Leaf TS stain-trial, Safranin vs Fast-green - the battle for supremacy!

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mrsonchus
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Leaf TS stain-trial, Safranin vs Fast-green - the battle for supremacy!

#1 Post by mrsonchus » Sun Apr 03, 2016 11:46 pm

Hi all, as you may know I'm currently working to improve my staining technique; at this time I'm concentrating on the stain/counter-stain combination of Safranin (pink/red) with Fast-green. This is a very, very well-known combination and one that I particularly like for the differentiation of tissues comprising my Botanical sections.

The ideal result generally, is to use the 'basic' (in that it stains acid-tissue elements) Safranin (pink-red sometimes orange-red) to stain nuclei, chromosomes when condensed during cell-division and other elements also acid in their exposed (to incoming stains) tissue-chemistry, then to 'counter-stain' the basic (chemically speaking) elements with the acid-stain Fast-green.
Practically this means with perfect differentiation of the stain/s that the Fast-green will stain only cytoplasm and not any acidic structures etc that the Safranin has already stained... ideally that is! :oops:

Anyway, the balance isn't an easy one to get right let alone perfect, and like all staining techniques in my limited experience is variable across different tissue types, thicknesses etc - many factors effect staining results. This makes staining a very difficult thing to learn without a lot of experimentation, and these pictures are from tonight's 'session' in my teeny lab (dining-room :oops: )..

I just bought a new cheap condenser for £15 with some of my slide-income and have seen a noticeable improvement of in particular images taken with my Toupcam camera. They seem a lot sharper and of a better resolution than they were with my 'old' rather tatty condenser. The new one is similarly a very basic abbe 1.25 but I think is of a better quality or maybe condition - hard to be sure though as these are the first and only images I have of these particular slides. Time and more pictures of different slides will tell I suppose. Anyway, enough talk, here are some pictures..

This is a section stained primarily with fast-green and a tiny (unintentionally so I hasten to add :oops: ) measure of Safranin which is all but invisible. I still like this result as the Fast-green has made a fine and rather delicate image to my eyes, with pretty good detail despite the rather low contrast of this stain when used alone... This is a 2-picture stitch of a Daffodil (again!) leaf TS and has most leaf-morphology within..
ws_edit1-x20-stitched-daff-.jpg
ws_edit1-x20-stitched-daff-.jpg (218.05 KiB) Viewed 1182 times
Here's a rather better single image of the same tissue with the same objective, and a few labels,
ws_edit2-3-pic-FFDSSD-stack.jpg
ws_edit2-3-pic-FFDSSD-stack.jpg (279.08 KiB) Viewed 1182 times
The labels are:
p = 'parenchyma' - a 'filler tissue' in the center of the leaf that also stores substances, large volume cells..
vb = 'vascular bundle' AKA leaf-vein running parallel to long-axis of leaf and so seen in TS...
sm = 'spongy mesophyll' - (mesophyll is another name for parenchyma, when found in a leaf) a hardly if at all photosynthetic type of parenchyma with an open 'spongy' cell arrangement, facilitates gaseous exchange within leaf tissue by virtue of this structure also...
pm = 'palisade mesophyll' = 'long' chloroplast-filled and therefore photosynthetic cells found immediately below the epidermis...
s = 'stomate' (pl stomata), a pore that opens & closes to regulate gaseous exchange - often has an large chamber behind it, the 'sub-stomatal chamber'...

I think the very-predominant Fast-green has made a reasonable job of staining, many structures & tissues are readily discernible it seems...

Now, here's a picture of how the tissue looks when the Safranin & Fast-green work together in their intended stain/counter-stain roles - to me a far superior result....
ws_edit1-x40-FFDSSD-stack-3.jpg
ws_edit1-x40-FFDSSD-stack-3.jpg (274.72 KiB) Viewed 1182 times
The Safranin has stained the cuticle very nicely (the red strip on the epidermis' surface) and shows quite clearly the way this cuticle is continuous within the stomate-linings that form the pore itself.. The Safranin has just started to catch the chloroplasts also.

