Three Plant-Section Thicknesses Compared

[mrsonchus]

Hi again all, following my earlier post, I've now got a set of 3 sections over a range of section-thicknesses, cut with the delectable Shandon microtome... I've some 5µ, 10µ and 15µ sections that are stained similarly in Safranin and permanently mounted in 'Numount' - a modern substitute for 'Canada Balsam'.

Here are some pictures of each, lets see if there's any relevant difference as the section thicknesses change over the range mentioned;
Here's a 15µ section to start;

ws_x20_15micron_carex_pendu.jpg (308.89 KiB) Viewed 12013 times

This looks pretty good to me, but perhaps seems a little thick to give best definition and clarity?

this one's sectioned at 10µ;

ws_x20_C21_10mic_carex.jpg (203.81 KiB) Viewed 12013 times

This I would say is a big improvement (bearing in mind the purpose of the imaging being attempted), with more detail and information at the appropriate level of magnification for tissue morphology to be seen clearly. The image has, (by virtue of the stain not image pp) or appears to have, better stain-penetration and consequently more contrast and clarity, although the 15µ seems to have slightly better differentiation?

and finally here's a section taken at just 5µ;

ws_x20_5mic_carex_3.jpg (303.36 KiB) Viewed 12013 times

Well, there do seem to be more 'empty' cells appearing, which up to a point is I suppose a good thing, emphasizing the cell-walls more for our intended goal? The whole image appears more 'delicate' somehow, maybe evn sharper, but the apparent sharpness may be a photographic consequence.. A nice image and good for morphological information to a large extent. But, of the three, I personally rate the 10µ as the best and most informative image.

It may well be the case that the optimum, for this purpose, with this tissue, stained in the way it has been (all relevant factors certainly) lies between 5µ and 10µ, but I'd rather not run the comparison with 6µ, 7µ, 8µ and 9µ to try to find out! This quick & simple test will do me for now, I think I'll settle for say 8µ for further Carex tissue sections...

Well, what do you think? How do they compare, are there any appreciable differences given that I've cut them with the intention of simply giving a decent image of the cell arrangement and overall tissue morphology of the subject, the leaf of a 'Carex.pendula' - a sedge with tough leathery and sharp-edged leaves that's always trying to overrun my gardens..

Sorry to be so brief, I've such a lot going on with my adventures at the moment I've got to keep each topic a little more concise than I'd prefer, still I can always fill in any gaps later if need be.

I'm having a wonderful time, starting to be able to produce reasonable results consistently. This is going to enable me to start some botanical projects without needing to constantly fix recurring (and major) problems in the histological process. Thanks to the mighty Shandon! (again)

I'm going to make a separate thread for the staining-in-the-wax tests that I've been working on, and the introduction of a superb metachromatic stain called 'Toluidine Blue' (TB0) that I've started to dabble with... What fun!

p.s.
I've just been looking back over some earlier sections and have a couple from May that are interesting to compare! Pre-Shandon days!

Same tissue from the same actual paraffin-block - different time (3 months ago) different microtome (Cambridge Rocker)...

Somewhat lacking in integrity, but the best I could manage at the time, and better than no section at all...

ws_carex_section_may_2015_2.jpg (223.82 KiB) Viewed 12006 times

and;

Hmm... a bit tatty..

ws_carex_section_may_2015.jpg (124.8 KiB) Viewed 12006 times

Things have moved along since them! (I love my Shandon!)

[billbillt]

Hi John,
WOW!... Great work... Looking forward to your new thread!....

BillT

[mrsonchus]

Thanks Bill, more to come soon, the 'in wax' and 'Toluidine Blue stain' threads with some interesting and striking lateral-root endogenous-formation pictures incorporating both. Can't wait, should get them up quite soon, although I've a very busy day ahead tomorrow, lots of driving to do - yuk!

Glad you like the post!

[JimT]

I definitely think the 10 um image is the best. In fact, quite a bit better than the other two.

Have you tried other stains and alternative lighting techniques; Rheinberg. oblique , or UGF filters? They can bring out cell wall structure as well as the goodies inside.

Looking forward to more of your posts.

JimT

[mrsonchus]

I definitely think the 10 um image is the best. In fact, quite a bit better than the other two.

Have you tried other stains and alternative lighting techniques; Rheinberg. oblique , or UGF filters? They can bring out cell wall structure as well as the goodies inside.

