A quick adventure with a weed, another slight improvement

[mrsonchus]

Hi all, I've been pretty busy wrestling with my sections etc and am slowly improving my technique.... I've been looking for solutions to the main problem I'm currently up-against, poor sectioning in the form of 'tatty' sections. It definitely seems to stem from several areas of the tissue-processing workflow as rather poorly executed by me. But things are better, both with the chemistry of the multi-stage processing protocols and with the 'nuts & bolts' of good microtomy when sections are to be cut.
I'm now getting far better integrity and it's attendant improvement in imaging quality with a camera.

Here are a few quick pictures - I'm going to follow up pretty soon with a more detailed description of this particular specimen's processing.
The subject - a common garden 'weed' called a 'Willowherb', sections made at 12µ - a tad too thick for this tissue I think, probably better at 10µ but I have to make compromises at this stage in my learning and balance tissue integrity (usually means thicker sections) against the 'ideal' section thickness that will give an optimum amount of information - although my interest is more morphological (histological) than cytological, at this time anyway but who knows?
I'm simply very happy each time I see some tangible progress & improvement in my sections & slides..

This is the 'willowherb' - a weed found on the edge of a slabbed area in my garden.

ws_willowherb_collage_1.jpg (250 KiB) Viewed 3337 times

Top left - the plant between some slabs,
Top right, short (about 4mm) stem-sections,
Bottom left, sections undergoing 'fixation' for at least 48hrs in FAA (Formal Acid Alcohol) - kills and preserves specimen,
Bottom right, sections during clearing, tissues have alcohol used for dehydration removed by the introduction of an intermediate fluid that 'clears' the tissue of alcohol and also 'clears' it in the optical sense too. This clearing agent (I use 'Histoclear') is a wax-solvent that facilitates the next step of infiltration of the tissues with paraffin-wax in preparation for block-casting (embedding)!

Here's such a block, with a section of willowherb-stem embedded in it, ready to mount for the Microtome's close shave..

A stem section of Willowherb in a wax block

ws_willowherb_wax_block.jpg (227.09 KiB) Viewed 3337 times

This is the protocol I used for this tissue set - a whiteboard is a thing of beauty when nothing else will do!
(sorry the picture's a bit small):

My menu for good sections...

ws_willowherb_Protocol_Boar.jpg (381.23 KiB) Viewed 3337 times

Some half-decent results followed - better certainly than my previous attempts, things are taking shape as I gain experience (especially with the vital practice of using the microtome!) - tissue integrity is greatly improved, not perfect but closer. This stem cross-section has been stained for 60 seconds with 'Fast Green' prepared in 'Cellosolve' and added whilst the section was saturated with 95% IPA immediately following de-waxing and before permanently mounting between slide and cover-slip, as seen below. The stain has given very good contrast & clarity to most of the tissue but seems to have had very little, if any effect on vascular tissue... The addition of a 'counterstain' to bring-out the vascular bundles would undoubtedly fix this, but I'm not that advanced yet...

ws_wherb_stem_XS.jpg (314.07 KiB) Viewed 3330 times

The stain is a bit patchy and the tissue slightly damaged - the area of crushed-looking epidermis was I suspect caused by my clumsy handling of the material through processing - still, not too bad so far.

Finally a closer look, with some measurements made with a recently acquired stage-micrometer!

Cells with measurements and intra-cellular details, such as there are

ws_willowherb_measured_cell.jpg (339.86 KiB) Viewed 3337 times

The detail of protoplasm isn't bad where some can be seen, but far better nuclear detail is seen when using Methylene Blue or even Safranin 0 to stain, to combine stains is one of my next projects, whilst trying to improve my basic technique of course..
The 3D nature of the cells is apparent too - although I suspect this is a by-product of my somewhat oblique sectioning in this case . Not a nucleus to be seen though - sounds like a job for MB!

Better go to sleep now, sorry this isn't a more comprehensive post, but I though a quick snapshot of what I'm up to may be a bit overdue. I'll follow this up with much more details of problems and their solutions (to the best of my limited capabilities at least) encountered during the 'Willowherb Project' ASAP. Having great fun & learning loads! Onward!

