Colourful Exploration of Lichen Structure

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mrsonchus
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Colourful Exploration of Lichen Structure

#1 Post by mrsonchus » Fri Feb 12, 2021 10:13 pm

Hi all, well, finally got a few slides made of a foliose lichen!

These are they (or some of them) as on a twig from an apple tree in my garden)
Image



Having made a very quick test-slide the other day (see the story from the beginning in this earlier post for more detail on the process..) I had a little play-about today with a variety of stains, to see what I can bring out of the first slides, cut at a rather thick 10µ. I'll be sectioning to about 5µ probably over this weekend in an attempt to bring out more detail, but the nature of lichen isn't a regular structure of neat cells as found in true plants. Rather they have essentially a 'sandwich' structure of fungal-filaments and algal cells, sometimes in clumps, singly, chains etc depending on the variety of alga 'partnered' with the fungus of the 'whole' as it were - i.e. the lichen proper.

Also I know virtually nothing about lichen - so please add any detail of interpretation of these images to this thread if you have the knowledge. I've tried to label the parts as best I can at this time, but am highly likely to be in error in areas, except maybe for the identification of the algae and the fungal-hyphae - maybe - please bear with any errors seen here with my labelling - I'm a very new lichen-botherer!

Some colour and hopefully structure,


Here's a portion of the thallus of the lichen, sectioned then stained with a common combination of safranin and alcian-blue stains, both of which will be virtually permanent when mounted as slides - hopefully...
Image

It's pretty clear here that there's no regularity of structure, as mentioned above, within this tangle of fungal hyphae at the center or 'medulla' of this lichen!
Image


Fungal hyphae in medulla, closer-in.
Image



Interesting to see how the thallus edge is structured also...
Image



In an attempt to increase contrast of staining in some structures I then switched to the application of 'orange-g' stain, together (this is not a simultaneous stain protocol, the stains are applied, differentiated and assessed individually and serially) with 'rhodamine-B' (red) and a tiny touch of 'lacto-phenol cotton-blue' (which is barely seen). Overall the contrast is high however and colourful, which is always a nice feature I think.
The fungal elements have I think been stained green by the combination of LPCB and OG stains, both of which may have bonded with the algae to give green, an effect often seen with the use of a yellow and a blue stain in the same tissue - not always do they mix, but they often do. I think this is what the green is due to - the tissue was certainly not still green when sectioned and mounted!

Some of the algae also seems to have been stained red by the rhodamine-B rather than green as aforementioned.... Also it seems that algal filaments can be seen to wrap around some of the algae that are red-stained.... Seemingly a paradox but maybe the algae are different or in different states in each apparent staining result? I personally have no idea as I know nothing about lichen or indeed algae staining, this is my first attempt with this fascinating 'stuff'...
Image


Easier to see closer-in.
It occurs to me, having had a look around the net, that these red-staining algae (if that's indeed what they are) which seem 'packaged' almost, by fungal hyphae thread, may be a reproductive structure called 'soredia' (sing soredium). If they are, I wonder why they stain red and seemingly other algae in the same staining protocol seem to stain green? Of course my assumptions for this question may themselves be totally wrong - if anyone has any knowledge of this please feel free to add to the thread as I for one would be very interested to know more!
Image

Labelled as best I can - please correct errors - I'm learning about lichen as I go...
Image


I also noticed that, in the upper and lower cortyex there seem to be areas that haven't taken stain, and they are not random - perhaps crystals or birefringent structures/particles lie within? So, a quick look through crossed polarisers may help..
It seems to be the case as they seem to be glowing appropriately....
Image


Application of a full-wave retarder plate in addition to the crossed-polarisers should give diferrent colour-shifts in structures with a regular orientation such as strands or crystals may possess (or at least this is my very basic understanding of retardation-plates - please add info if you can) according to their orientation to the 'axis' of the retardation plate (I think...).
This effect does seem to be shown also, although I don't really have an interpretation for exactly what it shows I'm afraid, I'm virtally in the dark with lichens I'm afraid. A nice coloured image at least.
Image

I'm afraid that's all I have time for this evening, but even from these early slides it seems clear that these lichen thingies have a lot of hidden details worth exploring. I have 22 more tissue wax-blocks from which to make sections (see earlier post referenced in link above) and some include the reproductive 'cup-like' structures that lichens have, which I believe are called 'apothecia' (sing apothecium) - which may be very interesting to see sectioned and stained - looking forward to finding some in sectioning!
John B

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Re: Colourful Exploration of Lichen Structure

#2 Post by zzffnn » Fri Feb 12, 2021 11:28 pm

Beautiful work, John B, as always. Please keep them coming.
Last edited by zzffnn on Sat Feb 13, 2021 12:31 am, edited 1 time in total.

