Colourful vessels from an old block

[mrsonchus]

Hi all, I've been taking some longitudinal sections from an old (September I think..) block that still has some lengthwise-split Sonchus stems that I had trouble sectioning a while back, new blades have settled this problem. I've been studying the various tissue-types of plants and at the same time trying to start a set of half-decent representative slides, mainly using the trusty Sonchus tissue!

I've also taken many progressive sections right through the tiny florets and their 'seeds' below, both in XS and LS in order to study the structure of the ovaries, pappus, florets etc, with some success. Anyway here are a few pictures of longitudinal sections taken to try to reveal some vascular detail. The stain used is the marvelously metachromatic TBO!

Here's the block whence came the sections featured;
The vertical piece wouldn't section - very messy , the horizontal was fine, direction (orientation) is I now know a very important factor when sectioning tissue that varies in hardness such as the stem's epidermis, vascular and ground tissues. Another valuable lesson learned!

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Here's a cross-section view of the vascular bundles who's secrets are hopefully revealed in these pictures...
The stain used for this slide is I think Methylene blue, the dark middle of one of the large vessels is I think a 'slime-plug' that is precipitated from ever-poised 'protein bodies' within the vessels that act a little like our blood's clotting mechanism in that they clump together to form the sealing-plug if a vessel becomes damaged.... fascinating!
The vascular-cambium is a row of dividing cells that one way (towards the outside) form new phloem, and the other (inwards) way form new xylem (once these new cells differentiate that is). This gives the radial patterns so often seen...
The fiber-cap here is still adjacent to the phloem, unlike some that form bundles further outwards near a distant epidermis.

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Here are some longitudinal sections that the TBO has made a very nice job of staining and revealing quite fine detail;

The main vessels differentiated quite well by the superb TBO, generally the blue is xylem and the purple is mainly phloem.

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Here I luckily exposed an elusive sloping sieve-plate at the end of a phloem element, the xylem have ends at right-angles to the long-axis and no sieve-plates across the joins.

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Here ribs can be seen as they appear to be 'squeezing' the cell-walls - reminds me of our vacuum-cleaner's extension-tube that I use to clean the car! (or indeed a Human trachea....)

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That's my 5 used up! I've a few more to follow in the next post after this one, hope you're enjoying the adventure! (I know I am )
Back in next message

[gekko]

Very nice work . I very much liked the first (lower power) image of the longitudinal section. The differentiation by staining is very satisfying. Thank you for sharing this.

[mrsonchus]

Aha - here are a few more pictures of the vessels...

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This really does look like a trachea to me!

Here's a 'ladder' end plate of what I think is a tracheid, not quite a vessel! Please excuse the poor quality, this is the first one I'v actually seen in one of my own slides and I've cropped and enlarged (and attempted to sharpen! ) a little too much!

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The x100 gave me quite a detailed image of a broken end of a vessel-rib which seems to show that they are not circular in cross-section as they appear to be, unlike the springs on my car's suspension that they resemble so much!

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Finally here are some elements (with the pitted/porous) walls measured - the more I see the more fascinating these structures become!

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p.s. I'm really unsure of a lot of the identification and terminology I've used in these posts, please excuse any glaring mistakes! I'm as new to botany as I am to microscopy and clearly only have a tiny amount of technical knowledge - as I discover my mistakes I always try to correct them, bear with me I am still a beginner in the botanical world...

Having a great time,
oh, I've a picture left, so here's a quick picture of a XS through the bottom-area of the (composite) flower-head of yes, you guessed it, a Sonchus. It is a section still in the wax sitting on a slide awaiting dewaxing, staining, mounting etc, but the pattern is quite interesting - the objects are the 'seeds' (achenes/cypselas - fruits actually) below each floret of the composite flower-head. I've put the image to inverted to try to emphasize the pattern a little. I've many sections and mounted slides of this subject, when I get a chance I'll try to post a set of images as they are quite fascinating when compared in cross and longitudinal section...

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Hope you've enjoyed the adventure as much as I have - back soon!

[mrsonchus]

Thanks Gekko, pleased you like them, I'm having lots of fun and learning all the time!

[mrsonchus]

Found a couple of nice XS of the flower-heads stained a little too heavily with TBO..

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and

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To bed! To dream of leaves and stems!

[gekko]

Again, very beautiful, but also very instructive! Thank you, John.

