Stomates and cellulose acetate butyrate- any experiences?

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Kinetochore
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Stomates and cellulose acetate butyrate- any experiences?

#1 Post by Kinetochore » Thu Jun 01, 2023 10:35 pm

Morning all

Have a large number of species to image stomates from- and they're all tricky. The nailpolish/ stickytape tek definitely doesn't work, cyanoacrylate is too fragile. Some leaves are hairy on the underside, while others are too thick to visualise without embedding and sectioning on the paradermal plane ( I'd like to avoid that particular learning curve if possible ).

I read somewhere that 1/16" cellulose acetate butyrate ( CAB ) film can retain a good impression of leaf surfaces for light microscopy when leaves are pressed with acetone between solid plates

Anyone have any experience with this tek?

Also having trouble sourcing the CAB film in Australia

I'll try PVC wood glue while I'm waiting for other answers. Nothing is ever as easy as it says on the box, is it :lol:

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Re: Stomates and cellulose acetate butyrate- any experiences?

#2 Post by MikeBradley » Fri Jun 02, 2023 3:23 pm

Kinetochore, are you aware of this method using UV varnish and a pair of slides? I came across it on the Quekett Facebook page:
https://www.facebook.com/robert.ratford ... 2063797577

BTW, I purchased Replicating Tape from Ted Pella in the US, they have branches in Australia too.

Good luck

Michael
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Kinetochore
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Re: Stomates and cellulose acetate butyrate- any experiences?

#3 Post by Kinetochore » Fri Jun 02, 2023 10:03 pm

Kinetochore, are you aware of this method using UV varnish and a pair of slides? I came across it on the Quekett Facebook page:
https://www.facebook.com/robert.ratford ... 2063797577

BTW, I purchased Replicating Tape from Ted Pella in the US, they have branches in Australia too.
Thanks Michael! Between this site and the Ted Pella shop I think I've found my new happy place online :D

I'd seen references to the UV varnish but I've almost given up on casting methods not proven for my species ( for which there is no data ) as many of the formulations vary between manufacturers, product descriptions and countries- over time

The Ted Pella shop- wow- that tape price is so far below the prices I've seen that it makes a really viable fast alternative that will let me have a good crack at getting results for the preliminary visuals screen

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Re: Stomates and cellulose acetate butyrate- any experiences?

#4 Post by woodytia » Fri Jun 16, 2023 9:55 pm

I'm new to the forum but as a former pant chemist I have used CAB in various lacquer formulations, Eastman Kodak were the main supplier, it was much easier to use than nitro cellulose as it could be dissolved in single solvents, one of the biggest uses was as a component in the coloured layer on car bodies prior to the application of the clear coat (clear on base), it is a UV stable film former you may find folk who preserve ancient ceramic artifacts use it after they have put them back together, it might not be flexible enough to use on its own so other components may be required to plasticise it natural products like castor might work if compatible.

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Re: Stomates and cellulose acetate butyrate- any experiences?

#5 Post by Kinetochore » Tue Jul 04, 2023 11:13 am

The Ted Pella replicating tape is an absolute pearler. All sorts of things are now visible to me :D

Thanks everyone

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iconoclastica
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Re: Stomates and cellulose acetate butyrate- any experiences?

#6 Post by iconoclastica » Mon Jul 31, 2023 12:57 pm

I have been using cyano-acrylate a lot for this purpose. Mostly it works well, but indument on the leaf is an issue indeed. Also the slow curing times tend to allow the production of air bubbles arising through and getting fixed on top of the stomata. After I had made all these measurements, I also tried UV cured glue (Bondage). That looks promissing: curing faster and remaining more elastic, but I don't have enough experience with it as yet.

Could you post a link to this replicating tape?

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Re: Stomates and cellulose acetate butyrate- any experiences?

#7 Post by Kinetochore » Fri Aug 04, 2023 10:13 pm

iconoclastica wrote:
Mon Jul 31, 2023 12:57 pm
Could you post a link to this replicating tape?
Cyanoacrylate was a failure for me because all the brands I tried were too brittle and inflexible to remove from the leaf surface. Using adhesives ( incl PVC, PVCA ) to get stomata pics seems to be a brand thing in Australia, with a lot of products behaving very differently from each other for what look like identical compounds. Now I own a small cupboard full of adhesives that didn't work for me. Need glue? Drop round!