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Re: Colourful Exploration of Lichen Structure

#3 Post by Crater Eddie » Sat Feb 13, 2021 12:25 am

John, you come up with some of the neatest stuff. It is always a pleasure following your adventures.
CE
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Re: Colourful Exploration of Lichen Structure

#4 Post by MicroBob » Sat Feb 13, 2021 8:17 am

Hi John,
very interesting observations! It really is fascinating that fungus and algae find togehter to form a cooperation - something we humans often don't manage though we have hands and a brain. And this not only under an overhang inside a crater somewhere but in many and different places.
Your paraffin embedding obviousy gives a better result then my attempt with PEG, so I will soon give this a try too. In contrast to other botanical material lichen can probably harvested in winter without severe drawbacks.

Bob

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Re: Colourful Exploration of Lichen Structure

#5 Post by mrsonchus » Sat Feb 13, 2021 12:30 pm

Hi Bob!

Must agree with you there, the more I see of lichen the more intriguing I find it. Everytime I look over the slides or read on a little more re lichen in general I end up with a new plethora of questions and plans for further sectioning. I'm just about to select a few of the 22 blocks in my fridge for roughing - sectioning into them to see what they have for us. I'm after some more complete sections through solid (or as solid as lichen tissue gets anyway...) thallus and the apothecia that I know are in the pieces. Should be some interesting slides to com if these starter-sections are an indication.

Hopefully back soon with an update and more images.

Thanks for the encouragement and following-along Bob, pleased you are enjoying this adventure! :D
John B

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Re: Colourful Exploration of Lichen Structure

#6 Post by MikeBradley » Sat Feb 13, 2021 10:57 pm

John, talking about intriguing lichen, there’s a good chapter devoted to Lichen in Merlin Sheldrake’ recently published book about Fungi - “Entangled Life”. In a chapter called “The Intimacy of Strangers” he explores the puzzles that are posed by the lichens. It was my reading of this chapter,in a book about Fungi that started me off exploring lichens through the microscope. The whole book is a fascinating read overall, too.
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Re: Colourful Exploration of Lichen Structure

#7 Post by mrsonchus » Sun Feb 14, 2021 12:05 am

Hi Michael, looks like a good read, I may well get a copy for myself, it's a very reasonable price.
Buried naked in a pile of damp and musty woodchips to imagine what it feels like to decompose! Now that's intriguing - and of course from said book - nothing to do with me! :D :D

I took a fresh set of sections today from another block with hopes of finding some of those reproductive structures, but have really only just scratched the surface of the material I'm afraid. I sectioned at 7µ, 5µ and 4µ with a view to increasing resolution and detail of the sections and staining. Well, this evening I was able to stain and mount one quick 4µ slide with the following results. A lot more detail and resolution as hoped, but the choice of staining - safranin and alcian-blue, was not the best I think.

Here's an image of the 4µ section, at the edge of the thallus, an improvement over the 10µ for certain, but a long way to go yet....
Image


The increased resolution and detail of the thinner section becomes pretty noticeable at highr magnification - the decreased depth of focus (a consequence of the higher n.a.?) of the higher magnification objectives is offset by the decrease in section thickness which at 4µ from 10µ is significant and removes almost all Z-axis overlap.
Image



This looks like a fold in the thallus rather than a protruding structure - but I may be totally wrong...
Image



The fungal cells (red circular larger structures) have sectioned pretty cleanly but lack detail - if there's any available, but I suspect that discrete chloroplasts should become visible, with better staining - a work in progress!
Image



Another similar to the above,
Image



More detail, unsure of the identity of the blue-stained feature that seems to protrude at the top of this image - I need to know more about lichen! If anyone has any knowledge of what's shown in these images please add to this thread.
Image

That's all I have tonight, more to follow tomorrow with a variation in staining also. I also need to section some more of the blocks in my search for the so-far elusive (but definitely present in the tissue samples, hopefully at a good sectioning orientation too) apothecia...

Back soon with further examination of this interesting subject - I need a Lichenologist!

p.s. A quick comparison of section thickness, 10µ top and 4µ below.

The 10µ section.
Image


The more detailed 4µ section.
Image


Hmmmm I think I may well try a 2µ section tomorrow - better make sure the blocks are nice and cold for that! :D
John B

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Re: Colourful Exploration of Lichen Structure

#8 Post by mrsonchus » Sun Feb 14, 2021 2:05 am

Crater Eddie wrote:
Sat Feb 13, 2021 12:25 am
John, you come up with some of the neatest stuff. It is always a pleasure following your adventures.
CE
Hi Eddie! Long-time-no-see, great to have you back, pleased you like the adventure!
John B

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Re: Colourful Exploration of Lichen Structure

#9 Post by mrsonchus » Sun Feb 14, 2021 2:06 am

zzffnn wrote:
Fri Feb 12, 2021 11:28 pm
Beautiful work, John B, as always. Please keep them coming.
Hi zz', good to know you like them, thanks for joining the adventure!
John B

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Re: Colourful Exploration of Lichen Structure

#10 Post by mrsonchus » Sun Feb 14, 2021 1:10 pm

Hi all, here are a few images from the single 4µ slide I made last night, an HDR composite from 4 images and the rest are monochrome with green IFF550 filter used to aid resolution, but then they're imaged through apochromats so the filter may make little difference anyway?