[vasselle]

Bonjour
Très belles séries images et bien commenter.
Merci pour le partage.
Cordialement seb

[75RR]

Nice stuff!
As gekko says it is most useful that the stains differentiate between structures.
Your explanatory arrows are very welcome!

Interesting that the Phloem sieve plate is sloped.
Have you tried diagonal sections?

[mrsonchus]

[quote="75RR"]Nice stuff!
....deleted for brevity.......
Have you tried diagonal sections?

Arrggghhh..., sorry,
Not yet, I am however going to make some tangential sections within my next batch to further explore structure. The diagonal would possibly be of limited value but may of course result in a section-plane parallel to sloping sieve-plates which would be interesting.
I'm still waiting for replacement clearing-agents and alcohols as about a week ago I was sent Histoclear II labelled as my usual Histoclear(I) - the two are not interchangeable as Histoclear II isn't compatible with the mountant that I routinely use - 'Numount'. The result of this has been many spoiled slides (that I should be able to re-mount hopefully) and spoiled & unusable clearing-series including the alcohol mixed with them - a cost to me of about £60!
Also spent about 6hrs tracking down the source of the problem. Turned out to be the supplier's mistake and I'm still waiting for replacements... Meanwhile I've been unable to process new tissue and have just about used-up my few remaining blocks , one of which is the source of these stem LS sections. To add to it all, we're away today for a week in the hills of Yorkshire, my lab will be strangely quiet without me..

However, I'm planning a study of local bryophytes while I'm there, the cottage in which we're staying is next to an ancient churchyard and stream. My darling Wife has insisted I take the stereo 'scope with us! I had no choice but to agree! What a woman..

Back soon from Yorkshire (which at this moment is flooded), get ready for a few stereo 'scope moss pictures....

p.s. Pleased you're enjoying the adventures I think the arrows are a little OTT but I always label and interpret my sections as a great way of studying, the pictures are a great form of note-taking for me too. Such fun!

[75RR]

Arrggghhh..., sorry

Not at all. Just thought it might be interesting.

... we're away today for a week in the hills of Yorkshire ...

sounds very nice. Enjoy your holiday.

[mrsonchus]

Arrggghhh..., sorry

Not at all. Just thought it might be interesting.

... we're away today for a week in the hills of Yorkshire ...

sounds very nice. Enjoy your holiday.

I think you're right, it would be interesting as that's an uncommon orientation, and I can see no reason not to give it a try just to see the tissues from that aspect - I'll include some diagonals in my next batch!

We've in a cosy 300yr old cottage now, with a roaring fire, stone walls over 60cm thick, and a good WiFi signal (oh, and I'm just unwrapping the trusty stereo 'scope) - tomorrow the churchyard! Mosses abound!

[Raul]

Fantastic pictures, book quality there I'm glad to see you're still excited by your work. Keep up the good work.

p.s. the methylene blue pictures seems to have a little metachromatic effect on it, do you know a method of emphasizing the effect?

Raul.

[Naphthalene]

Indeed, great images, mrsonchus! Post more!

Have you tried the alternative fixation methods? I wonder if more details can be seen by fixing the tissue sugars using periodic acid-lysine-formaldehyde (PLP) fixative or lipids by using the dichromate based fixatives. Though I havent seen these utilized with plant samples (I have very little experience with these) , I have tried the latter type on the animal tissue samples and they greatly improved the cell shape and consistency. So I wonder if some improvement can be introduced by fixation method?

[mrsonchus]

Indeed, great images, mrsonchus! Post more!

Have you tried the alternative fixation methods? I wonder if more details can be seen by fixing the tissue sugars using periodic acid-lysine-formaldehyde (PLP) fixative or lipids by using the dichromate based fixatives. Though I havent seen these utilized with plant samples (I have very little experience with these) , I have tried the latter type on the animal tissue samples and they greatly improved the cell shape and consistency. So I wonder if some improvement can be introduced by fixation method?

Hi Naphthalene, good to hear from you!
I've really only just started to get reasonable results consistently and am still very inexperienced. All my fixation is with FAA at this time although I've just started to explore protocols of my own with reasonable success in terms of shortening the entire processing time when assessed for different tissue types and conditions - all botanical of course...