Looking forward to more of your posts.

JimT

Thanks Jim, I've only just started to master staining at this stage - I've a pretty good selection of stains to try, I need to learn to use each one effectively in isolation and then start the investigation of both combination-mixtures (e.g. Masson's trichrome) and the serial application of multiple stains (e.g. Gram and counter staining).
It's all relatively new to me (histology and the microscopic technique/s) and I'm picking it up as I go at the moment, although the recently-acquired ability to virtually remove section-quality as a limiting and obstructive factor is enabling an acceleration in my studies to some extent.

As for lighting techniques I do intend to experiment with some such as Rheinberg, oblique, incident, dark-field etc but feel that they may be of greater use when studying thicker sections such as hand-cut live tissue for example. The thinner 'top-down' tissues that I am studying may possibly lack sufficient cell-wall depth and present a sub-optimal perpendicular profile to these methods, that seem perhaps to fundamentally exploit 'shadows' within thicker and possibly, slightly at least, obliquely-cut samples?

A technique I will definitely pursue when the time's right is that of polarization, as structural-orientation and crystalline (probably ergastic?) substances can be tricky using stains alone - I think anyway.....
One step at a time is my mantra, I really need to perfect and (vitally I'm certain) to understand the processes & techniques as I approach them.

I only got my first 'scope (an old 'Swift Collegiate 400') in November last year - it's early days for me still.
I bought a microscope to 'get a better view' of 'wild' British plants that I enjoy learning about, especially the systematics of them - fascinating now to be able to pick & choose what to examine and to be able to do it! The endogenous origin of lateral-roots is a fascinating subject that I have only been able to section, stain, mount and study since Friday! I have some very nice images of those sections to post ASAP in the next topics referred to in the post before this one..

Hmmm - I don't know what a 'UGF filter' is, but I gather it's a 'Universal Gradient Filter' having had a quick look online.. It also seems to work principally with lower-power and more 3D subjects such as living aquatics? I don't think (although I'm only surmising) that it has much to offer my area of microscopy.
Mind you, thinking about it, these methods may be useful for my adventures in palynology - lots of 3D material there... never say never!

Thanks for your kindness and input, it's really interesting and stimulating to consider such things!

The birth of my hobby....

new_swift_scope-(1).jpg (46.07 KiB) Viewed 12000 times

swift_1.jpg (32.46 KiB) Viewed 12000 times

And here I am! Having a huge amount of fun and learning loads too!

[einman]

I, for one, will be observing your experiments with polarization. I have an American Optical Polstar scope that has been begging me to put it to use.

[mrsonchus]

I, for one, will be observing your experiments with polarization. I have an American Optical Polstar scope that has been begging me to put it to use.

Hi einman, the 'Polstar' looks to be an absolutely beautiful instrument!
I'll be a while yet I'm afraid before starting off with polarization but may attempt some basic observations as a 'side-project' or maybe even improvise a bit of kit to start with, although it seems the 'scope I now have (a 'Brunel-Microscopes SP200') may be fitted with polarization for a very reasonable cost.

The more I read about it, the more interesting polarization becomes to me...
Thanks for your interest einman - watch this space.

[apochronaut]

Just wondering what you feel are the principle main difference is accounting for the quality variation between the Shandon microtome and the rocking microtome? Is it primarily the sharpness of the blades or is it a case of precision or other?

[mrsonchus]

Just wondering what you feel are the principle main difference is accounting for the quality variation between the Shandon microtome and the rocking microtome? Is it primarily the sharpness of the blades or is it a case of precision or other?

Hi apo' - a good question,
I would say the main criterion is the sharpness of the blade (availability of replacement blades notwithstanding). The Rocking Microtome in my opinion, and (considerable) experience of it's use, undoubtedly has the potential to cut 'good' sections through what I have found to be the optimum-range for botanical subjects, between about 5µ --> 12µ, maybe 15µ too.
It must be remembered that a significant factor limiting the quality of my sections, cut when the blade was new and at it's best, was that of my own inexperience and lack of ability in using the Rocker to it's true potential.
When it performed well it did cut some good sections, it's very easy to use but cannot approach the accuracy of the Shandon - not even from a distance...

