Extreme microtomy - I scared myself!

[mrsonchus]

Hi all, amazingly you may think, but I haven't been staining slides this evening - I've been putting the 'Mighty Shandon' through it's paces and trying some very, very thin sections - with super results!

I've been practicing and aiming for quality rather than quantity tonight, with a doubly-embedded anther in TS, and an ovary in LS, here are a few pictures of the sections floated onto slides and still 'wet' - they'll need about 48hrs to dry and stick to the slides sufficiently to then process and mount, but here's a taster of what occurred in 'the lab' this evening,

This is a Daffodil's anther at 6µ - happily this one seems to contain more pollen than a lot of my previous 'open' anthers and may have more structural detail also as the tissue of the anther hasn't gone through as much degeneration as an open anther may have.

ws-6micron-anther.jpg (189.74 KiB) Viewed 4595 times

After giving my blade a good clean with my newly-acquired super-soft 'camel brushes' (OK - cheapo paintbrushes then... ) I went a bid bonkers and cranked down the Mighty-Shandon's dial to 4µ, all was well, then to 3µ, all still good, then to a nervy 2µ - argghhh - guess what - the MS simply churned out a nice ribbon of perfectly-cut 2µ sections, the sight of which made me sit-up and take notice I can tell you!

Here's one of the thinnest sections I've ever cut successfully, so delicate were they that I had to 'scoop them up' with my favourite (chilled first to avoid the wax sticking to the forceps) 'super-fine' forceps and place them extremely carefully onto the waiting water-bath surface which was at a healthy 42 deg C.
All went well and about 30 sec of stretching of the wax and sections therein later I had them on a slide safe and sound!

The rationale for trying this extra-thin sectioning is to enable more/maximum detail to be gleaned from the very dense tissues of the ovaries that I am currently studying - I find that the more dense a tissue is, and ovaries and ovules are very dense it seems, the thinner a section may need to be to maximize detail beyond the overall morphology, for example the cellular details of the dividing and developing egg-sac seen in earlier posts..

Anyway here's the 2µ section, looks similar to the others but I'm looking forward to staining and mounting a few of these beauties when they're dry...

ws-x20-bS4-2micron-stitched.jpg (306.03 KiB) Viewed 4595 times

Imagine this, the pollen grains are about 35µ in diameter, the sections at 2µ would therefore give about 17 'slices' per pollen-grain! Amazing really - the 'Mighty Shandon' just gets better!

Here's a nicely-cut 4µ section of an ovary in LS,

ws-4micron-ovary-LS.jpg (474.39 KiB) Viewed 4595 times

Finally, here's a row of sections from tonight ready for drying...

ws_sundday-sections.jpg (137.12 KiB) Viewed 4595 times

Can't wait to stain & mount these beauties - I'm going to be very careful with these though!

Back soon.

[charlie g]

Gulp...two(2) microns...I hope gravity waves don't hamper your blade (joke,joke). It will be terrific to see any mature pollen sectioned at 'mid girth'. And LS of the ovules embryo sacs is perhaps better than CX of embryo sacs for the position of the nuclei.

Please be rested when working two micron ribbons, and thanks for this developement, John B.. charlie guevara

[rnabholz]

You starting things much smaller and you will have a mushroom cloud over your home.

Amazing! You are hitting on all cylinders with this project. Keep the reports coming and congratulations.

Rod

[mrsonchus]

I wonder if 1µ is possible..... ....

[billbillt]

Give it a try, John... I would very much be interested in seeing the results!...

BillT

[gekko]

Superb photomicrographs of your super thin sectopms!

[mrsonchus]

Thanks Gekko, I tentatively de-waxed, stained & mounted a few slides tonight, I'm leaving them overnight to dry as while wet they will not be anywhere near their best...