I dehydrate with IPA, of various strengths of course, and clear with 'Histoclear (I)' at various ratios with IPA as expected. I suspect that the friability of tissues when sectioning is intimately but not solely connected with any fixing and dehydration regimes.... I have in addition found, perhaps not surprisingly, that my results in this respect have improved in parallel to my (slowly and modestly) increasing expertise as I practice the fine art! (I'm still however a very inexperienced beginner at this time.. )

My next planned explorations of the chemistry/ies of processing (fixation & dehydration primarily) are in the duration and formulation of FAA fixation & fixative - I'm certain that simple steps based upon previous acceptable results are the way forward in what could easily become a confusing maelstrom of chemistry, timing, interrelated procedures etc.... I need to keep organised and not overreach myself if I'm to acquire a good knowledge of and capability to execute the basics!

Of the chemistry you recommend I have no knowledge whatsoever - I also work literally in my kitchen and dining-room at home and am only able as a private amateur to acquire a limited range of chemicals & reagents etc - I'd definitely like to explore for example the methods you mention of course but don't feel anywhere close to being ready to approach such an unknown yet - I just don't know enough about it I'm afraid.

I've also immediate plans to further explore the double-embedding techniques as my first attempts (with gelatin as the 'inner medium) were very encouraging and promise to greatly enhance my ability to orientate tissues in a precise manner that will augment my study of the morphology and anatomy of plants when considering their systematic classification and taxonomy (British wildflowers & 'weeds') - this is where my core interests lie, it all started with a picture of an unknown weed taken with an embarrassingly archaic mobile 'phone a few years ago...

It becomes ever clearer to me that orientation is crucial - oblique sections can give very unpleasant and unhelpful images that appear 'blurred' or perhaps 'smeared' as some texts so perfectly describe this effect, until looking closer reveals the cause to be the slanting views of cell-walls that should ideally be perpendicular to the viewpoint. That and the quality of staining, again in it's infancy as far as I'm concerned, seem to me to be extremely important factors concerned with information-content and quality of finished and mounted (and of course photographed) permanent sections.

So much to study, even before any botanical studies are followed! A fantastic adventure of which I love every minute
You've been a great help to me several time Naphthalene, please keep the pointers coming, they're always valuable and helpful to me, and of course greatly appreciated!

[mrsonchus]

Fantastic pictures, book quality there I'm glad to see you're still excited by your work. Keep up the good work.

p.s. the methylene blue pictures seems to have a little metachromatic effect on it, do you know a method of emphasizing the effect?

Raul.

I know that it (the metachromasia) may be enhanced (increased that is) by ensuring that the Methylene blue stain is allowed to 'mature' for several days by exposure to air in order to facilitate oxidation before starting to use a working batch. This enables three different (isomers I think) 'versions' of the stain's molecules to form, each of which stains slightly differently and the mixture exhibits metachromasia as a result - at least that how I think it acts...

[mrsonchus]

I find that the dense tissues, such as those of ovaries and cypselas as above, are better in terms of detail when sectioned quite thinly, about 4/5µ seems to work well, whereas at about 10µ, which is I'm certain the optimum thickness for leaf XS and seen in some of my earlier posts of Carex.pendula leaves, the definition is rather 'confused' (although the surrounding leaves that enclose and protect the flower-head have good detail as do the Carex sections)...
The TBO seems a rather blunt instrument here also, although I've clearly overstained these XS! I find Methylene blue to be superior with the ovary tissue, rather more refined in my experience, although staining is a very dark art indeed! Great fun and very rewarding when good sections rise from the depths of a wax block after the mighty Shandon has had a nibble at them!

Can't wait to get home and start a new investigation! Oh - enjoying my holiday of course, currently it's blowing a gale and horizontal rain is battering the tiny wooden windows of the charming cottage we're in - oh, the table (only place available) upon which I reverently place the stereo 'scope when we arrived is nowhere near a power-socket, theres only 1 double in the room! Flagstone floors and solid stone walls - I feel like Fred Flintstone! Still, off to buy a lonnnngggggg extension lead tomorrow if I can find a shop within 50 miles! Ahhh..... holidays, such joy!

[mrsonchus]

Back home! Had to abandon cottage when all power failed at 1pm, just got out of the village as 2 of the only 3 routes out were under 3-4 ft of water and the last one, the one we managed to pass thanks to our 4wd, was at about 18 inches as we crept through, back along a dale, over some tops and home! I still need to return to collect the rest of our belongings hopefully on Wednesday if possible.....

What a day - no energy left really to do much other than sleep....

Back soon.