Ovule and Ovaries, glorious colour, mysterious cells

Here you can post pictures and videos to show others.
Post Reply
Message
Author
User avatar
mrsonchus
Posts: 3788
Joined: Tue Feb 03, 2015 9:42 pm
Location: Cumbria, UK

Ovule and Ovaries, glorious colour, mysterious cells

#1 Post by mrsonchus » Sat Feb 27, 2016 4:53 pm

Hi all, I've a few pictures of ovules of the Daffodil sections again, these are a mixture of stain-types I've tried. Also I've concentrated of the embryo early stages in particular the first couple of cell-divisions of the embryo as it begins to form...

Here's a few quick pictures, I'll try to add some info later and hopefully some more regarding what the sections are actually showing! I'm still learning this plant embryology as I go, great fascinating fun. :D

This is my first dalliance with my newly arrived 'Harris Haematoxylin' nuclear stain, the idea being to use this as a 'speciality' nuclear stain together with Safranin and Fast-green, two excellent & complementary stains that you've seen in many of my latest slides since I've managed to get them working together well.
Here is an attempted tri-stained ovule within the ovary (in TS) of the Daffodil seen in many of my current set of sections detailing the male (anthers and pollen) and female (ovary and ovules) reproductive processes and anatomy of a Daffodil.

This is a 39-image stitch of x100 objective pictures, I love the x100, it is really bright and always gives me excellent resolution - it's my favourite objective for sure.
It is possible to see the Fast-green in the cells to the far right, the HH is the dark-purplish of the dense cells of the ovule (nucellus tissue) and nuclei. It seems here that two cells of the actual embryo-proper (central) have each taken one stain each, one seems to have taken Harris-Haematoxylin the other is the pink of Safranin (which or course is a nuclear-stain in it's own right).. Very interesting and I would like to find out if this is accidental or indicative of a difference between these two nuclei?
ws-edited-x100-tri-stain-ov.jpg
ws-edited-x100-tri-stain-ov.jpg (399.87 KiB) Viewed 1215 times
Here's a cropped version of the x100 stitch, the resolution remains pretty good I think,
ws-cropped-x100-tri-stain-o.jpg
ws-cropped-x100-tri-stain-o.jpg (307.01 KiB) Viewed 1215 times
This is a stacked image, not a lot of difference in detail seen in the above cropped (single focus without stacking) image of the stitched master-pieces,
ws-ovule-stacked.jpg
ws-ovule-stacked.jpg (264.15 KiB) Viewed 1215 times
Here are some cells of the nucellus, that tissue from within which the ovary begins do grow, again with the tri-stain method,
some of these cells also apper to have 'atypical' nuclei and I suspect things are happening in this tissue that I have yet to understand or recognize! Another case of 'to the books'! See what you think it all means...:D
ws-tri-stain-0021.jpg
ws-tri-stain-0021.jpg (325.62 KiB) Viewed 1215 times
Finally, you may wish to shield your eyes or grab a polarizer for this one!
This is an 'enhanced' (with ammonia-water) section stained with the savagely-fast and efficient (basic) Fuchsine stain in 0.5% aqueous preparation after 5 minutes of alkali-'enhanced' staining! The idea of these tests was to achieve pure strength of staining, no subtlety was attempted (or indeed achieved! :oops: ) at these early stages - this is pretty evident in this monstrously vivid almost frighteningly bright image! This slide almost glows in the dark it's that vivid!
ws-fuchsine.jpg
ws-fuchsine.jpg (242.71 KiB) Viewed 1215 times
Apologies if that last image has made your head spin! :D

Anyway, sorry to be so brief, I hope to post some more analytical information perhaps later tonight if I get the chance! Hope you like these and find them interesting. :D
Back soon. :)
John B

User avatar
Raul
Posts: 90
Joined: Tue May 12, 2015 4:55 pm
Location: Bradford, England

Re: Ovule and Ovaries, glorious colour, mysterious cells

#2 Post by Raul » Sat Feb 27, 2016 5:20 pm

John B that is very beautiful, I especially like the complementarity of haematoxylin and fast-green. Superb pictures,great clarity, please do post the protocol and all the information from the slide.

