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 Post subject: Leaf-veins in TS and LS
PostPosted: Fri Dec 08, 2017 12:05 am 
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Hi all, just a few images you may like from this evening's slide-surfing of some nice (Daffodil) leaf TS made a while back using one of my 'extreme' protocols - extreme in terms of the short length of the protocol from fresh tissue to sectioned tissue floated onto slides. In this case I tried a 10hr protocol and it worked very well with the Daffodil tissue.

Anyway, re-visiting these slides to assess their condition after nearly two years as far as I can remember - coupled with the fact that I've been away for the last week and these images are taken with my 5mp Toupcam eyepiece camera using my trusty 'portable' SM-LUX while I was away from the lab and the Orthoplan - and the fact that I just love slide-surfing! :D :D

These are TS of Daffodil leaflets from about Feb 2016 I think. I was looking at them and considering how nice the vascular bundles of the leaflets have sectioned.
The 'bundle sheath' cells that surround the vessels, both those going 'along' the leaf and those traversing the leaf are easily seen around the veins and veinlets in these sections. These sheath cells support, to some small extent, the bundles through the more open tissue and are a way for substances (water and dissolved minerals maybe) from the roots to move from the xylem out into the leaf cells and so-called 'phloem loading' of sugars produced by the chloroplasts to move into the phloem and so be distributed, used or stored elsewhere in the plant's tissues and organs.

Anyway, details aside, the veins and their surrounding bundle sheath cells are seen in transverse section and the lateral veinlets that are 'sideways' across the axis of the leaf have, together with some bundle sheath cells above and below, been caught in longitudinal section (that is LS of the veinlets, the leaf section is in all cases TS).

Nice colours too, so I though I'd post them up for your perusal.

Here are a few sections freshly floated onto slides, still of course in the sectioning wax. The boomerang-shaped sections are the daffodil leaf sections seen in the following images,
Attachment:
ws_10_hour_extreme_protocol.jpg
ws_10_hour_extreme_protocol.jpg [ 138.51 KiB | Viewed 296 times ]


Here are a couple of bundles and their sheaths caught in TS,
Attachment:
ws daf leaf veins.jpg
ws daf leaf veins.jpg [ 209.61 KiB | Viewed 296 times ]


In closer the xylem, phloem and bundle sheath cells are clearly discernible,
Attachment:
ws daf leaf veinlet.jpg
ws daf leaf veinlet.jpg [ 158.26 KiB | Viewed 296 times ]


Here's a lateral vascular bundle (leaf vein) and sheath caught in LS within the TS of the leaf section,
Attachment:
ws daf leaf lateral veinlet (2).jpg
ws daf leaf lateral veinlet (2).jpg [ 69.86 KiB | Viewed 296 times ]


The last image is a funny shape as I had to rotate it as image is from part of the section that is in one of the 'arms' of the 'boomerang-shaped' leaf sections and so not originally horizontal....

Hope you like them, great fun just to slide-surf some evenings - I always find something new or a better view or image of something seen many times before - great fun and very satisfying. Such complexity as seen within plants never fails to teach me something wonderful every time I take the time to look and see through the trusty Orthoplan's glassy eyes!

John B. :D :D :)

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PostPosted: Fri Dec 08, 2017 9:33 am 
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Great images, John
... and your annotations are exemplary.
Many thanks for sharing these.

MichaelG.


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PostPosted: Fri Dec 08, 2017 10:31 am 
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Very instructive and beautiful.
Please give a link to the protocol? Surely the stains you used are specified there?
Thanks!

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PostPosted: Fri Dec 08, 2017 10:52 am 
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MichaelG. wrote:
Great images, John
... and your annotations are exemplary.
Many thanks for sharing these.

MichaelG.


Thanks Michael, I seem to have made the images a little on the bright-side... :oops:
Great fun to do, and always great to know others like 'em my friend. :D :D

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PostPosted: Fri Dec 08, 2017 11:13 am 
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Hobbyst46 wrote:
Very instructive and beautiful.
Please give a link to the protocol? Surely the stains you used are specified there?
Thanks!


Many thanks Hobby' - you're very generous.

The 10hr protocol is my own, I've run many protocols and the optimum is so often on a per-tissue or even batch level it never ceases to keep me on my toes! :D :D

Here are some of my lab notes from then,
Attachment:
ws_10_hour_extreme_protocol (1).jpg
ws_10_hour_extreme_protocol (1).jpg [ 77.52 KiB | Viewed 266 times ]


and the protocol itself,
Attachment:
ws_10_hour_extreme_protocol (3).jpg
ws_10_hour_extreme_protocol (3).jpg [ 48.81 KiB | Viewed 266 times ]


and,
Attachment:
ws_10_hour_extreme_protocol (2).jpg
ws_10_hour_extreme_protocol (2).jpg [ 42.69 KiB | Viewed 266 times ]


The staining - a very difficult thing to give a precise protocol for as small, sometimes counter-intuitive factors are very often of important...
In a nutshell - the red stain is aqueous Safranin - 1% w/v (i.e. 1g of stain-powder to 100g/ml of distilled water) - slides are hydrated then stained in Safranin for between 2 and 30 minutes depending upon the balance between red and green staining and of course the level of specificity/differentiation required between the tissue elements that stain best with Safranin (such as lignified 2ndary cell-walls of xylem vessels, nuclei..) and those that stain best with the green (often looks more blue in images) stain (Fast-green) such as cytoplasm.
The Fast-green stain is a very fast-acting stain that is added to the slide with a pipette for about 5 to 6 seconds before being gently. evenly and quickly removed with OH. The Fast-green is a 0.5% solution in 85% alcohol.

