Three Plant-Section Thicknesses Compared
Posted: Sun Aug 23, 2015 7:50 pm
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; This looks pretty good to me, but perhaps seems a little thick to give best definition and clarity?
this one's sectioned at 10µ; 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; 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)... and; Things have moved along since them! (I love my Shandon!)
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; This looks pretty good to me, but perhaps seems a little thick to give best definition and clarity?
this one's sectioned at 10µ; 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; 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)... and; Things have moved along since them! (I love my Shandon!)