User avatar
Raul
Posts: 90
Joined: Tue May 12, 2015 4:55 pm
Location: Bradford, England

Re: Ovule and Ovaries, glorious colour, mysterious cells

#3 Post by Raul » Sat Feb 27, 2016 5:31 pm

Also it looks to me that the cell which does not seem to have cytoplasm and the red nucleus (stained with safranin) is undergoing apoptosis (programmed cell death)
http://labs.bio.unc.edu/dangl/pub/pdf/C ... e_2011.pdf
Here's a paper that I found on this. (the image is taken from that paper).
Attachments
apoptosis.jpg
apoptosis.jpg (11.98 KiB) Viewed 1212 times

JimT
Posts: 3247
Joined: Fri Oct 24, 2014 1:57 pm

Re: Ovule and Ovaries, glorious colour, mysterious cells

#4 Post by JimT » Sat Feb 27, 2016 6:29 pm

John, those are as usual fantastic. I can see why you like the X100. The resolution is tack sharp!

billbillt
Posts: 2895
Joined: Mon Nov 10, 2014 10:01 pm

Re: Ovule and Ovaries, glorious colour, mysterious cells

#5 Post by billbillt » Sat Feb 27, 2016 6:43 pm

Hi John,
You could publish a textbook from your wonderful work!..

BillT

User avatar
mrsonchus
Posts: 3788
Joined: Tue Feb 03, 2015 9:42 pm
Location: Cumbria, UK

Re: Ovule and Ovaries, glorious colour, mysterious cells

#6 Post by mrsonchus » Sat Feb 27, 2016 7:42 pm

Thanks fellows, I'm very happy you like them, they interest me greatly and I've about another 100 where those came from still stored in wax on slides until I check through them and select another promising set to stain and mount.
Sectioning results in literally dozens of viable slides (thanks to the 'Mighty Shandon') and they can be stored un-sectioned as wax-blocks or as sections in wax that have been floated onto slides and dried! A fabulous resource for those times when material is scarce or the season is just plain wrong!

Raul - the planned cell-death is something I haven't considered and know absolutely nothing about other than it happens! That's a very good idea of yours old chap and I'm going to enjoy consulting the tomes tonight on this topic, fascinating and a great topic for study - the ovary and it's contents is surely a hugely dynamic location, so much goes on, so much growth, differentiation, division both mitotic & meiotic, cell-death and degeneration, even digestion if you consider the journey of the pollen-tube towards the ovule, sometimes it even 'gnaws it's way' through the wall rather than entering gracefully via the micropyle it seems!

Having a great time, my 10hr protocol is going to be so useful for these relatively small and permiable tissues! Such fun and mystery!
Thanks chaps for your interest and very generous comments, I really appreciate your support and company. :)

Look what I've got 'in the cupboard for a rainy day'...!

A selection of blocks containing tissue that will keep virtually indefinitely...
ws_DSCN3437.jpg
ws_DSCN3437.jpg (66.36 KiB) Viewed 1196 times
Sections on slides ready to de-wax, again good virtually forever!
ws_DSCN3438.jpg
ws_DSCN3438.jpg (60.19 KiB) Viewed 1196 times
and more in dryer-trays,
ws_DSCN3439.jpg
ws_DSCN3439.jpg (88.6 KiB) Viewed 1196 times
and
ws_DSCN3440.jpg
ws_DSCN3440.jpg (69.81 KiB) Viewed 1196 times
I'm hoping to get some sectioning done this evening but not sure if I'll get the time...
ws_DSCN3442.jpg
ws_DSCN3442.jpg (60.84 KiB) Viewed 1196 times
What a superb treasure-trove! :D

Back soon-ish....
Last edited by mrsonchus on Sat Feb 27, 2016 8:21 pm, edited 1 time in total.
John B

User avatar
mrsonchus
Posts: 3788
Joined: Tue Feb 03, 2015 9:42 pm
Location: Cumbria, UK

Re: Ovule and Ovaries, glorious colour, mysterious cells

#7 Post by mrsonchus » Sat Feb 27, 2016 7:51 pm

JimT wrote:John, those are as usual fantastic. I can see why you like the X100. The resolution is tack sharp!
Thanks Jim, yes the x100 is a real beauty, I'd dearly love to supplement it with a correctable (collar) dry x60 - I'll need to save-up for quite a while for one of those! At least I've got my micrometer now and am able to measure my coverslips! The x100 is just so bright and as far as I can see clear.
I love it, from that exciting instant when the lens encounters the oil and a 'twinkle' of light appears as if to confirm the meeting of these two old friends has gone to plan!
The 'cranking' (very carefully of course) of the table up towards focus, more excitement, then the quite dramatic increase not in magnification as much as clarity and brightness achieved when remembering the use of the dry x60 as my usual precursor and 'finder' for the dazzling star-of-the-stage, the x100!