Replicating tape link
https://www.tedpella.com/replicat_html/44840.aspx

Of course, since I successfully had it working first time, the tek hasn't worked subsequent times. Typical science :lol: :lol: .

Think it's because I used a different species where the underside was a little furry. And I didn't remove the midvein on the leaf like I did in the image below. And I used open-cell neoprene to pad out the sandwich, re-using that means there was some residual tape that adhered and interfered... waiting for some proper closed cell neoprene tape to arrive. I need to dedicate a week or two to finessing the visualisation as we head into what looks to be a busy Spring season. This is going to take a while

If I can get the tek to work reliably for a few species I'll do a write-up. It's not as easy a process as described on a lot of the online tutorials- most of which seem to use houseplants or plants with large, shiny leaves.

Here's a phone pic of the first working image I got using the tape. Visuals at higher resolution than in pic were great at 400x, but imaging with the phone was not

After removing the leaf, I stained it with some seriously old tol blue, added a crop of glycerine before whacking on the cover slip. The glycerine also made a big difference. I Macgyvered the image with a camera phone as I haven't optimised the Canon yet, there is no scale. I'll worry about all that later. Right now the priority is getting clear stomate images, then I'll formalise the documentation to allow comparison

ImageHirsuta stomata by Chirp Osaurus, on Flickr

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Re: Stomates and cellulose acetate butyrate- any experiences?

#8 Post by iconoclastica » Sat Aug 05, 2023 1:33 pm

If I understand you well, you are using a different CyAcr technique than I do. I normally don't peel off the hardened glue.

I thourough clean a slide with alcohol. Then I apply a thin film of CyAcr, over 1 to 2 square cm. Than I place the leaf on the glue, followed by a second slide (that as well might be a bit greasy). I clamp the sandwich for about an hour with two paper clamps. The upper slide is removed and the leaf pried away. This is the most critical stage. Often it comes away well, sometimes not at all, frequently some parts won't come off. That is why I use quit large areas of glue: then often there's enough surface of good impressions to study it under the microscope.
The leaf is prepared by cutting a section of it that fits the slide, possibly without projecting veins.

I wrote about this (applied to other leaf surface characters in the pteridologist
the slide sandwich
the slide sandwich
slides.jpg (48.52 KiB) Viewed 3550 times

some results:

I had a major disaster with my harddisk last month, therefore I am not sure about the metadata. Probably 20x and 40x objectives, transient light, and leaves of the fern Dryopteris. The photos have been optimized for measurements, not for showing the fines details.

a larger area at lower magnification
a larger area at lower magnification
f2052309808.jpg (158.88 KiB) Viewed 3550 times


f1405313456.jpg
f1405313456.jpg (114.63 KiB) Viewed 3550 times


f482470896.jpg
f482470896.jpg (87.23 KiB) Viewed 3550 times

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Re: Stomates and cellulose acetate butyrate- any experiences?

#9 Post by Kinetochore » Sun Aug 06, 2023 7:45 pm

Thank you- your pics are exquisite!

Yes indeed- I had been following the standard youtube and written protools the wretched algorithm has been throwing at me for months, which simply coated the underside of the leaf and peeled the CA, nail polish or PVC-A when dry.

No I hadn't thought of this variable and I'll give it another crack for CA and PVC-A ( which held the impression but distorted permanently on removal )- as well as CAB tomorrow

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Re: Stomates and cellulose acetate butyrate- any experiences?

#10 Post by Kinetochore » Tue Aug 15, 2023 8:38 pm

iconoclastica wrote:
Sat Aug 05, 2023 1:33 pm
I place the leaf on the glue, followed by a second slide (that as well might be a bit greasy).
Interested to hear which grease you used to keep the top slide from adhering to the CyAcr slide. I tried both avocado oil and vasoline in smeared and in larger amounts. All my slides stuck together

Silicon grease? How do you remove any grease remnants?

Still trying. Waiting for my neoprene tape so I can have another crack at the cellulose acetate

Many thanks

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Re: Stomates and cellulose acetate butyrate- any experiences?

#11 Post by iconoclastica » Wed Aug 16, 2023 11:55 am

Nothing technical, really. I repeatedly use the same top slide and take great care not cleaning it. When it gets lost, I take a new one and wipe it off the skin of my nose...

That said, there isn't normally much contact between the glue and the upper slide, for the blob of glue is smaller than the plant tissue that covers it. On the photo you can see work off-centre at the left side. Thus, escaping glue is at the far left end and to pry the slides apart I work a dissection needle in from the right side. It can be helpful not to have the short edges aligned, but sticking out 1-2 mm.
It is rare for the slides to break when working them apart.