In the following picture we see how the chloroplasts may also be made to stand-out clearly with a little extra Safranin....
ws_edit1-x60-palisade-FFDSS.jpg
ws_edit1-x60-palisade-FFDSS.jpg (318.54 KiB) Viewed 1182 times
The chloroplasts (strange to think they are naturally green :D ) are really easy to see here, together with the way they 'pack' the palisade cells to maximize photosynthesis - the palisade cells' nuclei have also appeared as if by magic - their acidic contents stained by the basic Safranin - lovely to see. Also the cells (parenchyma/mesophyll) behind the palisade layer may be seen to have small amounts of what I think are chloroplasts only, and these are thinly lining the cell-walls rather than filling the cell (see left-edge of image just above the text-box)! Amazing what stains are able to show when they're feeling cooperative! :D

Well, that's about all I've time for tonight, I hope this hasn't been a little long-winded, it's a habit of mine I'm afraid. I just thought that this small example of one of my explorations of a couple of slides may give folks an insight into the reasons that I love Botany and histology so much! The mysteries are endless, the surprises constantly appearing, the adventures always exciting!

Anyway, I'd better get some sleep, the dog wants to go out also!

I hope you enjoy this little adventure as much as I have! :D
John B

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vasselle
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Re: Leaf TS stain-trial, Safranin vs Fast-green - the battle for supremacy!

#2 Post by vasselle » Mon Apr 04, 2016 1:42 am

Bonjour.
Très belles séries images.
Et les commentaires sont clair.
Merci pour le partage
Cordialement seb
Microscope Leitz Laborlux k
Boitier EOS 1200D + EOS 1100D

JimT
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Re: Leaf TS stain-trial, Safranin vs Fast-green - the battle for supremacy!

#3 Post by JimT » Mon Apr 04, 2016 10:43 pm

The Safranin has stained the cuticle very nicely
.

Reminds me of my Mother's painted fingernails. Like the way Safranin brings out extra detail but have you tried desaturaing the red channel in post processing?

JimT

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mrsonchus
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Re: Leaf TS stain-trial, Safranin vs Fast-green - the battle for supremacy!

#4 Post by mrsonchus » Mon Apr 04, 2016 10:55 pm

JimT wrote:
The Safranin has stained the cuticle very nicely
.

Reminds me of my Mother's painted fingernails. Like the way Safranin brings out extra detail but have you tried desaturaing the red channel in post processing?

JimT
I do in a hit & miss manner, I often however need to lower the default colour saturation (128) of Toupview to about 100 though - it can be a little 'overenthusiastic' otherwise! :D

The Toupcam isn't very keen on reds I notice, it's much better with blues I find in general. I'll have a little tweak of the red channel and maybe overall saturation, may save the need for sunglasses! :D

Watch this space for a couple of toned-down versions, I find it often brings detail out to an extent in the images. I wish I had a camera that would give me what I see through the 'scope! Is there such a camera of a manageable cost?
:)
John B

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Re: Leaf TS stain-trial, Safranin vs Fast-green - the battle for supremacy!

#5 Post by mrsonchus » Mon Apr 04, 2016 11:17 pm

Here are a couple with the saturation turned down a little and also the red channel turned down in highlights, mid-tones and shadows, a little better I think, not so garish on screen..

The original,
ws_edit1-x40-FFDSSD-stack-3.jpg
ws_edit1-x40-FFDSSD-stack-3.jpg (274.72 KiB) Viewed 1153 times
Edited a little, perhaps I shouldn't have lightened it though...
ws_edit2-x40-FFDSSD-stack-3.jpg
ws_edit2-x40-FFDSSD-stack-3.jpg (285.9 KiB) Viewed 1153 times
I think there's a slight improvement in the detail of the stomate's outer-'teeth' and the lining (cuticle continuation) of the pore in this one, very hard to say really.