Pure pleasure, pure delight! Ahem.., anyway, I really like my x100! :oops:
John B

User avatar
mrsonchus
Posts: 3788
Joined: Tue Feb 03, 2015 9:42 pm
Location: Cumbria, UK

Re: Ovule and Ovaries, glorious colour, mysterious cells

#8 Post by mrsonchus » Sat Feb 27, 2016 7:52 pm

billbillt wrote:Hi John,
You could publish a textbook from your wonderful work!..

BillT
That's very generous Bill! It's delightful when a staining-protocol goes well - it fills me with happiness!
John B

User avatar
mrsonchus
Posts: 3788
Joined: Tue Feb 03, 2015 9:42 pm
Location: Cumbria, UK

Re: Ovule and Ovaries, glorious colour, mysterious cells

#9 Post by mrsonchus » Sat Feb 27, 2016 7:54 pm

Raul wrote:Also it looks to me that the cell which does not seem to have cytoplasm and the red nucleus (stained with safranin) is undergoing apoptosis (programmed cell death)
..........
Downloaded and ready to go Raul, many thanks for your effort in finding this for me. :)
John B

User avatar
rnabholz
Posts: 3075
Joined: Thu Jan 08, 2015 10:11 pm
Location: Iowa USA
Contact:

Re: Ovule and Ovaries, glorious colour, mysterious cells

#10 Post by rnabholz » Sat Feb 27, 2016 8:07 pm

More amazing and informative images from you John,

Thank you,

Rod

User avatar
Crater Eddie
Posts: 1829
Joined: Wed Nov 12, 2014 4:39 pm
Location: Illinois USA

Re: Ovule and Ovaries, glorious colour, mysterious cells

#11 Post by Crater Eddie » Sat Feb 27, 2016 10:52 pm

The Haematoxylin seems to have given some very interesting results, I have been looking forward to seeing what it would do. Gorgeous images, as usual.
CE
Olympus BH-2 / BHTU with Olympus E-P1 MFT camera mounted
LOMO BIOLAM L-2-2
LOMO POLAM L-213 / BIOLAM L-211 hybrid
LOMO Multiscope (Biolam)

User avatar
mrsonchus
Posts: 3788
Joined: Tue Feb 03, 2015 9:42 pm
Location: Cumbria, UK

Re: Ovule and Ovaries, glorious colour, mysterious cells

#12 Post by mrsonchus » Sat Feb 27, 2016 11:19 pm

Raul wrote:John B that is very beautiful, I especially like the complementarity of haematoxylin and fast-green. Superb pictures,great clarity, please do post the protocol and all the information from the slide.
My pleasure Raul, which protocol would you like to see, are you referring to the staining or the tissue-processing?

In a nutshell, the tissue started the staining protocol here for the tri-stain seen in the pictures, from DI water, having been de-waxed, the wax-slovent removed with OH and then the OH removed in stages and replaced by water until rehydration was complete and DI water was reached. This was the 'launch-pad' as I think of it, for the staining routine/s.

So, all stains applied straight onto sections (dropper into bottle of stain) wet on slide horizontally on my desk - quicker than using Coplin-jars and more controllable when exploring new techniques I find..

start from water (DI or tap-water - not seen any difference really)
Harris Haematoxylin aqueous used as-is straight from the bottle as supplied by Brunel Microscopes (BM), stain for 5 minutes then remove excess stain with tap-water, the slight alkalinity slightly augments the stain (sometimes called 'bluing' with some stains)..
brunel harris haematoxylin.JPG
brunel harris haematoxylin.JPG (22.96 KiB) Viewed 1188 times
Now you have a nicely-contrasted and stained section, ready for the Safranin, used as a 1% aqueous mix for 2-5 minutes and rinsed in the same manner as the HH, this I buy as powder (again from BM) and mix and filter my own concentrations (simply put, stonger or longer?),
brunel safranin powder.JPG
brunel safranin powder.JPG (20.56 KiB) Viewed 1188 times
Mix and filter, it stores for months!
ws_filtering_stain.jpg
ws_filtering_stain.jpg (135.03 KiB) Viewed 1188 times
Incidentally, if you think the HH is a little strong, remove some with a rinse or dip of acidified OH or water, I acidify both with simple 'white vinegar' - about 1% v/v is what I use,

Then we need to switch from aqueous to alcohol-based staining for the appropriately-named 'Fast-green' - I buy it in powder-form and use an aqueous mixture also, but for sheer speed and strength and I find controllability, I use, you guessed it - BM's ready-made bottle of Fast-green (OH) with cellosolve,
brunel fast green cellosolve.JPG
brunel fast green cellosolve.JPG (19.13 KiB) Viewed 1188 times
This involves dipping the at this stage 2-stained slide into 75% OH for about 5 seconds, letting it drain for a second or two, then similarly into 95% OH for about 5-10 seconds. This renders the section ready for the Fast-green, if any water is around this Fast-green will let's just say, 'turn inconveniently nasty'!
So, put the slide flat on the desk as with the first 2 stains, drop enough Fast-green onto the section to swamp it, wait between 5 and 10 seconds - no more, remember the name... then rinse away the excess Fast-green to your satisfaction, that is to say you have a nice balance between the three stains, with 95% OH - staining is complete!