As an introductory instrument it is surely very good, but the inexperienced user will probably (I know I certainly did!) misuse it horribly and damage the blade very quickly indeed! Then the problem of sharpening blades becomes tangible - and it's a 'game-changer' - to get the blade sharp, at the exactly-correct angle and alignment, using all the necessary paraphernalia (and there's a lot) is for me and I would imagine many others, an unrealistic aim in the home environment with a 'normal' budget and a 'normal' amount of available time, not to mention the disinclination to perform this irksome task well.

The Rocking-Microtome's shortcomings can be avoided completely with regards to the blade by fitting a replaceable-blade system (holder) as found in the Shandon - universal replaceable blade holders are available for rotary microtomes but not for the Rocker however, and given that such a holder would cost in the region of twice the cost of the (rocking) microtome, probably never will, commercially at least!

Now, in terms of precision, quality etc, I would estimate the full potential of the Rocker to be able to deliver sections of about 60% of the Shandon's best, quite a good performance considering just how good the Shandon has revealed itself to be in use...

The Shandon has of course the precision, but whether a section has a 10% or a 50% margin of error is probably not a major factor for someone carrying out the sort of 'work' (It's a hobby after all, I'm a total amateur with a total amateur's needs) that I am.

Now the retraction feature of the Shandon - this I consider to be a very large advantage indeed, the Shandon retracts the specimen-block by 40µ on each upstroke, eliminating the possibility of the block contacting the blade's back-edge during this return-stroke, the Shandon can cut up to 30µ thick and will therefore not go anywhere near (within approx 10µ at least) the blade with this feature in action. This really has a profound effect on the ability and tendency of a 'good ribbon' of sections being delivered from the blade. This ribbon-production is very important when 'moving through' a specimen to reveal related features as the depth into the specimen increases or approaches a region of interest therein.

The replaceable blade system is simply superb - a brand-new blade with a cutting-region of up to 80mm long approx whenever needed, for a cost as low as about £1.50 per blade for a 'std' blade (about £60 for 50 blades, delivered) up to about twice that - is very affordable indeed. To change a blade takes me about 30 seconds, enabling the routine use of one blade for 'roughing' and another for the fine-cutting of the sections to be used. Also the orientation of the blade remains completely consistent as the locking and carriage systems are extremely sturdy and stable when 'locked down' in use.

The Shandon is as a result of superb design and manufacture a very easy instrument to use well, far easier than the Rocker. The Rocker also cuts sections through a necessarily curved path as the arm 'rocks' with a circular travel, albeit not very noticeable, it can be seen when the cut face of a block is examined and presumably therefore has the effect of giving sections that are thinner in the middle than at the start and finish edges of the wax-block.

Put briefly, the Shandon's advantages, and they are significant, are in my opinion due to the blade's potential to always be at it's very best, combined with the sheer quality and precision of it's construction & performance in all respects, not to mention the considerably superior ease of use and resultant increase in quality and consistency of sectioning.

With the use of steel blades requiring re-sharpening the Rocker soon becomes unusable and brings sectioning to a grinding halt - this is exactly what I experienced, leaving microtomy as an unattainable goal for the hobbyist without the £1,500 or more needed for the purchase of a used rotary, or perhaps about £700 for a copy of an old rotary design such as the old Reichert model first considered by myself...

I simply had an amazing piece of luck when I acquired an unused brand-new Shandon (complete with 10 replaceable Shandon blades!) for a ridiculously low price (I actually paid £100 above the cost of a copied Rocking Microtome for it...).

This piece of luck has got me to where I am now but must surely be very, very rare. My advice to you apo', is simple, if you can afford it, buy a rotary (used) such as the Shandon, that takes the simply superb replaceable blades. Ultimately I think a rocking microtome will become a heart-breaker...

p.s. I'm currently working (slowly) on a replaceable-blade holder that will fit into the rocker and take the same blades as used in the rotary microtomes, it's certainly working, the principle was demonstrated (see my earlier post re the use of a std razor-blade in my first trials) when I stuck a razor-blade to the rocker's ruined knife blade with sticky-tape, held my forefinger against it to keep the middle of it firm, and watched in amazement as the rocker produced several beautifully cleanly-cut sections where before I'd only managed tatty, ripped, holed and fragmented sections with the original knife!
This led me to the conclusion (correct thank goodness....) that the blade was the major factor ruining my sections, not the histological processing that was carried out on them. the rest is history, one amazing piece of luck later I'm up and running producing some great sections!
The reason I'm pursuing the replaceable system for the rocker is that I would love to produce something that's cheap (and at this time It costs about £3 to make one) to make and can be made by anyone, giving the rocking microtome back to folk that are starting out and can't afford a rotary microtome or expect the amazing luck that I've had... That would make me very happy and I intend to keep working on this until I succeed with something that's demonstrably effective. No surrender with this one!

