A few pictures from this evening's Safranin trials
A few pictures from this evening's Safranin trials
Hi all, I've been working with Safranin this evening both to practice my use of this beautiful stain and to make my first ever slides with a new variety of chemistry. Basically I've changed from Histoclear I (the original clearing-agent/wax solvent) to Histoclear II - a far less odorous formulation that also requires the use of a different (resinous) mounting medium from the trusty (also resinous) 'Numount' that I've used with Histoclear I up 'til now. I now use 'Omnimount' - specifically designed by the manufacturer (National Diagnostics) of all the Histoclear formulations (there are three as far as I know) for use with Histoclear II - although I have also found it to be compatible with Histoclear I.
Anyway, in a nutshell the new protocols are working well and I've just been able to stain & mount a few trial-runs sectioned a couple of days ago. I've used mainly Safranin alone for the ovary slides and a little Fast-green also for the stem sections shown here....
A few colourful pictures, some labelled for content - sorry if I've overdone the labels, I get a little carried away and label everything I see sometimes!
This is a nice result I think - it's (yes - you guessed it! ) a LS of the bottom of the flower-head of the Sonchus.oleraceus 'weed' otherwise known as the 'Smooth sowthistle' here in the UK. It's similar to a Dandelion in that it has (yellow) 'flowers' that actually consist of dozens of tiny 'florets' sitting atop a 'capitulum' - a sort of platform if you like.
Anyway this is a section (LS) through the base of one such capitulum and shows a row of floret-bottoms - actually each floret's ovary is seen sectioned with the single ovule within.
The Safranin really has shown a remarkable degree of metachromasia here and has, all on it's lonesome, differentiated various tissue-types quite distinctly to my eye I think...
If the brightness, intensity and metachromasia here is in any way connected to the new mountant or Histoclear II then I'm very pleased indeed!
and another section similarly stained, vascular bundles within the base of the capitulum are clearly differentiated and almost appear to be approaching a violet tone... The colours here all match what I see through the 'scope - I used a custom WB set from the blank background for this series of images just to make sure.
Here's a close-up of the interestingly-stained nuclei of an ovule - the nuclei have themselves differentiated into 'blobs' and the main nuclear material - I think the 'blobs' may even be pores and the main nuclear envelope differentiated around them - but it's only a guess without looking at the tomes! Great to see such detail though at this early stage - the slides are still wet as usual!
and, (actually I think the 'blobs' may be too large to be pores - I'll take a look tomorrow or when the slides are set, with the x100 oil...)
This section is of the stem (TS) of a Sonchus.asper, the close relative of the above S.oleraceus, although I've since learned of many microscopic differences during my study and slide-making of these two fine fellows! Here's part of the stem TS of said S.asper, stained with Safranin and just a touch of half-strength Fast-green. Apologies for the rather unrestrained labelling - I got a little overenthusiastic!
Here are some details, a vascular bundle (epidermis of stem is towards bottom of picture, pith is towards top)..
A closer-in look at the vascular-cambium/phloem boundary - with a few sieve-plates that were luckily close to the sectioning plane
I've many more but this is all I've time to post tonight - it's now 1:30 am here in the UK..
I hope you like them - I had great fun making them.
Anyway, in a nutshell the new protocols are working well and I've just been able to stain & mount a few trial-runs sectioned a couple of days ago. I've used mainly Safranin alone for the ovary slides and a little Fast-green also for the stem sections shown here....
A few colourful pictures, some labelled for content - sorry if I've overdone the labels, I get a little carried away and label everything I see sometimes!
This is a nice result I think - it's (yes - you guessed it! ) a LS of the bottom of the flower-head of the Sonchus.oleraceus 'weed' otherwise known as the 'Smooth sowthistle' here in the UK. It's similar to a Dandelion in that it has (yellow) 'flowers' that actually consist of dozens of tiny 'florets' sitting atop a 'capitulum' - a sort of platform if you like.
Anyway this is a section (LS) through the base of one such capitulum and shows a row of floret-bottoms - actually each floret's ovary is seen sectioned with the single ovule within.
The Safranin really has shown a remarkable degree of metachromasia here and has, all on it's lonesome, differentiated various tissue-types quite distinctly to my eye I think...
If the brightness, intensity and metachromasia here is in any way connected to the new mountant or Histoclear II then I'm very pleased indeed!
and another section similarly stained, vascular bundles within the base of the capitulum are clearly differentiated and almost appear to be approaching a violet tone... The colours here all match what I see through the 'scope - I used a custom WB set from the blank background for this series of images just to make sure.
Here's a close-up of the interestingly-stained nuclei of an ovule - the nuclei have themselves differentiated into 'blobs' and the main nuclear material - I think the 'blobs' may even be pores and the main nuclear envelope differentiated around them - but it's only a guess without looking at the tomes! Great to see such detail though at this early stage - the slides are still wet as usual!
and, (actually I think the 'blobs' may be too large to be pores - I'll take a look tomorrow or when the slides are set, with the x100 oil...)
