Measuring chromosomes on 1µ sections

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mrsonchus
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Measuring chromosomes on 1µ sections

#1 Post by mrsonchus » Sat Mar 05, 2016 1:46 am

Hi all, nearly forgot about the 1µ sections cut a couple of days ago. I stained and mounted a few slides from these terrifyingly-thin sections, after ruining several or having them simply fly-away and stick irretrievably to the dog! Anyway they're needless to say a little tricky but not impossible to handle, and here are a few pictures of them stained with Toluidine-blue. They show chromosomes well in the ovule-tissue containing cells dividing in mitosis...

Here's part of a section that folded-over while mounting - this happens occasionally and whilst it's a bit of a pain, it does give an opportunity tho measure the thickness of the folded-over tissue and therefore the thickness of the section, as compared to that set with the 'Mighty Shandon' at the time of sectioning. In this case the sections were cut at a nominal 1µ set on the MS's dial! The thickness measured supports the fact that the MS has indeed faithfully sectioned at the absolute minimum of it's operating-range, that of a single, lonely, micron... :D
ws_1-micron-measured.jpg
ws_1-micron-measured.jpg (111.98 KiB) Viewed 964 times
It can be seen also that at 1µ the walls of un-strengthened 'ordinary' cells, such as parenchyma for example, are a little bruised and battered by the 1µ cut.
This is no surprise to me and isn't important as I'm not targeting the overall morphology at this thickness (for that I would section at between about 6-10µ) rather discreet chromosomes and details of strengthened-tissue such as xylem-vessels, and to this end I'm quite pleasantly surprised by the fine details the 1µ has apparently given the slide.

Here then is a 1µ section showing chromosomes in varying stages of mitosis. The detail is fine but I can achieve a similar level of detail at a far easier-to-cut 4/5µ!
ws_daffodil_chromosome-meas.jpg
ws_daffodil_chromosome-meas.jpg (177.78 KiB) Viewed 964 times
This one's a section at 1µ through a xylem-vessel's reinforced walls and really does show fine details that would be more difficult but not impossible to achieve at say 5-10µ... I think the thinness of the sections is making focus with the camera more crisp, no stacking needed at 1µ I suspect.. :D
ws_1micron_ovary_xylem.jpg
ws_1micron_ovary_xylem.jpg (129.58 KiB) Viewed 964 times
Sorry to be brief with these but I've no time left tonight. I think the 1µ may have a limited use but is a good practice-run for the nerves and steadiness of technique! :D I suspect my favoured thicknesses will be between about 4 and 10µ as these have both given me superb results recently. :D

Back with some more adventures soon. :)
Last edited by mrsonchus on Sat Mar 05, 2016 3:16 am, edited 1 time in total.
John B

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Re: Measuring chromosomes on 1µ sections

#2 Post by rnabholz » Sat Mar 05, 2016 2:10 am

And the Gold Medal goes to........John for his one micron sections....Outstanding demonstration of skill.

Congratulations on some really spectacular work.

Rod

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Re: Measuring chromosomes on 1µ sections

#3 Post by mrsonchus » Sat Mar 05, 2016 2:19 am

Aha - you're a very kind fellow Rod! :D

I'm having a lot of fun with these chromosomes but I particularly like some of the very fine details; an example of which is seen in the spaces between the xylem-vessel-'corners' in the picture, the side of the vessel is also visible. I'm sure the thinness of section is significantly augmenting if not enhancing the focus of my 2mp Toupcam - at 1µ and through my trusty x100 objective everything seems amazingly sharp and bright... :)

Thanks for your generous compliment Rod. :)
John B

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Re: Measuring chromosomes on 1µ sections

#4 Post by JimT » Sat Mar 05, 2016 11:04 pm

And the Gold Medal goes to....
Not only the gold medal but automatic induction into the sectioning hall of fame.

John, fantastic as usual. When do we get to see 0.5 um sections :D

JimT

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Re: Measuring chromosomes on 1µ sections

#5 Post by mrsonchus » Sun Mar 06, 2016 12:00 am

JimT wrote:
And the Gold Medal goes to....
Not only the gold medal but automatic induction into the sectioning hall of fame.