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Re: Stomates and cellulose acetate butyrate- any experiences?

#12 Post by Sure Squintsalot » Wed Aug 16, 2023 10:04 pm

"I had a major disaster with my harddisk last month, therefore I am not sure about the metadata".

Any chance you were using a SanDisk (or WD) 4 Tb drive? I'm reading that these things are crapping out all over the place, and I have 5 of them running on 2 computers.

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Re: Stomates and cellulose acetate butyrate- any experiences?

#13 Post by iconoclastica » Thu Aug 17, 2023 9:17 am

No.

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Re: Stomates and cellulose acetate butyrate- any experiences?

#14 Post by Kinetochore » Thu Aug 17, 2023 11:18 pm

iconoclastica wrote:
Wed Aug 16, 2023 11:55 am
Nothing technical, really. I repeatedly use the same top slide and take great care not cleaning it. When it gets lost, I take a new one and wipe it off the skin of my nose...
Lol, no catalogue number for that then

I'm a goose, I took your grease vs CyanAc to heart, and used a lot of both. I'll try your way and report back. Thanks

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Re: Stomates and cellulose acetate butyrate- any experiences?

#15 Post by Kinetochore » Fri Aug 18, 2023 1:48 am

Victory!!!

Thank you!

The trick for me is to use minimal superglue, the thinnest smear of vaseline, to offset the slides as you mentioned- and not to wait an hour. Ten - fifteen minutes is plenty here to prevent the glue from hardening too much. Slides can be carefully separated using a blunt scalpel blade and left to air dry further for a few minutes without affecting quality of the cast. A lot of the leaves I work with are delicate, or otherwise tricky

Pics taken quickly on camera phone, the visuals through eyepieces are of course much better

I still need to work out a way to fix the tissue so all the stomates are closed, for accurate count and measurement- a few options, but I'd hate to do anything that could upset the superglue ability to accept a cast

I'll try this next on my target species, which is a lot furrier on the underside. And set the camera and scale up for accurate measurement

ImageIMG_20230818_111526 by Kinet Ochore, on Flickr

ImageIMG_20230818_111425 by Kinet Ochore, on Flickr

Happy dance!

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Re: Stomates and cellulose acetate butyrate- any experiences?

#16 Post by iconoclastica » Fri Aug 18, 2023 1:00 pm

Congratulations! Nothing as good as getting results when you've worked hard for them (an nothing more frustrating if you don't, but that's not for now ;-))
Kinetochore wrote:
Fri Aug 18, 2023 1:48 am
The trick for me is to use minimal superglue, the thinnest smear of vaseline, to offset the slides as you mentioned- and not to wait an hour. Ten - fifteen minutes is plenty here to prevent the glue from hardening too much.
I had noted the optimal time varies, but haven't yet worked that one out. Probably influenced by air moistrure an temperature. I spoiled more by not waiting long enough than by leaving the preps longer, but indeed, the optimum should work out best.

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Re: Stomates and cellulose acetate butyrate- any experiences?

#17 Post by Kinetochore » Mon Nov 06, 2023 7:39 pm

*sigh* finally got back to the sampling, figuring I'd sorted this and other problems. Turns out the change in humidity here- back to almost 100%, fluctuating between 90% and 60% in the work room over the course of the day- totally blows the previously successful protocol out of the water. Could also be the humidity when sampling, and a higher water content of samples.

Cellulose film not adhering to slides, even when I clean them with acetone prior to use. Tried different brands of slides. Can't seem to find optimal curing time for superglue- it either peels off the slide, or the leaves won't lift

I've tried untreated freshly harvested leaf samples allowed to dry 10 min, leaf samples treated w ETOH: GLA and with 10mM calcium chloride ( to make sure stomates are all closed )

I think I could be going back to learning embedding and sectioning, which is probably a painful learning curve in its own right, and success there may even be subject to the same variability under changing ambient temps and humidity

All part of the wild and whacky world of science and absolutely standard I suppose- but so frustrating! ( At least we had enough rain to put the bushfires out here, seriously grateful )

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Re: Stomates and cellulose acetate butyrate- any experiences?

#18 Post by iconoclastica » Tue Dec 05, 2023 10:47 am

Have you seen this thread. As you can see, results vary though.

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