I hope this goes some way to answering your questions einman, these are of course only my own experiences and opinions, but are definitely based upon real-world experience. Good luck.

[apochronaut]

Thanks for that detailed answer. I certainly makes shopping a lot easier. I had considered a sliding microtome , but what you are indicating is that it becomes difficult to maintain precision on the less automated designs. I'll have to re-think that.
I guess Shandon has become one of the major makers but there aren't too many on the aftermarket..... and each maker has peculiarities of design. Here in North America , luckily, AO rotary microtomes are fairly common , and aren't too expensive. I will have to check and see if the blades made for the Asian copies will fit the original AO( Reichert) 810,815 or 820 because I suspect the electronic versions of it Leica are now making aren't using the same blades.

[mrsonchus]

Thanks for that detailed answer. I certainly makes shopping a lot easier. I had considered a sliding microtome , but what you are indicating is that it becomes difficult to maintain precision on the less automated designs. I'll have to re-think that.
I guess Shandon has become one of the major makers but there aren't too many on the aftermarket..... and each maker has peculiarities of design. Here in North America , luckily, AO rotary microtomes are fairly common , and aren't too expensive. I will have to check and see if the blades made for the Asian copies will fit the original AO( Reichert) 810,815 or 820 because I suspect the electronic versions of it Leica are now making aren't using the same blades.

Pleased to help a bit apo', you'll have no problems with blades, all the models have I believe the ability to use the de facto standard replaceable system, there are several brands (of blade) all fulfilling this purpose. Basically there are two 'types' (sizes really) the 'high-profile' and the 'low-profile', the former is simply slightly the thinner (from top cutting-edge to bottom edge that is). If like me you require a (very) good and versatile microtome I'd certainly go for a rotary, no doubt in my mind at all apo'.
The 'sledge' microtomes are good for cutting harder tissues but nowhere near as versatile as the rotary type (they don't really produce 'ribbons' either) - but of course the sledge is not designed for exactly the same task as the rotary, you would probably be able to pick up a sledge microtome at a later date to compliment your rotary, great if you're interested in sectioning say bone, wood (very good for this application) or materials with similar physical properties.

Don't bother trying to get a Shandon in particular, I believe all the top names are equally as good - make sure you buy one with what I've found to be the most important features:

retraction
'std' replaceable blades
specimen-block holder orientation-adjusters (very useful indeed) for fine adjustments during sectioning

Oh - the specimen-block holder is an assembly that screws onto the actual 'up & down' arm (forgotten what it's called ) and comes in two 'versions', the 'quick-release' version that is essentially intended for use with the 'Tissue-Tek' style specimen cassette/mould combination (I use these, they're simply unbeatable), and the 'universal' specimen-clamp version- as found on my Shandon.

The universal clamp is in my opinion easily the best, it's very flexible and versatile, all manner of specimen-blocks (wax) can be used with it, including of course the 'traditional' wooden blocks and, very conveniently, the aforementioned cassette system blocks that fit the QR head!

I could go on with a lot more but some of my previous posts show a lot of the features mentioned above, also the Shandon actually in action, it may be worth a look.

p.s. I think personally I would resist the urge for a fully-automated electronic version as unless the whole of your histological-workflow is standardized to the point of producing very consistent and predictable results (tissue-processors etc) you may find the manual version to be much more flexible in use and able to be used with far more judgement and skill, as appropriate (and definitely stimulating and enjoyable) when working with the very diverse and essentially unique preparations (specimens) that an amateur such as myself working at home will be interested in and therefore working with. Just a thought.

Anyway, I hope this helps a little apo', please ask again if you would like any more feedback or discussion.

[apochronaut]

Thanks, John. Simply due to the sheer volume of them around, I think I can get an AO 820 for very little. I guess they almost dominated the market for quite a while. I will be asking questions for sure. The help is appreciated.

[mrsonchus]

Thanks, John. Simply due to the sheer volume of them around, I think I can get an AO 820 for very little. I guess they almost dominated the market for quite a while. I will be asking questions for sure. The help is appreciated.

