plant leaf stem cross sections

[Charles Krebs]

Sliced up a few more plants into skinny small pieces
First we have the leaf stalk (petiole) of a lilac bush (Syringa vulgaris). I cut these sections just at the start of the leaf base, where the leaf blade is just beginning to "flare" out. This is what you see as sort of "ears" in the top three images. I tried several simple stains to see the difference. All four of these are darkfield images.









These next images are from the stem of a foxglove plant (Digitalis purpurea). The stem has prominent trichomes as seen in two of these images. The lighting was (partially) crossed polarizers, darkfield and brightfield.

[Ken Ramos]

As usual your work is to be envied Charles. You're pretty good with that knife!

[Sumguy01]

Very nice set.
Thanks for sharing.

[sebba28]

Beautiful.What stains did you use?

[Charles Krebs]

Beautiful.What stains did you use?

Thanks.

The stains were toluidine, safranin, and a little combo with Janus green on the 3rd one.

[WalterD]

Very nice colorful set, thanks for sharing.

[iconoclastica]

Is it acquired skill or special equipment that results in such perfect sections? (Probably both...)

[micro_pix]

.

Beautifully prepared and photographed.

David

[Charles Krebs]

Is it acquired skill or special equipment that results in such perfect sections? (Probably both...)

Thanks for the comments. I'm just starting to play around with this subject for myself. I have used a Vibratome at a lab where I work, but at home here all I have is one of those small, inexpensive hand microtomes I bought years ago. Working with the Vibratome, while immensely different than a small manual hand microtome, made it very clear that there were a couple universal key elements you need to get decent sections. The subject must be constrained so that it can't move during the cutting. A good, extremely sharp, blade used properly and at the proper angle is very important. But expensive special equipment is not needed for the images I show here. Keep in mind that my goals are very simple. I want to use fresh live material. I'm interested primarily in visual patterns and overall structure, which I find quite beautiful. The sections here are much thicker than those needed to study the finest botanical details. In that case a precise mechanical microtome and rather extensive subject preparation (fixing, infiltration and embedding) are needed to cut the very thin (perhaps 3 to 10 micron) sections. (And then additional processing is needed after sectioning).
I can walk in my front door with a new sample and have it under my microscope (wet mount) in just a couple hours or less.

Depending on the subject matter you can actually cut similar section to what I have here completely hand-held with only a sharp razor blade. But my normal configuration right now is a hand microtome to hold the subject, and "low-profile" disposable microtome blades held in a blade holder from Radical Instruments from India. The way I am set up my typical sections are usually somewhere in the 50-200 micron range. With certain subjects I can get down to about 20 micron or so, but many fresh subjects just won't give good results below about 50 microns. Many times it is necessary to put the subject in paraffin to hold it steady enough to cut (but no subject fixing or infiltration... the paraffin is simply used as a support medium).

[ChrisR]

Hi Charles,.. I've always wondered, does a Vibratome tend to cut with less need for suport of the material? I would assume so, especially if the settings were just right.
Can one alter the frequency and stroke of the commercial machine? It wouldn't be too difficult to make something with both being variable.

[Macro_Cosmos]

These are incredible, love the colours! Nicely stained.
I've seen manual microtomes pop up locally, the prices are usually very very high. Automatic ones... well I'd rather buy 10 more lenses with those used prices.

[Charles Krebs]

Hi Charles,.. I've always wondered, does a Vibratome tend to cut with less need for support of the material? I would assume so, especially if the settings were just right.
Can one alter the frequency and stroke of the commercial machine? It wouldn't be too difficult to make something with both being variable.

With the one I use you can adjust the "amplitude" (the amount of movement the blade makes as it moves side-to-side). You can also select the advance speed... the speed at which the blade moves into the subject. The other adjustment that you can make (besides the depth of the cut) is the angle of the razor. This can make quite a difference. The settings used can make a very large difference in the quality of the section. In my case the subject was mounted on a small pedestal (with cyanoacrylate). Some subjects could be mounted directly, others (most) were embedded in agarose to provide support. The cuts are made under water (or some other appropriate liquid). The thickness adjustment was fine enough to get sections down to 10 micron or so, but that only seemed feasible with "ideal" subjects. Many fresh soft subjects (not treated in any manner) just will not hold together for sections that thin. Often 50 microns (or considerably more) were the best I could do.

I'm finding about the same limits with my hand microtome. Both approaches need a well supported specimen, I would say about equally so. I usually cut by hand with a diagonal slicing stroke while trying to keep the cutting surface wet.. Seems to work pretty well. Neither one seems to be very good with hard subjects (for me anyway).

[ChrisR]

Thanks CHarles.
It sounds like your machine didn't have oscillation frequency adjustment, though some do. This Leitz document gives some numbers:
https://drp8p5tqcb2p5.cloudfront.net/fi ... ies_EN.pdf

The frequencies are much lower than I would have expected (typ 85Hz). Some wood cutting tools are much higher.
From college days I recall playing with the fracture-toughness testers. The faster you cut, the cleaner, and the less energy absorbed. Turning all the knobs up and trying various expendable specimens at hand, produced a neat round hole in one of my socks.
Loudspeaker voice-coil and razor blade lash-ups beckon.


MacroCosmos - the go-to microtome in the UK is the "Cambridge rocker", which often appear used around £100. They're calibrated down to 10µm.


Those are lovely clear images Charles. I shall show them at school; we have a couple of keen 14yo botanists.