The Microscopic Beauty of Plants & Tree

[RobBerdan]

I just posted an article on my web site that I think some members might be interested in called the The Microscopic Beauty of Plants and Trees. I have been hand sectioning plants and trees and tried a wide variety of disposable razor blades, shaving blades and microtome blades. I am using hand microtomes or cutting the sections by hand with a razor and I can get sections about 30 microns thick which is OK, but in order to photograph these thick sections I have to focus stack. I am going to buy a sliding microtome for wood and plants in the future. Some of the pictures I also photographed from prepared slides which of course were easy. I have included links to original science paper with PDF links to them and numerous videos, some by Oliver Kim which are excellent.

Article: https://www.canadiannaturephotographer. ... ants1.html

[WhyMe]

Those are awesome!

[MicroBob]

Hi Rob,
your article is very comprehensive gives a very good overview over the available sectioning techniques from simple to complicated. Also the images are very good. You must have spent lots of time to write, prepare, photograph and compile this article!

Somewhere in the lower third you showed a microtome knife with handle. You might add that the cylindrical sleeve on the back of the knife is used to incline the knife away from the stone or leather for sharpening the bevels. These sleeves are actually quite rare and it is much easier to sharpen a knife when you have the proper sleeve for it. Nice to have the complete set!
Very useful for plant sections is a hand microtome together with a blade holder like this: viewtopic.php?f=6&t=7013&p=62098&hilit= ... ria#p62098 The blade holder keeps the edge away from the table surface and it stays sharp much longer. It is also quite easy to maintain even thicknesses of cut. This has been in use here in Germany for 30 years or so.

Bob

[RobBerdan]

Thanks for the suggestions I had a look at the device you made out of plastic I will try something similar. You say you make 7- mcron sections - I would like to make 1015 micron sections so I am looking at purchasing a sliding microtome. You have some very nice sections - I still have a lot to learn and need more practice cutting the sections.

By sleeve I think you mean something to hold the angle of the blade constant? I have a leather sheath and boxes to store the microtome blades. I am also testing the disposable microtome blades and holder - but it takes time to experiment and I will work hard to get really thin sections. Even the stains are difficult to get - I just purchased some Safranin and Astra blue from a biotech company in California so will be testing them shortly.

I appreciate the advice, I also looked at the other links you included but the documents are in german which I am not alble to read.

Is the blade holder available for sale anywhere? If not can you provide specs so that I might make one, or would you sell me one?
Thank you kindly for your comments
Robert Berdan

[MicroBob]

Hi Rob,
with fresh plant stems I try to get 50µ sections (0,05mm). If this doesn't work I cut 100µ and work down from there until I get the least thickness of section without torn cell walls.

With sleeve I meant the cylindrical component that sits on the back of the knife. A Type C knife is basically a steel wedge. They are not sharpened by taking material of the flat sides, too much work. Instead a secondary bevel is ground, at a some degree blunter angle. To acheive this a cylindrical guide is pushed onto the back of the knife. The knife glides over the stone with the edge and the guide then. The guide is removed before sectioning of cause.

To translate text or even whole websites google translator is usefull. DeepL is sait to give even better results. For botanic sectioning especially the webstite of the MKB Bonn is great.

For plant sections Etzold FCA (fuchsin, chryosidin, astra blue) and Wacker W3A (acridine red, acriflyvin, astra blue) give nice differentiated colours.

Bob

[RobBerdan]

Hi Thanks the web site is amazing and has beautiful photomicrographs.

Today I tried an older AO Hand microtome from 1950s I purchased on Ebay and a new disposable microtome blade from India and right away I was able to cut sections between 10-15 microns - I might not need to buy a sliding microtome - I just need more practice and will now experiment with some different stains. I am attaching a few pictures of a popular tree that was sectioned fresh and stained with Safranin or Toludine blue.

[Hobbyst46]

Excellent images !
May I ask:
1. Were the cuts fixed prior to staining ?
2. How much would such microtomes cost on eBay, order of magnitude ?

[MicroBob]

Hi Rob,

very nice sections! I like the safranin stained ones best. That microtome is very nice too and very practical. It is for sure much better than the new cylinder microtomes that are available today. Was that metric 10-15 µm? That is very thin for a cut from fresh material.

Bob

[RobBerdan]

In answer to some questions. The branch was cut fresh, clamped and sectioned as is no fixing or softening as I did in the past. Softening the tissue by boiling in 10% glycerol and water seems to cause the bark to separate. The microtome cost me $300 US on Ebay, but I have never seen another one like it. I downloaded the manual online. The section thickness was estimated by focusing on the top and bottom of the slice - my microscope fine focus offers I micron increments. I adjusted the thickness by hand with looking at the verniers and tried to cut the thinnest sections I could. I also used the disposable microtome blade from india with the Feather low profile disposable blades (cost $100 for 50 purchased on Amazon). I will be testing cutter blades used for paper knives. I will try to cut thinner sections in the future and experiment with some different stains in particular combining Safranin and Astra blue. Took me a long while to find astra blue from a biotech company in California yet many of the research papers I read use this combination. I will also try some fluorescent microscopy. To my surprise some of the chemical companies won't sell or mail to residential addresses - even though the chemicals are not restricted.

Thanks
RB

[MicroBob]

Hi Rob,
I usually fix my plant samples with AFE. It contains ethanol, acetic acid and a bit of formaline and is a proven fixative, but I don't know how important this step actually is. According the thickness of section I have made the experience that when I choose it too shallow, I just scrape over the specimen. With the next advance I then cut twice the intended thickness.
One year ago we had an extremely well attended group meeting (to our surprise) on the topic of plant sectioning. A guest brought such a microtome like your one with him. I haven't used it myself but it looks very well made and is beautiful. My experience with cutter blades is that the blades from OLFA are quite a bit sharper than all others I tested.
For fluorescence Rhodamin and Acriflavin are nice. For chemicals you might try to build up a contact to a local university. Your activities prove that you would use them properly and know what you do. And especially stains are only needed in tiny amounts.

Bob