You're very welcome apo'
The AO 820 looks perfect to me, virtually the same as the Shandon - the US availability (and consequently the prices) of microtomes is enough to make anyone here in the UK weep, they're everywhere!

Go get one apo', you'll be very surprised indeed by how simple they are to use. Since getting my Shandon I've really been able to concentrate on the studies that I actually want to do, rather than constantly problem-solving and fiddling with the rocker - firing-up a rotary is a great feeling, you'll really start to get the most from your microscopic endeavors with an AO on your bench!

Exciting times are coming up for you apo' ! keep in touch if you need any quick pointers that may save you some time or difficulty, I've learned a lot from my adventures and you will without doubt enjoy doing the same!

[billbillt]

Hi John,

Have you got anything new to post?... You have started a very interesting thread!....

The Best,
BillT

[mrsonchus]

Hi John,

Have you got anything new to post?... You have started a very interesting thread!....

The Best,
BillT

Hi Bill, yes I've been working on a couple of areas of my workflow; I've been investigating the tendency of some sections to have areas that 'ruck-up' in the manner of a carpet on a slippery floor, and fail therefore to stain and mount well. This involved a series of trials with 75 slides that revealed an undeniable link between the temperature of, and the water used in, the wax-stretching water- bath stage.

Also I've been studying, practicing and perfecting (or at least trying to) staining with aqueous stains, and the attendant travel of the sections from Histoclear (from dewaxing) through an OH series to 95% OH, through a hydration-series to DI water, into staining, quickly through an OH series to OH then again quickly through another series back to Histoclear, which is miscible with resinous mountants (I use a synthetic Canada-Balsam substitute called 'Numount'), the ultimate destination of the now-stained section/s. Mounting is very quick, simple and reliable with practice, and I have certainly practiced!

I've been using positively-charged Leica slides also that have proved to be very good for preventing the sections from simply falling off the slides during processing - a very frustrating problem that I've succeeded in eliminating thank goodness! The final piece of this jigsaw was the making of a 'subbing-solution' using Chrome-alum, this sticks sections to slides so well that they need to be scrubbed off when/if the slide is to be re-used! However if too-strong a solution is used, background-staining will occur and this of course spoils a slide, at least in aesthetic terms if not practical...

I've also been working on a slightly enhanced image-stacking method that has really yielded good results (i.e. an improvement in my own stacking results) - this is very recent (yesterday) and still being tested at this time, but looks good.

Practicing with various section-thickness/blade-angle combinations when sectioning has also been revealing although my conclusion here was that the optimal position was the one I'd been using from the start - as mentioned in the Shandon manual, i.e zero degrees, leaving the blade's manufactured angle to work it's own magic (the blades are made at 35 degrees I believe).

Microtomy practice has given me the best sections that I have ever had! I've just mounted some Aloe-leaf (yes, from the original blocks!) sections that are quite simply beautiful - perfect tissue integrity and very good staining, with trouble-free mounting too.

I've literally just nipped into the other room to move 2 tissue cassettes from 2 hrs of 75% IPA (from FAA) into an overnight 95% IPA dehydration step, these 2 cassettes are my next batch of blocks! One contains Sonchus.asper stem and root, the other Aloe leaf (again). I'm using these specimens again as it's a good method for comparison with earlier results, using my current, hopefully improved workflow, should prove very interesting indeed.

Finally, I'm looking at the orientation of truly tiny tissues during embedding in wax, they can be very difficult to 'get to stay put' as it were! I'm going to work with the so-called 'double-embedding' technique that uses agar as an initial mount as it stays workable for a long time due to its physical properties (mainly it's manner and temperature of state-change from liquid to semi-solid). The resultant agar-block is then trimmed and embedded within wax in the usual manner! A superb technique if I can get it to work, but I'll have to wait for the arrival of the agar-powder I ordered yesterday!

Oh, there is one other thing I'm looking at but I'm still reading a couple of books on the subject at this time, it's too early to start any practical work - maybe in a couple of months time - that's tissue micro-propagation, (plant that is) - when my Wife enquired about this she said she hoped I wasn't intending to "create a monster from spare parts" .

I've bought several excellent books also that are an absolute goldmine of histological knowledge that really helps. One's a cracking book on the fungus-plant relationship.