[gekko]

John, as usual, exceptionally clear, detailed, and illustrated presentation and beautiful photos, taking us step-by-step through the exacting, painstaking work. While the final results you obtain justify all the preparatory work, I have a feeling that you also enjoy the many, never-ending challenges you encounter along the way.

[75RR]

Agree with gekko, comprehensive is the word I was looking for.
Am more than ever convinced that microtome paraffin specimen section preparation is a vocation.

[mrsonchus]

Many thanks for your kind words gekko & 75RR.
The process is getting easier (less difficult may be a better term... ) with repetition, careful recording, analysis and above all practice. The articles I'm going to try to produce should hopefully help others to avoid very many of the setbacks, mistakes and problems that I have encountered and been stupid enough to make as I progress.
Having achieved a reasonable but very basic capability to carry out the entire process, necessarily with subjects chosen because they seemed (at the time! ) to be quite good as 'std' or 'control' choices to start with (i.e. easier ), I'm now ready to go to the next level. That's why I've started with the 'averagely sized, shaped, etc' specimens of the Willowherb stem and the trusty old Aloe leaf (taken from a long-suffering tiny plant that sits on the corner of my work-table - poor thing keeps getting a 'haircut' every now and then ). I'm really pleased with the successes that are starting to come, 1 good slide is definitely worth the production of 100 poor ones!
These two subjects were processed under the tightest and most closely-controlled & monitored, not to mention analysed, protocols that I've carried out thus far - I said to my dear Wife that I needed to 'play hardball and get as thorough as I could with my approach' - this has given me a jump in quality that is enough for me to feel able to share with those interested. I share all the problems of course, but without any real end-product any post is I feel in danger of becoming a tedious and dull list of moaning and excuse-making.
A problem needs I think to be presented with it's (partial at least) solution to have much of a meaning to others considering trying these activities. Anyway, the 'next level' is I think the ability to achieve a higher standard of consistency and of course quality. It has become crystal-clear to me by this stage that this will definitely mean the consideration of each individual specimen-type (e.g. the Aloe leaf and the Willowherb stem) and it's requirements for a successful outcome - the results absolutely tell me that this is vital.
Different protocols (not radically-so however, more matter of 'tweaking' as appropriate) are needed for certain subjects.... So to cut a long ramble short - I need to either adapt and produce 2 slightly different protocols for the Aloe leaf and Willowherb stem, or use only one for further study - I'm going to continue with the longer option of keeping the 2 specimen types as I really don't want to lose the ability to compare & contrast as I continue. Sorry to ramble on, thought a bit of extra info may be interesting to some. As previously mentioned, I'll follow-up with a lot more detail and explanation ASAP.

p.s. just bought a 'closing-stock' pristine & totally unused stereo-zoom 'scope with transmitted and incident lighting and a beautifully clear & smooth range from x10 to x40 with the supplied x10 WF eyepieces. I got this for a very reasonable price indeed and am feeling like a 'dog with 2 tails' at this time .... This 'scope will also augment my specimen-preparation and crucially monitoring throughout the processing of samples, saving me a lot of to-and-froing from my work table to my trinocular! Also I won't have the worry about getting chemicals on the big 'scope and it's delicate parts - the new stereo has a lovely big ground-glass dissection plate! Luxury! I'll post a quick picture of it ASAP - I've got to cut the grass now, my Wife is as usual being extremely patient with my hobby!

Back soon with some more pictures!

[mrsonchus]

Here's a couple of pictures of staining with trusty Methylene Blue - a good stain for nuclei and most other tissue & cell components.

This is a stoma with it's attendant cells..

wsmethylene_blue_stoma_cells_stain.jpg (238.93 KiB) Viewed 3310 times

This is a Methylene Blue image of nuclei and nucleoli, the nuclei are a bit shrivelled - they've been through a lot of chemistry!

ws_methylene_blue_nuclear_staining.jpg (268.96 KiB) Viewed 3310 times

Much better than the Fast Green nuclear staining.

Back soon....