure applied at the bottom of the sandwich causes the top of the sandwich to open a bit, so creating gaps on the sides of the sample. That of course is bad. How do you, experienced microtomists, deal with this? 

Thank you!

Mike

[Bob]

Hi Mike,
I have a few suggestions for your observed problem:
- I cut the carrot to a rectangular block, split it in the middle and put the halves together with the pith outside. This seems to help to keep the carrot halves in suitable shape while they are used and dry out over time.
- I leave no gap between the blocks but whittle a notch into them that just clamps the object when the halves are pressed together. There are small woodworking gouging chisels that work well for this job.
- Sometimes I cut the inner surfaces a little concave to concentrate clamping pressure at the top and bottom of the carrot halves.

Cheers
Bob

[Michael Cytrynowicz]

Thank you Bob, appreciated.

I will try what you are suggesting (pith outside). The chisels are for stems, right?

How are you making the concavities - longitudinally (most concave at mid-height) or radially - I suppose the former?

Mike

[Michael Cytrynowicz]

Like so...

[Fahrenheit]

Hi Mike,

yeah, that's some sort of a problem, that comes up with the way cylinder microtoms usually clamp the specimens.
I try to give the carrot (I don't use styrodur, as it damages the blade) a form that fits the clamps better.

And there is an old trick with the carrot: it's central cylinder containing most of the vessels is harder and not as moist as the root parenchyma surrounding it. If You cut the carrot block (with the cylinder in the middle - use small carrots ...) that should hold the specimen in two parts and turn each half at 180 degrees, the cylinder part is at the outside and the parenchyma part at the inside, were the specimen is to be placed.

Now wait some minutes: the parenchyma looses some of its moisture and shinks a little bit, but the cylinder keeps its size. So each part bents a little bit inwards - and that's it.
When You are cutting leaves, that will do the job, but for me it also works without the trick most of the time.

Sprouts I usually cut unembedded, which normaly is no problem, if they are more than let's say 2,5 mm thick.

Best
Jörg

[Michael Cytrynowicz]

Hi Jörg!! - I had sent you an email a week ago, I wonder if I had an older email address.

Anyways, what a pleasure to hear from you! - and a pleasure to see your new Yuzu Orange :-)

To everyone: Jörg was the one who got me in love again with botanical microscopy, 10 years ago - I had to put it aside for work and family reasons, but now that I am retired and very free here in Portugal, I'm back. Thank You Meister Weiß.

I will try to follow very correctly what you and Bob suggest, abut the carrot, and I am happy you explained the anatomical reasons. Also, very interesting that about waiting a few moments for the Parenchyma to dry a bit. I suppose it makes it a bit more spongy, and grab the microtome claws better as the 2 halves bend inwards. Smart.

I will try also and cut the sprouts free-standing (if they are ~ >2.5 mm thick). I think I will try and secure them close to the perimeter of the metal tube, and hold them with something like Bob's holder.

Jörg, do you find it easier to cut fresh or is it easier to cut fixed (and etOH 70%)?  Of course, fresh allows different observations.

By the way, I got myself a new used tischmikrotom, right now it's in California.

Hearty Salutations from Porto!

Mike