That's about it at the moment, I'll post details of some of this work as soon as I've rationalized my notes and have a form that I can post that isn't a great horrible mess! So many things to learn, the beautiful slides are definitely worth the effort, I love comparative botanical morphology and indeed histology and am having a great time learning the ropes! Such fun!

Sorry this reply is a bit mixed-up, I've just thrown it together quickly in answer to your kind question - I'll post some better organised material as soon as I get time - I'm also practicing the use of Microsoft's free 'OneNote' system for my journals and notebooks, I found 'Evernote' to be rather limited and a bit too keen on attaching strings to it's use for my liking. My initial use of the blogging infrastructure went pretty well but OneNote et-al give me a greater sense of being in control of my own material, rather than needing to trust a far-away server 'somwhere'.... Anyway, I'm going on too much and I need to have my supper.

Back soon with a few new threads - work in progress! Thanks for your interest.

[gekko]

That's about it at the moment,

This has got to be the understatement of the century!

[JimT]

I echo Gekko.

So Mrsonchus, what is your serious hobby ?

BTW, I would be interested in your experience with;

"I've also been working on a slightly enhanced image-stacking method that has really yielded good results (i.e. an improvement in my own stacking results) - this is very recent (yesterday) and still being tested at this time, but looks good."

Looking forward to more of your travels.

JimT

[mrsonchus]

That's about it at the moment,

This has got to be the understatement of the century!

Ha! Gekko nice to hear fro you - yes I suppose it's actually quite a list, there's just so much that's intriguing & fascinating, here's a quick look at a stitched pair of stacks of the vascular-bundles near the epidermis of the stem of a Sonchus.asper I've been looking at, the clarity is an improvement, due in part I think to the slightly improved stacking method I now use, but also the workflow is gradually improving too with practice - I like this image, the tissue-structure is pretty clear;

ws_slide_24_sonchus_asper_v.jpg (486.57 KiB) Viewed 11885 times

Here's an image of the dreaded Aloe - I've cleaned the image up a bit with PSE9 as the slide was still wet and still had dust & debris on it as I have to wait 'till it dries to polish it up with some lens-cleaner... The tissue integrity is finally sound - this specimen has been a great test-subject as it's presented numerous problems at all stages up to this point, but now I'm starting I think to get the better of it. I think the gelatinous-filling of the Aloe's leaf has been quite a complicating factor, but here the cells containing the 'gel' can now be imagesd well too. Not a bad result, improvement is clearly evident (thank goodness! );

ws_slide_5_aloe_leaf_XS.jpg (126.08 KiB) Viewed 11885 times

I'll post some 'proper' material as soon as I can. Sorry this is so brief.

[billbillt]

Well, I think you are doing a great job... These photos are wonderful...

Regards,
BillT

[gekko]

I agree with BillT. And one must always remember that those images are the culmination of a great deal of very exacting and time consuming work.

[mrsonchus]

I echo Gekko.

So Mrsonchus, what is your serious hobby ?

BTW, I would be interested in your experience with;

"I've also been working on a slightly enhanced image-stacking method that has really yielded good results (i.e. an improvement in my own stacking results) - this is very recent (yesterday) and still being tested at this time, but looks good."

Looking forward to more of your travels.

JimT

Hi Jim, well, microscopy has been an 'offshoot' of my main hobby of the identification and study of British wildflowers, most folk call them 'weeds' - but they are wrong, although my lovely Wife did take a little persuading when I preserved a good strong patch of nettles for study... I particularly enjoy the mystery of a plant's ID and detailed structure - everything about a plant, from the macro- to the micro-scopic is that way for a reason - the 'form vs funtion' relationship I suppose. I've been studying for example a tiny little plant growing in the shady back-edge of a flower-bed in my front garden, behind a monstrously vigorous hebe, that is most intriguing and beautiful, with a very complex structure.

Here's the sort of thing;

ws_100_3980.jpg (62.72 KiB) Viewed 11880 times

and

ws_100_3986.jpg (63.44 KiB) Viewed 11880 times

Incidentally this turned out to be a 'Pale Toadflax' (Linaria.repens)...

One thing leads to another as they say, and suddenly I've bought 2 microscopes, 2 microtomes, a room full of chemistry apparatus and a desk the size of an aircraft-carrier, not to mention about 50 not-inexpensive books!

It all started with a picture of a wildflower taken about 5 years ago with an old mobile 'phone!

