When 2µ seems far too thick - the Mighty Shandon roars...

[mrsonchus]

Hi all, I've some perturbing news...... I had a little sectioning-session this evening during which I...I.... cranked the MS down to 1µ whilst sectioning anthers at what now seems almost a door-step thickness of a massive 5µ!

Anyway they're not perfect but I'd estimate them at about 75% perhaps... It was a very brief try at the bizarre 1µ - almost like a wall between two dimensions!

Ahem, here's a very quick couple of pictures, hopefully I'll get a few more posted later.
On the rack and looking very thin.... See the back-row!

ws_lonely-microns.jpg (85.24 KiB) Viewed 4247 times

back soon....

[Crater Eddie]

I felt a few rumbles, I thought it was distant thunder but now it is clear to me that it was the MS coming close to cleaving a hole in the space-time continuum.
Be careful there John.
CE

[JimT]

CE, it was just the deep space Ripple effect.

John, at 1 um how can you even be sure they are there?

JimT

[mrsonchus]

Haha fine fellows both!

I floated a few 1µ ovary TS and have a few pictures. They're a little 'wrinkled' I'm afraid - they were as you can imagine extremely difficult to handle... They don't look very impressive at this stage but then they are literally still wet and will need at least 48hrs to dry onto the slides enough to be reasonably sure of good adhesion. If I rush into de-waxing material is almost certain to be lost..

I did actually manage to get a ribbon forming quite well with a little applied 'tug' of the ribbon, away from the blade as the cuts were made - a very delicate operation but not particularly difficult to achieve.

Here are a few pictures of the goings-on of this evening...

The Mighty-Shandon never fails...

ws-1-micron-ribbon.jpg (100.16 KiB) Viewed 4237 times

and stretching - they're far from perfect but I'm still amazed that they made it this far!

ws-1-micron-sections-floati.jpg (60.53 KiB) Viewed 4237 times

and onto a slide they go..

ws-1-micron-wax--sections-o.jpg (104.2 KiB) Viewed 4237 times

A quick picture - had to be very careful my 'scope's halogen didn't compromise the still-wet sections, took these through a blue-filter and corrected WB at time of capture...

ws-1-microm-section-slide.jpg (277.51 KiB) Viewed 4237 times

So, that's a 1µ section - a bit mucky but not bad for a first and unplanned attempt - these weren't even taken with a new blade, these were cut with my least-used 'best-blade' and they are intriguing. At this stage I'm not entirely convinced of their usefulness without an improvement in the tissue-preparation of the block to be sectioned at this rather extreme thickness. This looks awfully like paraffin-sectioning pushed slightly beyond it's natural limits - which I suspect may lie at about 3/4µ for maximum integrity and demonstration of tissues embedded in wax - perhaps plastic embedding would take this limit down to the 1µ but I can't see myself needing or particularly wanting to go down that route in the near future if at all...

A good exercise though - even the ability of wax itself to section at 1µ is according to the books a matter of particular formulation....

Give me a few days and I'll return with some pictures of 1µ stained & mounted sections - something I would like to see! I think I need to be extremely careful with these, they are horribly delicate and vulnerable...

Great fun though, hope you find this as interesting as I do - onward-ho!

Back soon!

[charlie g]

Careful, John B., ease up slowly to the thicker realms of macrophyte tissue reality. To dwell too long at the Shandon's 1 micron fancied space...and you may be dragged down by that blade to a frightful Zeno's paradox of space and closing distances. I fear you may in slow motion struggle to climb out of that paradox of spaces between spaces..no matter how much thickness you attempt to again realize...eegads..don't have a wall mirror by the Shandon just yet...get away from that one micron 'flat land' to which Edwin Abott first charmed me with...please don't look at your reflection when standing sideways to any wall mirror...first break free of Zeno's arguments! charlie guevara

[mrsonchus]

Ha Charlie-G - maybe the 1µ is a case of a true reductio ad absurdum! I'm running back to the multi-micron realm ASAP!

A great exercise but hmmm.... not sure it'll catch-on in my lab!

[Yvan]

.

[mrsonchus]

Very nice!
Have you tried to cool the paraffin block down to about 4°C (39°F) prior to sectioning? It will largely improve the cutting properties of the paraffin, making this type of "semi-thin" sections a piece of cake.

Hi Ivan, yes I've subsequently been able to routinely section at 3µ both with and without cooling of the block overnight.
I find, having run several trials with different tissues that temperature is one factor of several for the successful sectioning at the 3-1µ levels. The tissue type is a major factor (type as in species and tissue-type within the same plant).
Some tissue will section far more successfully at room-temp (21 deg C approx) I find, some at 3-5 deg C as from refrigerator.

I'm sectioning such a tissue right now, comparative stem TSs of Sonchus.asper and Sonchus.oleraceus. I've found that the S.oleraceus is better suited to chilled sectioning as it is a far 'tougher' and more 'gritty' tissue than it's close relative S.asper, which sections better at room-temp. These sections are at 2 & 3µ.

Surprisingly I find that blade-angle is virtually irrelevant (within reason of course) also, and this is true for the two blade-types I use, 'Feather' blades and 'Edge' blades depending upon which is cheapest at the time of re-stocking!

I also (occasionally) use various softening-agents directly upon the block's cutting-face and have found the most effective to be household fabric softener!

Many many factors of varying degrees of importance - certainly makes sectioning as much an art as an algorithm!

Thanks for your interest and input old chap.