Sectioning and slide making - Sonchus (Sow-Thistle) flower-heads

[mrsonchus]

Hi all, I've been busy comparing tissue-processing protocols for the inclusion of a 'clearing' stage using a wax solvent such as 'Histoclear' and for the exclusion of a clearing stage altogether, the dehydration with alcohol progressing directly to wax infiltration rather than via Histoclear.
Anyway, that trial is in progress and I have slides stained and mounted, but I haven't written-up my results yet - but when I do I may well put another post in the forum to show folks what happened..

As an off-shoot of the above trial I have about 20 or so wax-blocks to 'play with' and in this post I aim to show the sectioning of some conventionally-processed composite (i.e. comprising dozens of tiny florets on one flower-head, which look like a single large flower but is actually the tiny florets en-masse as it were) flower-heads, including a clearing-stage.

These sections are both transverse - across the flower-head, and longitudinal, through the flower-head 'length-ways' and are quite interesting as progress is made 'down into the flower-head' as sections are cut during transverse sectioning in particular. The spiral arrangement of the florets soon becomes evident as the top of the flower-head is breached and sectioning moves deeper into the florets...

The Sonchus.asper with it's composite yellow flowers, looks like this;

ws_DSCN1342.jpg (43.53 KiB) Viewed 5301 times

the big yellow head is made-up of dozens of tiny 'florets' that look like this.

ws_sonchus asper floret.jpg (59.28 KiB) Viewed 5301 times

For the longitudinal sections I divided a flower-head 'down the middle' to allow the inner face to be presented to the microtome's blade as sectioned, here's a fresh flower with the tiny florets all lined-up inside,

ws_sonchus flower head (3).jpg (41.48 KiB) Viewed 5301 times

Here's a (closed) flower-head with it's top leaves peeled back to reveal the tiny florets within from above - this is the face to be sectioned for the transverse sections, the flower-head will be presented 'head-first' to the microtome's blade for this orientation...

ws_sonchus flower head (2).jpg (39.34 KiB) Viewed 5301 times

This is the wax-block to be sectioned longitudinally - even in the wax-block the florets are clearly visible...


This is the partly-sectioned transverse flower-head wax-block - this is the 'top-down' face with it's top taken off as sections have already been taken at this stage...


Here's a quick video of the cutting of the transverse sections(6µ) - one hand for the camera, the other to pick up the section-ribbon... tricky!


The results...


I bought a few 'double-sized' slides and cover-slips a while ago now, with a view to perhaps using them for long-ish series of serially-cut sections to show structure as the sections 'drill-down' through the tissue, much as they do with these flower-head sections. Anyway, as well as many normal slides, I floated some sections onto these to 'see what happens' - and was able to get a whole series onto each 'super-slide'.
Here are a few images of some of these sections floated fresh onto slides and onto a couple of those big-blighters....


The super-slide with normal single-sized slides - twice the room!



Some nice sections resulted,
some LS sections,


Here are some nice-looking TS sections,


Closer-in the wonderful spiral patterns of the tiny floret positions upon the capitulum is visible, just as they were laid-down as primordia from the (now defunct) apical-meristem whence they came....


Close-in the individual florets that have been transversely sectioned are visible with their 5-way organography evident even in this 'still-in-the-wax' stage...
Can't wait to get these de-waxed, stained and mounted onto slides!


So we have longitudinal stages,


and transverse stages,


So, just these 'in the wax' versions tonight, but tomorrow the fun starts, as I should, given the time, be able to get a few stained and mounted! I suspect these will be great for staining practice!

Back soon with some far more interesting and colourful images of the resultant slides from these little beauties!

Hope you like them. I've included all the early images as many may not have seen how slides come from the raw material of the plants - although I must also apologise for repeating in essence what I have posted before...

Back soon!

[zzffnn]

Very nice work, John B. I especially like the above slide/image. You know flower heads are my favorite plant subjects. Please save a slide like that one for me, I would like to buy it from you; maybe we can slowly assemble a few slides, then have you sent them together. Thank you!

[charlie g]

Fantastic work ( as you often share with us,Mr.Sonchus!), thank you, thank you. I especially drop into the Fibonacci sequence of nature which manifests in your transverse composite flower heads in early development.

How stunning, to gaze down into the transverse flower head which unleashes a beguiling spiral sequence ( yes, yes...you have captured a Fibonacci sequence with your craft...as deep a calling to microscopy views as if a beacon to songs of nature...I'll calm down now.). Wonderful botanical explorations, I sense my sleeping meadows flowers in a richer light due these slides you share here!

Charlie Guevara, finger lakes/US

[mrsonchus]

Hi Charlie my friend - good to hear from you. Yes, as you know the FS crops-up throughout nature - in plants it's always present in arrangements pf multiple structures and organs, for example phyllotaxis - the most efficient arrangement of leaf generation series in terms of the photosynthetic function. The instances go on forever in the Botany it seems to me!

Pleased you like the work old chap, I managed to get a chance to quickly make some slides today - see next post for images....

John B. Having so much fun with plants it should surely be illegal!