Hmm, that nutshell seems to be quite large.... sorry, here're the 'bare-bones' of it! :D :oops:

hydrate to de-ionised water (battery top-up water)
Safranin 1% aqueous for 2-30 mins
dehydrate to 95% alcohol (the strongest available isopropanol) and perfectly suitable
Fast-green 0.5% in 85% alcohol (I use isopropanol as it's cheap and easy to get)

If you need fine details just let me know and I'll give some more info.

John B. :D :D :)

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PostPosted: Fri Dec 08, 2017 3:09 pm 
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mrsonchus wrote:
Thanks Michael, I seem to have made the images a little on the bright-side... :oops:

Perhaps a little, John ... if you're looking for maximum visual information from the image.
But I think these images of yours are more equivalent to the very best hand-drawn illustrations; and they are informative in that rather special way.

MichaelG.


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PostPosted: Fri Dec 08, 2017 3:41 pm 
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Thanks a lot.

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PostPosted: Fri Dec 08, 2017 5:34 pm 
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MichaelG. wrote:
Perhaps a little, John ... if you're looking for maximum visual information from the image.
But I think these images of yours are more equivalent to the very best hand-drawn illustrations; and they are informative in that rather special way.

MichaelG.


Very true - it's a decision that must be made, although we're all lucky enough to have the decision available with DPP these days of course. The choice of the more 'natural'-looking photographic version or the processed-away-from-that image towards the high-contrast almost 'poster-like' version that as you rightly say appears almost hand-drawn.

I always find, following this theme, that the very best, as in informative of structure and detail, images in my many Botanical books are the hand-drawn ones, especially the monochrome line drawings. These images allow the artist to pick details to represent and 'put them into the image' rather than the purely photographic guides that are packed full of high colour photographs, each lacking often the one particular character that one would actually like to clarify.

Thanks for your input and generosity - much appreciated my friend. :)

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PostPosted: Fri Dec 08, 2017 5:36 pm 
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Hobbyst46 wrote:
Thanks a lot.


You're welcome old chap - any info I can help with, just ask - I've been through an awful lot of work to try to learn the art of microtechnique over the last couple of years and it's always great to pass-on some of what I've been up-to.

John B. :)

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PostPosted: Thu Dec 14, 2017 7:20 pm 
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Very nice! Work like yours is always a joy to observe.


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PostPosted: Thu Dec 14, 2017 9:18 pm 
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einman wrote:
Very nice! Work like yours is always a joy to observe.


Thanks einman, pleased you like them - although the more I look at a couple of those images the more embarrassed I feel - I may just take some fresh images of the same slide.... :oops: :D :D

I've been back to the pollen exines tonight, sectioning some nice 2µ and 4µ to compare to the earlier featured 6µ sections.

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PostPosted: Thu Dec 14, 2017 9:40 pm 
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Fantastic. Makes me impatient for Spring - not even officially Winter yet.

Always enjoy your images and explanations.

JimT


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PostPosted: Thu Dec 14, 2017 9:40 pm 
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Sorry for the duplicate reply. Don't know how to delete messages.

JimT


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PostPosted: Thu Dec 14, 2017 9:50 pm 
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JimT wrote:
Sorry for the duplicate reply. Don't know how to delete messages.

JimT


Hi Jim! Thanks my friend - yes, can't wait for everything to burst back into life! Here in my part of the UK it's alternating between cold & icy and very wet & soggy....

I've some nice Schlumbergera and Cyclamen floral buds in OH right now - hoping to get them through processing and into blocks over the weekend.

John B. :)

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PostPosted: Thu Dec 14, 2017 11:15 pm 
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Yes well one day I will break my Leitz 1512 microtome out and actually use it. Every time I see your work I keep telling myself I need to start following you more closely so when I do get started ...LOL

I have slowly been accumulating supplies. I acquired 10 Coplin glass staining jars with lids the other day. Add that to the slide warming table, bath, melting pot and the incentive to get started is mounting.

My wife just stares at teh boxes when they arrive.


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PostPosted: Thu Dec 14, 2017 11:20 pm 
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Einman, go for it! John B is an excellent teacher and mentor.


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PostPosted: Thu Dec 14, 2017 11:29 pm 
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einman wrote:
Yes well one day I will break my Leitz 1512 microtome out and actually use it. Every time I see your work I keep telling myself I need to start following you more closely so when I do get started ...LOL

I have slowly been accumulating supplies. I acquired 10 Coplin glass staining jars with lids the other day. Add that to the slide warming table, bath, melting pot and the incentive to get started is mounting.

My wife just stares at teh boxes when they arrive.



Go-on einman - you know it makes sense my friend! :D :D

You need very little to start you off - glacial acetic acid, isopropanol 95% alcohol, 'formalin', de-ionised water, Histoclear (original), Safranin powder, fast-green powder - the basics!

You're almost there!..... :shock: :D

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PostPosted: Thu Dec 14, 2017 11:34 pm 
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How much do you pay for Histo-Clear there? I will move this thread.


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PostPosted: Thu Dec 14, 2017 11:57 pm 
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einman wrote:
How much do you pay for Histo-Clear there? I will move this thread.


See you on the new thread old chap.

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PostPosted: Fri Dec 15, 2017 11:31 am 
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Folks
Reminder - glacial acetic acid is corrosive to the skin and to the respiration organs. Use with care, gloves and proper ventilation.

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