Don't let the slide dry-out, place (dip for about 30 seconds) into a 50/50 mix of OH and your mountant's solvent, in the case of the 'Numount' resin I use this is 'Histoclear', then into pure Histoclear (for Numount) for about 30 seconds minimum to prepare for the mountant's attention - the slide may be left in the Histoclear for at least 10-20 minutes if you, like me, like to clear-up at each stage and basically think about the next stage before I rush into action!

Mount cover-slip etc and enjoy!

Sorry this is a little condensed, the full procedure written-out clearly, accurately and without ambiguity would I think be about 10 pages long to make it solid.

Good luck, try just 1 stain if you like, I started with Methylene-blue and ruined so many sections as I learned how to tame stains I lost count! I had a great time learning though! (and still do - so much to learn... :D )

:)

Here's the one I made with this method, I think I may have under-stained the Fast-green component but at least the combination has produced a result!
ws-tri--stain-1.jpg
ws-tri--stain-1.jpg (317.79 KiB) Viewed 1188 times
Hope this helps Raul.
John B

User avatar
mrsonchus
Posts: 3788
Joined: Tue Feb 03, 2015 9:42 pm
Location: Cumbria, UK

Re: Ovule and Ovaries, glorious colour, mysterious cells

#13 Post by mrsonchus » Sat Feb 27, 2016 11:45 pm

Crater Eddie wrote:The Haematoxylin seems to have given some very interesting results, I have been looking forward to seeing what it would do. Gorgeous images, as usual.
CE
Thanks Eddie, yes I'm pleased with the 'first-go results', they seem to have good nuclear detail and a real sense of the sheer density of the cytoplasm and nuclear 'granularity' in a manner different to Safranin - a good nuclear-stain I find also.
I've a slide or two I made using the other 'version' of Haematoxylin that I purchased at the same time as the Harris, that's the Ehrlich's, but I haven't had time to make a proper appraisal of it's singular effect yet, before using it as a primary nuclear stain as I've just tried with the Harris - can't wait to start to investigate protocols incorporating the Haematoxylins, they seem to do a good job with Botanicals judging by these early results.

Lots of tinkering to come! Great fun. Supremely interesting too! :D
John B

User avatar
zzffnn
Posts: 2713
Joined: Sat Jun 20, 2015 3:57 am
Location: Houston, Texas
Contact:

Re: Ovule and Ovaries, glorious colour, mysterious cells

#14 Post by zzffnn » Sun Feb 28, 2016 3:18 pm

Wow, John B.

I rarely see a private "lab" that is so well organized and equipped.

Our previous research lab in university has more fancy equipments, but looking at that lab, you can tell it is for work, not for passion / love (hobby).

User avatar
Raul
Posts: 90
Joined: Tue May 12, 2015 4:55 pm
Location: Bradford, England

Re: Ovule and Ovaries, glorious colour, mysterious cells

#15 Post by Raul » Sun Feb 28, 2016 8:50 pm

Thank you John B for answering, yes that was exactly what I wanted to know, beautiful results, very good staining protocol.
Also why did you filter the Safranin solution? I also bought it from BM and it did not have any problems with it of impurities( I bough the powder as in your image).
Congratulations for your good work and magnificent slides, the effort you put in it is well spend and well rewarded with the results.

User avatar
mrsonchus
Posts: 3788
Joined: Tue Feb 03, 2015 9:42 pm
Location: Cumbria, UK

Re: Ovule and Ovaries, glorious colour, mysterious cells

#16 Post by mrsonchus » Sun Feb 28, 2016 9:24 pm

Raul wrote:............
Also why did you filter the Safranin solution? ....
Thanks Raul, pleased to help. The Safranin that I filtered (seen in picture) was the aqueous solution that I made using the powder, Brunel's ready-made cellosolve version is 'ready to go' although I do transfer these into bottles with built-in droppers for ease of use.