[Hobbyst46]

Mr. Sonchus - I cannot do what you do, but I share your feelings about the plants. There are similar yellow dandelion flowers (or a very close relative) around here - now I will get to know their anatomy!

[mrsonchus]

Mr. Sonchus - I cannot do what you do, but I share your feelings about the plants. There are similar yellow dandelion flowers (or a very close relative) around here - now I will get to know their anatomy!

Great to hear my friend - yes, I've grown to love Botany, and after a couple of years into microscopy and learning the basics of Botanical microtechnique I now find myself in the extremely fortunate position of being able to make my own sections - sheer pleasure and to say the very least fascinating!

I've a new post with finished slide images - posting here in about 5 minutes....

Thanks for looking, John B.

[mrsonchus]

Aha!
Had a little time today and managed to get a few early slides mounted of these transverse sections with the beautiful patterns - see Mr G's post above...
I have some images from two thicknesses of section, namely what I call 'morphological' sections at a mighty 12µ. Great for showing overall structure and at this thickness the tissue greedily sucks up stain and gives very vivid images, and at a much thinner 5µ that I call my 'histological' sections as they show much tissue detail right down to the intra-cellular levels - you'll see the differences in the following images I think...

All the following images are stained with my own 2-in-1 stain worked on a while back now, comprising Safranin and Alcian-blue mixed to give a readily differentiated (simply alcohol then acidified alcohol) one-shot stain which the more I use the more I like.

It's an incredibly efficient not to mention convenient mixture, needs very little staining time (in fact only 5 minutes for 12µ sections and 12-13 minutes for 5µ sections - thinner sections, perhaps counter-intuitively, require longer staining times than thicker sections to 'build up the density of stain' as I see it) and is simplicity itself to use.

Here are some nice images of the previously pictured sections last seen 'still in the wax' yesterday, after a night drying, dewaxing, staining and mounting today as permanent slides in resinous mountant ('Numount') - I hope you like them....

Here's a 12µ section that has just breached the (top) outer-most leaves covering the composite flower-head within. The myriad and minute immature florets begin to be seen - a very exciting stage in sectioning!


A little later we start to see the whole population appearing, which will be at varying stages of development as the florets upon such a composite capitulum develop at different rates according to their distance from the centre of the flower-head - the outer florets develop earliest and onward as they get nearer to the least-developed florets at the centre - a phenomenon also and more commonly seen/noticed in a close relative of these little compositae - the Sunflower.

Again at 12µ,


Closer-in still at 12µ, individual florets, (how about a suit made of this patterned material! )...
These are sectioned at the bases of the florets - through the actual ovaries and ovules by the look of things. Below the level of pappus-hairs (which come from the upper-rim of the ovary that will be the 'seed' when mature). The red structure is the actual seed - surrounded by the outer-covering that we tend to think is the seed, but is in fact a hard fruit that looks like a seed! What look like numerous vascular-bundles are just that - when the flower atop this ovary is living (as now) these vessels run through the fruit and supply the floret structures with water etc - these ovaries are below or 'inferior' to the flower (floret) - similar to a Daffodil where the bulge below the flower is such an (far larger of course) inferior ovary. I must put up some labelled images when I get the chance.....


As may be seen with the above 12µ sections, they really take stain well and have differentiated between the red Safranin and the Alcian-blue very nicely and quite accurately.

Here are some nice thin and finely-detailed 5µ sections - again they've stained and differentiated nicely, at 13 minutes staining-time as opposed to the 5 minutes used for the above 12µ versions..

A complete transverse section with again florets at various (developmental) stages and sectioned at various depths too - as not only do they vary in maturity with position but they vary in height, as the capitulum upon which they form is quite concave when viewed from the side, meaning the outer florets will not only be at a more mature stage than those closer to the centre, but they will be sectioned 'further down' the floret as they are slightly higher (and so encounter the microtome's blade higher-up their structure) than those closer to the centre also...


Closer-in and also at 5µ, these florets are sectioned across a plane that includes the tiny hairs that will form the 'parachute' of the seed, and may be seen surrounding the floret as tiny 'bunches' of cells - each of which is a cross-section of a single hair ('pappus').


Here's a single floret.
Even closer-in the periphery comprising 5 (fused into a single 'strap shaped' 'ligule') petals stained red by Safranin is seen, then as we move inwards the inner (sexual) structures become apparent - the anthers (also fused into an 'anther-tube' at their tops), stigma etc - all becoming visible as the Mighty Shandon moves through the tissue - truly fascinating and rather beautiful I think!


I've got to go and do other things now, but I'll be back tomorrow perhaps with some of those longitudinal sections also - interesting to put them side-by-side to trace the floret structures as sectioning-planes deepen....

Back soon, hope you like them so-far!

John B.

[Bufo Bill]

Fabulous slides and images as always John. I feel a well prepared slide is one of those items one could refer to as "artisan", and you definitely fit that bill.
Regards from Bill.

[vasselle]

Bonjour
Superbes images avec une démonstration bien détaillées.
Très bon travail
Cordialement seb

[mrsonchus]

Thanks Bill & Seb' - pleased you like them - great fun to do! More to come!

John B.

[zzffnn]

Even my 7 year old son likes those images, John B! Keep up your nice work!