That's the basic story!

Oh, the stacking, yes it's really embarrassingly simple and obviously nothing new to most folk, but to me it's a new method!
I've always found it pretty difficult & inefficient (sub-optimal anyway) to try to 'move up' or 'move down' the subject whilst keeping an eye on various points within the focusing image as I go, Z-order needs to be preserved of course for a decent stack I believe.

All I do now (well since yesterday that is! ) is look for exactly the points I want to be sharp within any intended stack, focus on them individually at pretty high magnification withing the preview-panel of the software (I use 'Toupview') and capture a component image of the stack-to-be, but in no particular Z-order.
I concentrate on point-selection rather than a supposedly smooth Z-ordered series of 'slices'. What I do however as each picture is captured, is just note down the position of the 'scopes fine-focus wheel (it's finely indexed making this easy, although an index could easily be added with a tiny brush and some paint) as each picture is captured, then feed them in Z-order to the chosen stacking algorithm.
From others' fine stacking I learned that the number of images wasn't always the main criterion for a good stack, rather the selection of focus-points (planes) when capturing the component images - this 'new' method has shifted the emphasis entirely over to this factor, a 'smooth progression' seems to include a lot of sub-optimal or just plain poorly-judged images when I use the 'old' method, for me personally that is.
Simple and nothing new, but it really has improved my stacking results significantly! Sorry this is nothing really new, but it has helped me a lot.

Back soon with new threads, thanks for your interest.

[mrsonchus]

Thank you Bill & Gekko, you are very generous and kind.

[mrsonchus]

Here's a look at some of the next sections to come, more Sonchus I'm afraid..

The sections (will soon) come from here;

ws_DSCN2143.jpg (87.29 KiB) Viewed 11878 times

Here's a stack from the hand-held Kodak-C120 8mp compact that I use with the stereo 'scope - far from perfect of course but workable at least - interesting ends on these glandular hairs, a longitudinal section of a hair would be fascinating, but tricky I suspect. Maybe a good test for the soon-to-be-attempted 'agar double-embedding' method!

ws_glandular_trichomes.jpg (176.74 KiB) Viewed 11878 times

Some 'quick-preview' hand sections under the stereo 'scope;

ws_100_3968.jpg (93.83 KiB) Viewed 11878 times

Some cut-pieces of stem ready to go into processing, starting of course with FAA fixative for 48hrs or so...

ws_DSCN2158.jpg (74.5 KiB) Viewed 11878 times

They and the Aloe sections will probably take me a week or so by the time I've prevaricated somewhat as is my usual manner!

As soon as they're ready I'll post perhaps a quick 'walk-through' of their journey from garden & plant-pot to the glories of immortality on a slide!

[billbillt]

Hi John,

I never tire with the clarity and completeness of your work.. You always present a very interesting study whatever the subject... Always look forward to the next installment!...

BillT

[mrsonchus]

Thanks Bill, I'm really pleased you're enjoying the adventures.

[Peter]

Hi Mrsonchus,
The " 'ruck-up' " of sections my be corrected by the following method; after flattening the sections on the slide, drain off the water, place the slide on the bench cover with water saturated filter paper (I recommend Whatman No. 50 (usual disclaimer)), then firmly roll over with a hard rubberised roller, peel off the filter paper and allow the slide to dry as usual. This will normally give a well flattened section firmly attached to the slide, the only problem which may occur is if the section folds on its self in which case there is no way of unfolding it.
Hope this helps.
Peter.

[mrsonchus]

Hi Mrsonchus,
The " 'ruck-up' " of sections my be corrected by the following method; after flattening the sections on the slide, drain off the water, place the slide on the bench cover with water saturated filter paper (I recommend Whatman No. 50 (usual disclaimer)), then firmly roll over with a hard rubberised roller, peel off the filter paper and allow the slide to dry as usual. This will normally give a well flattened section firmly attached to the slide, the only problem which may occur is if the section folds on its self in which case there is no way of unfolding it.
Hope this helps.
Peter.