Similarly, original,
ws_edit1-x60-palisade-FFDSS.jpg
ws_edit1-x60-palisade-FFDSS.jpg (318.54 KiB) Viewed 1153 times
Edited slightly to tame the reds..
ws_edit2-x60-palisade-FFDSS.jpg
ws_edit2-x60-palisade-FFDSS.jpg (327.98 KiB) Viewed 1153 times
I think there is a slight reduction of textural information of the nuclei in this one though, hard to tell with only 2mp - but better than 0mp! :D

A little better, depends what you're trying to show also of course, different versions for different reasons often. Personally I prefer the toned-down versions.
:)
John B

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billben74
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Re: Leaf TS stain-trial, Safranin vs Fast-green - the battle for supremacy!

#6 Post by billben74 » Tue Apr 05, 2016 9:47 pm

Lovely images and I agree about the toned down effect.
Garish has its place, but not thats not here I think.

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Re: Leaf TS stain-trial, Safranin vs Fast-green - the battle for supremacy!

#7 Post by mrsonchus » Tue Apr 05, 2016 10:17 pm

billben74 wrote:Lovely images and I agree about the toned down effect.
Garish has its place, but not thats not here I think.
Hi Bill, I thought you may have more sections to follow the first ovary pictures? How are things going old chap? :)
John B

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Re: Leaf TS stain-trial, Safranin vs Fast-green - the battle for supremacy!

#8 Post by billbillt » Tue Apr 05, 2016 10:32 pm

Wonderful photos... As always top shelf work!...

BillT

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Re: Leaf TS stain-trial, Safranin vs Fast-green - the battle for supremacy!

#9 Post by billben74 » Tue Apr 05, 2016 10:49 pm

On the ovary front I haven't had chance to process more. I will look at the previous ones at x1000 soonish (and post when I do).
Spinning lots of plates at the moment...

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Re: Leaf TS stain-trial, Safranin vs Fast-green - the battle for supremacy!

#10 Post by mrsonchus » Tue Apr 05, 2016 10:51 pm

billben74 wrote:On the ovary front I haven't had chance to process more. I will look at the previous ones at x1000 soonish (and post when I do).
Spinning lots of plates at the moment...
Yes, you've a busy time of it Bill. Always on the look out to see how you're going! :D
John B

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Re: Leaf TS stain-trial, Safranin vs Fast-green - the battle for supremacy!

#11 Post by mrsonchus » Tue Apr 05, 2016 11:04 pm

billbillt wrote:Wonderful photos... As always top shelf work!...

BillT
Thanks Bill, you're very kind and encouraging my friend. The leaf-sections, taken from my stock of slides in wax, have really been quite a revelation. I keep finding new information within them, I suppose the more I learn re Botany the more becomes recognizable within my sections! :D It's always an adventure, every time I have a 'slide-surfing' evening, pure pleasure.

I've some doubly-embedded trials coming-up, the tissue seen in my earlier post for the trial to improve my previously 'bitty' agar are ready to section! It seems as though I've completely eliminated the problems I was having, the agar is now smooth as silk with no inclusions whatsoever!
John B

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Re: Leaf TS stain-trial, Safranin vs Fast-green - the battle for supremacy!

#12 Post by Raul » Thu Apr 07, 2016 9:29 pm

Great photos wow, I cannot remember seeing such good photos in textbooks (which are supposed to be the best of the best) John you have evolved very much, there is a lot of hard work and trial and error but you have done it.
Congratulations for all that you've done and thanks for sharing your work on the forum.

Regards,
Raul

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Re: Leaf TS stain-trial, Safranin vs Fast-green - the battle for supremacy!

#13 Post by mrsonchus » Thu Apr 07, 2016 9:49 pm

Thanks Raul, that's a very generous thing to say. I'm so pleased folk like my adventures, I certainly love my slide-making. :D
John B

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