I quite like the mono-look!

Image

[with reference to the above image]
Almost forgot to mention - can you see the out of focus areas as per the red arrows? These are folds within the section, there after sectioning and floating onto slide. They're an artifact of the actual sectioning, the surrounding wax was not wrinkled like this, it was the tissue only. Looking at the section with polarisers soon revealed the cause - crystals in this area that the microtome blade has either skipped-over or just been unable to cut cleanly.
These correspond to those birefringent particles seen in the polarisation images I posted from the thicker 10µ sections in my first-look sections of an earlier post. In the 10µ there was a lot less wrinkling here, perhaps the granules were able to be accommodated of even missed/pushed aside with the extra thickness?

So, how to avoid or preclude this? Well a couple of approaches are available. One is to use a surface (of the block being sectioned's that is) application of perhaps NaOH or whatever works best as a pre-section every few sections, as used with grass tissue which is full of calciul-oxalate crystals.
The other is simply to select those sections where this is least or closest to acceptable - O.K. unless each section is precious for one reason or another and discarded sections are undesireable or even unacceptable...



Out of focus areas in the following are simply not in the shallow DOF of the image from the objective....
Image


At 60x with a dry 0.90n.a. apochromat the detail isn't bad - also used a green interference filter (Olympus IF550) to try to glean a little extra detail.
I'd love to be able to see more detail, or at least have more knowledge of what I'm actually seeing - but I'll need to read a lot more about lichen biology and get the immersion 100x out I think! Where are the Lichenologists when you need one! :D
Image

Finally a 3-stop HDR with scale, used a didymium-filter also to enhance the reds a little....
Image

Off to make some more 4µ slides from the couple of dozen in my dryer, and hopefully to go down again to perhaps 2µ - just to see if it adds anything really....
Last edited by mrsonchus on Sun Feb 14, 2021 2:14 pm, edited 1 time in total.
John B

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Re: Colourful Exploration of Lichen Structure

#11 Post by MicroBob » Sun Feb 14, 2021 2:06 pm

Hi John,
great slides with lots of detail and contrast! I liked especially the visual demonstration of the clearer image the thinner sections give, very convincing. Yesterday we went to the coast to pick up some microscopy stuff and have a walk along a cliff. In the harsh climate 20m above sea level I also collected a rotten piece of wood with lichen. I hope it keeps well until it goes into FAA.

Bob

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Re: Colourful Exploration of Lichen Structure

#12 Post by mrsonchus » Sun Feb 14, 2021 2:17 pm

Go-get it Bob - my Darling Wife takes it as normal now when I bring old twigs and bits of leaf etc back with me!

If you haven't any FAA ready just put them straight into 70% alcohol - twigs and all, they'll be perfectly fine 'til your ready for them!

Nice to see a fellow twig-snatcher out there! :D :D I just updated the last images a little.
John B

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Re: Colourful Exploration of Lichen Structure

#13 Post by mrsonchus » Sun Feb 14, 2021 10:57 pm

Now then, this afternoon I stained and mounted a few more of thos 4µ sections.

This time I used a tri-stain (sequential) of safranin, orange-g and fast-green.
The results are really quite nice, and do indeed show some nice detail with the stain and thinner section combining nicely I think.

The structure seen in this first image is I think a soralium, a chamber that develops from the 'middle layer' of the thallus of the lichen (the 'medulla') and bursts up and through the upper cortex as it forms bundles of algal cells which are wrapped in fungal filaments! The 'bundles' are ready to grow and are an asexual reproductive form called soredia.... I think that's what this is although with my very, very limited knowledge of lichen I can't be certain.

Image


Closer-in more detail is visible, although I hesitate to label details at this stage!
Image


This image shows a lot of the fungal filaments found in the medulla.
Image



Again, closer-in.
Image



Here's part of the section that includes an edge of the thallus, I've tentatively labelled some parts.
Image

Hope you like these, I didn't have time to make a fresh set of sections today, but hopefully tomorrow I can go even thinner and/or find some more interesting features within the remaining wax-block that I have to section...
John B

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Re: Colourful Exploration of Lichen Structure

#14 Post by ldflan » Tue Nov 08, 2022 10:31 pm

Wonderful series, just seeing these. The birefringent structures showing the maltese cross effect look like starch granules to me.

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Re: Colourful Exploration of Lichen Structure

#15 Post by Sure Squintsalot » Wed Nov 09, 2022 3:45 am

Agreed. Pretty stunning work. I may yet look at lichens in a wholly different light.

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