Hi peter and many thanks for your help - I've tried more or less exactly this procedure but it has several shortcomings that make it unusable, at least for the botanical sections I use... I have however succeeded in completely eliminating this problem after a recent series of trials regarding the water-bath (remembering that I have only a baking-tin and a thermometer at my disposal! ). In short, the main (i.e. relevant) factors were revealed to be:
1) The temperature of the water-bath is crucial, anything below about 37deg will nearly always give wrinkled and 'rucked-up' sections that have both failed to stretch adequately (fully) on the water's surface and to dry flat upon the slide... The optimum temperature at least for my workflow lies in the range 42->46deg inclusive, with an average stretching-time of about 2mins..
2) The water - DI water is definitely optimal, closely followed by pre-boiled water - if tap water is used the gases it contains will simply cause mayhem when stretching sections - air-bubbles, wrinkles & folds, 'blisters' - horrible!
3) Duration of stretching upon the water's surface - with the above factors in place the time I have found to be about 2 maybe 3 minutes, less leaves wrinkles and folds, more can disrupt and even separate tissues.. Incidentally if a cooler temperature is used, say 33-35deg and the time simple increased to say 10mins absolutely no improvement to the unsuitability of this temperature is gained - I've tested this extensively.

The procedure you have detailed has for me at least resulted in many problems, movement of tissues - vastly increasing the tendency of their loss when moving further into processing, dewaxing & staining for example, tissues 'snagging on the (necessarily wet) paper used... The biggest drawback that completely ruled-out this procedure for me was however the mechanical-damage to the tissues - they were, upon examining in detail after mounting in the usual way, 'crushed', with cellular-contents everywhere and a total loss of integrity and therefore usability. The creases also simply folded over upon themselves very often, leaving the expected artifact of a nasty line of mangled cells running across the tissue..

If wrinkling does remain, my standard response now is to reject the slide-to-be as unusable and throw it away. If it's a really desirable or indeed important section, then I will leave the slide and it's troubled wax-section in water for an hour or so at RT then carefully manipulate the loosened section with forceps and needle - this method has worked perfectly for me and allowed not only the correction and completion of the almost aborted initial stretching-attempt but the subsequent completion of the slide and a resultant permanent (relative to my life-span that is to say!) mount suitably stained and finished in the usual manner.

Many thanks again Peter for your help, your method obviously works but I just can't seem to get good results when I try it - I'm doing something wrong somewhere I think.

Just found this hideous picture of a 'ruck'.... yuk!

air_bubble.jpg (148.21 KiB) Viewed 11853 times

That's just plain horrible!

[mrsonchus]

Oh, nearly forgot - I've finished the processing of my latest batch and have cast 4 blocks this evening whilst attempting a shorter and slightly modified protocol rather than the very long 'Ruzin type' version. Initial results look promising, the root-sections that have been very tough and brittle when sectioning have begun to section, from these new blocks, very well indeed, without the previously seen gaps at tissue-type interfaces, and epidermal-damage... Basically I tried 2 shortened protocols, one with a reduced clearing phase, the other with a reduced imfiltration phase... I have removed overnight infiltration entirely as I think this has been hardening tissue and causing brittle-tissue problems when sectioning - results look good but I really have only had time tonight to take and stretch onto slides about a dozen sections.
In the next couple of days I'll post some detailed material with a full account of the new 'lean & mean' protocol used...

Backs soon with a new thread!

[Peter]

Hi Mrsonchus,
Sorry I was of no help, pleased to know you have overcome the problem in your own way.
Peter.

[mrsonchus]

Hi Mrsonchus,
Sorry I was of no help, pleased to know you have overcome the problem in your own way.
Peter.

Hi Peter - of course you have helped - I just haven't for the life of me been able to get the hang of your procedure, I just keep 'squishing' my sections.... I had a god go at it, but just kept failing so decided to 'go the long way round' and basically got lucky with the water-bath tests - thankfully as they took an awful lot of time and effort.

I very much appreciate your kind help, I'm a total amateur and am learning new aspects every day, for example today I started placing my microtome-blades (I 'work through' a set of 5 that I use usually sequentially from roughing to actual sectioning) onto the freezer-slab that I stand my blocks on during a sectioning-run. and hey-presto - a slight improvement in the uniformity of section-form (compression, smoothness etc..)!

I've received much help from folks in this forum, Naphthalene has advised me several times for example, and always find advice helpful, even if I don't use a suggested method the suggestion is always in some way helpful, and most definitely welcome and appreciated!

Some aspects of my histological workflow are at times 'running on fumes' - I've put in a lot of work, but at the same time a seemingly random piece of good fortune has very often helped me, as has the kindly-offered advice of others such as yourself Peter.