The reason I filter the aqueous mixture home-made is simply to avoid any unpleasantness from any stray fibers, lumpy-bits in the stain as I mixed it maybe incompletely etc, just for good practice really, I'm a bit of a fusspot! It's as you say, not really necessary I'm sure. :)

Let us know how you get on. :D
Last edited by mrsonchus on Sun Feb 28, 2016 10:51 pm, edited 1 time in total.
John B

charlie g
Posts: 1461
Joined: Mon Oct 20, 2014 7:54 pm

Re: Ovule and Ovaries, glorious colour, mysterious cells

#17 Post by charlie g » Sun Feb 28, 2016 9:35 pm

Thanks for informing us how ready mixed,and how with seconds (the Fast Green),or minutes.these store bought and ready to go.stains can be used to great results (err, in your lab techniques), John B.!

In this series where you tantalize us with the Heam. stain...your first image says it all:


1) Daffodils are a perenial flowering agiosperm plant ( we dig up our bulbs after four years and reset them).

2) Length of day is critical (usually) to to triggering the floral primordia in Daffodils...so we know this specimen of an embryo sac at multiple cell stage...this specimen was 'forced by human intervention'...bulbs placed in greenhouse flats...kept in dark till bulbs formed roots,at perhaps 45F.. Once roots formed, temp raised to 65 F, and flats of bulbs well watered. Once flowers about to open...the Daffodils placed in direct sunshine. Your locales hillside Daffodils are quite a ways from 'springtime awakening'...I'm guessing.

3) So your first image in this series excellently depicts a: near to mature megagametophyte at perhaps eight nucleus stage???I say this as this 'embryo sac ( not an embryo)' already shows nuceli at opposite poles of this embryo sac...the egg cell is near the bottom of your image...the polar nuclei near the middle of the embryo sac.

4) this first image shows the entire (post meiotic female gamete+ it's attendant few cells=mature megagametophyte )...shows the entire 'gametophyte life stage' plant (only seven or so cells total usually).


5) the anatomy of your Daffodil flowers ovary..has each ovule stalked off a central axis in this plants ovary...these are termed : axil placentationed ovules...the stalk attaching to the plants ovary axis is the funiculus..in your earlier series..the stalk/ the funiculus was quite evident...here I suggest that the funiculus is to the left side of your first image.

6) I'm suggesting that your first image of this series depicts a nubile gametophyte life stage /a lady in waiting for her suitor...for her pollen tube bold arriving : mature male gametophyte ( the complete male gametophyte stage comprized of a pollen spore germinated to an organism with one pollen tube nucleus+ two sperm nuclei.That is the entire male gametophyte stage in these flowering angiosperms.


We can marvel at the first image in this series and anticipate if it goes like: a little wine, a little song once sir pollen spore has made bold purchase on the Daffodils stigma!?? thanks for making it look easy, John B., thanks for our open entry to your lab. charlie guevara 60's F in finger lakes/US

User avatar
mrsonchus
Posts: 3788
Joined: Tue Feb 03, 2015 9:42 pm
Location: Cumbria, UK

Re: Ovule and Ovaries, glorious colour, mysterious cells

#18 Post by mrsonchus » Sun Feb 28, 2016 10:01 pm

Ah Charlie G - you don't know how valuable your expert input is; the information imparted and your superb insight & analysis is exactly what I need at this stage in my adventures. I've numerous very fine books that will explain, illustrate, diagram and demonstrate the botany, but the real treasure is knowledge such as yours and your extremely generous willingness to take the time to shine a light onto my sections that I and others here may understand and learn without uncertainty.

I can't thank you enough Charlie G - you are in danger of becoming out 'consulting Botanist'! :D
I peel your knowledge from my screen with my retinas Mr G, I am very, very pleased you posted your superb pictures of your Kalanchoe-plantlet slides so long ago - they had 'expert botanist' written all over them!

Thank you again Charlie, you have helped and delighted me with your generosity. :D :)
John B

User avatar
billben74
Posts: 1020
Joined: Sat Dec 26, 2015 10:33 pm
Location: Cambridge, UK

Re: Ovule and Ovaries, glorious colour, mysterious cells

#19 Post by billben74 » Sun Feb 28, 2016 11:43 pm

Just amazing...
Mitosis, apoptosis such insight into real, wonderful, biology.

User avatar
mrsonchus
Posts: 3788
Joined: Tue Feb 03, 2015 9:42 pm
Location: Cumbria, UK

Re: Ovule and Ovaries, glorious colour, mysterious cells

#20 Post by mrsonchus » Mon Feb 29, 2016 12:29 am

billben74 wrote:Just amazing...
Mitosis, apoptosis such insight into real, wonderful, biology.
Thanks Bill, you're up next! :D :D

12µ....10µ......8µ......5µ....3µ..... screaming and fainting.... and----relaxxxxx......! :D

Hussar! Let loose the sectioners' wildness, fire-up the mighty 'tomes! :D
John B

Post Reply