John, fantastic as usual. When do we get to see 0.5 um sections :D

JimT

Argghhhhhh..... Jim! urk! :D :D

Hmm, maybe if I were to sell a massive number of slides I could afford an 'ultra-microtme' for 'semi-thin' sectioning! Now look, you've made me drool.... :D :oops:
John B

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Re: Measuring chromosomes on 1µ sections

#6 Post by mrsonchus » Sun Mar 06, 2016 12:38 am

Hi all, I found some more 1µ pictures that I seem to have forgotten to post, I'm pretty sue I haven't posted these yet, apologies if I have but just in case here's another I found of chromosomes in a 1µ section,

1µ section stained with Toluidine-blue (TBO) stain - mitosis everywhere!
ws_1-micron-TBO-0008.jpg
ws_1-micron-TBO-0008.jpg (359.26 KiB) Viewed 926 times
I think I'd better check my pictures again. :)
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Re: Measuring chromosomes on 1µ sections

#7 Post by p3aul » Sun Mar 06, 2016 12:48 pm

Pardon a question from a neophyte John! In this last picture; is the darker purple, cell walls and the deep purple(blue?) strings, the chromosomes? BTW beautiful pictures! I can't wait to try this!

Thanks,
Paul
Paul Microscope: Amscope T400b Camera: Amscope MU300
Telescope: Orion xt6 classic Dob, Zhumell z10 classic Dob

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Re: Measuring chromosomes on 1µ sections

#8 Post by billbillt » Sun Mar 06, 2016 5:42 pm

More wonderful photos... It appears the Shandon was a wise investment!...

BillT

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Re: Measuring chromosomes on 1µ sections

#9 Post by billbillt » Sun Mar 06, 2016 6:40 pm

Hi John,

Here is some data on chromosome sizes:

Size of Chromosomes
The size of chromosomes varies from species to species. In the same species, all the chromosomes occurring in a given cell will not be of the same size. However, in most organisms, chromosomes fall in the size range of 0.1 to 30 μm in length and 0.2 to 2.0 μm in thickness. Chromosomes are relatively larger in size where the number is less. In general, chromosomes are larger in size in plants than in animals. Among plants, cells of monocots contain larger chromosomes than cells of dicot plants.

From this location:

http://www.tutorvista.com/content/biolo ... mosome.php#

BillT

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Re: Measuring chromosomes on 1µ sections

#10 Post by mrsonchus » Sun Mar 06, 2016 11:29 pm

p3aul wrote:Pardon a question from a neophyte John! In this last picture; is the darker purple, cell walls and the deep purple(blue?) strings, the chromosomes? BTW beautiful pictures! I can't wait to try this!

Thanks,
Paul
Hi p3', a good question that I've only been able to answer myself for about 6 months... If you look at the first image the red arrowed-line measuring 19.1µ is measuring between two cell-walls that have been stained in the mid-range purple of that image.

The next picture (i.e. the second down) has a pair of parallel lines that are measuring a chromosome's 'thickness' at an apparent 0.8µ - the chromosomes (not discretely visible when a cell is not actively undergoing division) are shown more 'strongly' stained with darker shades of blue rather than purple - the metachromatic property of Toluidine-blue stain is partially manifested in this slide and image....

The last image has nicely stained (lignified) xylem-vessel walls stained as light blue.
The variations between images of the intensities of the three colours is just an articact of the imaging process and settings thereof such as white-balance and saturation - the relative differences between the different cell-constituents does however persist and the metachromasia of the TB0 is I think showing as three clearly differentiated colours.

Hope this helps, good question that I've asked many times in this forum also! :D
John B

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Re: Measuring chromosomes on 1µ sections

#11 Post by mrsonchus » Sun Mar 06, 2016 11:31 pm

billbillt wrote:More wonderful photos... It appears the Shandon was a wise investment!...

BillT
Thanks Bill, hmmmm - it would be nice to think this but, well, even a small return would be nice and it would be good to know other's like my slides! :D It's a little early to be ordering a new set of optics I think! :D :D
John B

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Re: Measuring chromosomes on 1µ sections

#12 Post by mrsonchus » Sun Mar 06, 2016 11:38 pm

billbillt wrote:Hi John,

Here is some data on chromosome sizes:

........., chromosomes fall in the size range of 0.1 to 30 μm in length and 0.2 to 2.0 μm in thickness....................
From this location:

http://www.tutorvista.com/content/biolo ... mosome.php#

BillT
Very handy Bill, thanks - this seems to indicate that these chromosomes may indeed actually be about the size indicated by 'Toupview's' measuring system, at about 0.8µ (very) approximately...

I must admit I'm finding the investigation and study of mitosis extremely interesting and rewarding (in fact I'm delighted by these sections from the 'Mighty Shandon') - being able to actually see discrete condensed chromosomes and even spindles in my sections is almost making my head spin! I feel like a very luck fellow I can tell you! :D
John B

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