Here's the 'rig' for this evening's stain-fest!

ws_700x525_DSCN3453.jpg (170.8 KiB) Viewed 4554 times

And 4-slides later, a 'selection-box', mostly overstained but not too bad. I'll post some pictures hopefully tomorrow evening, we've a very long & busy day ahead of us tomorrow I'm afraid...

ws_700x525_DSCN3455.jpg (150.41 KiB) Viewed 4554 times

From left to right we have;
2µ doubly-embedded anthers in TS, stained with the classic pollen-stain Fuchsine - these as is often the case with Fuchsine, are I think very overstained! I'll need to do that one again!
Next along more doubly-embedded anthers this time at 6µ and stained with the classic Safranin + Fast-green combo, better I think than the 2µ which thus far I find rather uninspiring....
Then there's a 4µ ovary in LS, stained with Safranin + Fast-green - this slide I rather like - I think the 4µ is better than the rather extreme 2µ personally..
Finally there's a 2µ doubly-embedded anther stained with Harris-Haematoxylin for the nuclei and Fast-green for everything else - quite nice and I think a subtlety with the HH that seems to reveal the bi-nucleate nature of the pollen-grains I think... I need to ask Charlie G about this first...

So far I'm unimpressed with the 2µ sections, but it is very early to assess them usefully, the cover-slips were still floating like continents upon the Earths liquid innards!

Really need to sleep now, we've an early start in the mroning.
Back soon with some pictures!

Here's a rather awful one, it's all I have really at this time, the vicious Fuchsine has bludgeoned this tissue into a red haze! I can definitely improve on this minor-horror!

ws-2micron-pollen-sac.jpg (349.07 KiB) Viewed 4552 times

Mind you, I like the cell-details in this ovule at 6µ and stained with Safranin - an altogether 'more civilized' stain than Fuchine at times! I need Charlie-G's expert-eye to look this one over, there seems to be a lot going-on here with the egg-sac?

ws-6micron-ovule.jpg (355.28 KiB) Viewed 4552 times

Hope you like them, apologies for the rather ghastly pictures, my excuse is they were a little rushed and still wet and... and...

Back soon with some 'proper' pictures and a little attempted analysis..

[75RR]

Hi mrsonchus,

impressed with those very thin sections.
I imagine at 2µm and 3µm that an exceedingly stable table is needed, I take it you do not have problems with traffic induced vibrations?

[Charles]

Awesome job on the thin sections John! Maybe thinner sections need a more dilute stain?

[mrsonchus]

Awesome job on the thin sections John! Maybe thinner sections need a more dilute stain?

Hi Charles,
Most definitely this has proven to be correct in a lot of cases but there are as always variations, the main factor being I've found the actual tissue-type (variation within type is pretty insignificant I find) as defined by the species of plant or the tissue's origin structurally speaking, e.g. dense-ovary tissue stains a lot more 'enthusiastically' than say the ever-problematic Aloe-leaf tissue which can be very coy when it comes to accepting stains...

I chose Fuchsine here for a couple of reasons, the first being that is an accepted 'classic' pollen-stain, the other being that I like it's tone although it can be quite 'reflective' or literally dazzling if, as here used unfettered...

Tonight I plan to stain perhaps with the often fine Methylene-blue that made such a splendidly-detailed job of staining Sonchus ovaries and ovules a while back for me.
The Harris and Ehrlich's Haematoxylins (not in the same tissue of course) are looking very promising indeed also, quite fascinating to see just how 'focused' they can be on nuclear material when properly differentiated either progressively or regressively, my preference is for regressive staining at this time.

Thanks for looking Charles, I've more to come as I find this a quite fascinating exercise in exploration and adventure!

[mrsonchus]

Hi mrsonchus,

impressed with those very thin sections.
I imagine at 2µm and 3µm that an exceedingly stable table is needed, I take it you do not have problems with traffic induced vibrations?

Hi 75', surprisingly perhaps I find stability not to be any measure of problem when sectioning. The Mighty-Shandon weighs in at a gargantuan 40kg and has I find a very high inherent stability - it almost has a palpable gravity-field!

It's on my large metal-framed and very sturdy (also reinforced by myself - I've screwed various extra braces and supports to it) desk and basically whatever movement does occur, and I can't feel any in use, occurs I suspect throughout the 'system' in a uniform manner and therefore causes no discernible effect to either my senses or the sections produced.

Traffic is pretty inconsequential also where we live, thankfully, we experience far more noise from the often very powerful flow of the nearby river Lune.

As the song goes 'solid as a rock...'