This section is of the stem (TS) of a Sonchus.asper, the close relative of the above S.oleraceus, although I've since learned of many microscopic differences during my study and slide-making of these two fine fellows! Here's part of the stem TS of said S.asper, stained with Safranin and just a touch of half-strength Fast-green. Apologies for the rather unrestrained labelling - I got a little overenthusiastic!
Here are some details, a vascular bundle (epidermis of stem is towards bottom of picture, pith is towards top)..
A closer-in look at the vascular-cambium/phloem boundary - with a few sieve-plates that were luckily close to the sectioning plane
I've many more but this is all I've time to post tonight - it's now 1:30 am here in the UK..
I hope you like them - I had great fun making them.
John B
Re: A few pictures from this evening's Safranin trials
Exellent work.
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Re: A few pictures from this evening's Safranin trials
Visually very appealing. Great work, as always.
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LOMO BIOLAM L-2-2
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Cameras: Canon T3i, Olympus E-P1 MFT, Amscope 3mp USB
Re: A few pictures from this evening's Safranin trials
Very nice-looking and informative!
Re: A few pictures from this evening's Safranin trials
Bonjour
Superbe série images.
Et bravo pour votre travail
Cordialement seb
Superbe série images.
Et bravo pour votre travail
Cordialement seb
Microscope Leitz Laborlux k
Boitier EOS 1200D + EOS 1100D
Boitier EOS 1200D + EOS 1100D
Re: A few pictures from this evening's Safranin trials
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Last edited by Yvan on Sun Dec 04, 2016 4:53 pm, edited 1 time in total.
Re: A few pictures from this evening's Safranin trials
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Last edited by mrsonchus on Mon Nov 28, 2016 12:21 am, edited 2 times in total.
John B
Re: A few pictures from this evening's Safranin trials
Yvan for the tissue preparation, you said that isopropyl alcohol should be used instead of ethyl alcohol for tissue processing. Does this apply to both the fixating and dehydrating processes? If so, is there a distinction between fixating with ethyl or isopropyl alcohol.
Re: A few pictures from this evening's Safranin trials
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Last edited by mrsonchus on Mon Nov 28, 2016 12:26 am, edited 1 time in total.
John B
Re: A few pictures from this evening's Safranin trials
Guess I don't know any better, but I think your slides are outstanding, John, and will buy more when I can.
Arnold, Missouri
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Re: A few pictures from this evening's Safranin trials
Thanks Radazz - great to know you enjoy them - makes me smile!
John B
Re: A few pictures from this evening's Safranin trials
Hello John,
I see these as more of your stellar work!.. Wonderful!!..
BillT
I see these as more of your stellar work!.. Wonderful!!..
BillT
Re: A few pictures from this evening's Safranin trials
Thanks Bill my friend - the Mighty-Shandon lives-on!
John B
Re: A few pictures from this evening's Safranin trials
That is the important thingI had great fun making them.
Sounds like you and Yvan should continue to correspond.
Meanwhile after seeing these I can only offer my humble hand sections for what they are, fun to do
JimT
Re: A few pictures from this evening's Safranin trials
JimT wrote:That is the important thingI had great fun making them.
Sounds like you and Yvan should continue to correspond.
Meanwhile after seeing these I can only offer my humble hand sections for what they are, fun to do
JimT
Hi Jim,
I think your hand sections do the job.. They looked good to my untrained eye.. Keep up the good work..
BillT
Re: A few pictures from this evening's Safranin trials
Thanks Jim.JimT wrote:That is the important thingI had great fun making them.
Sounds like you and Yvan should continue to correspond.
Meanwhile after seeing these I can only offer my humble hand sections for what they are, fun to do
JimT
I love your hand-sections, they are a totally different perspective to the paraffin-wax sections and your technique makes my hand-sections look like slices of bread! Their sheer beauty is to my eyes unsurpassed by paraffin-sections also - I've never seen a paraffin-section with that stunning 'liquid-look' that your sections characteristically possess my friend - keep them coming, they're a treat for all of us here.
John B
Re: A few pictures from this evening's Safranin trials
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Last edited by Yvan on Sun Dec 04, 2016 4:54 pm, edited 1 time in total.
Re: A few pictures from this evening's Safranin trials
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Last edited by Yvan on Sun Dec 04, 2016 4:55 pm, edited 1 time in total.
Re: A few pictures from this evening's Safranin trials
Congrats again John B. for excellent botanical microtechnique...you really have me with microtome fever in a nice way.
Thank you Yvan for your keen eye and shared thoughts on this posted set of botanical tissues...intresting for me indeed.
1) I must ask you, Yvan, specifically where in this sequence of sections you sense tissue distortions to an extent that 'rotary sectioning' might be at play...vrs: 'sledge microtome sectioning' as a possible less distorting section method? To my untrained eyes..I sense only 'missed cytoplasm'; inside cell walls due section plane/section thickness...missed cell nuclei inside cell walls in areas due section thickness/section plane. Please offer comments on the distortion areas you notice, Yvan..it greatly enhances this threads botanical images for me and perhaps many others.
I am not hijacking Mr.Sonchas'es thread here.., my coments here are a part of John B's current thread..I have sincere and burning querry on how: when the blade meets the tissue block..with settings of bevel and attack angle of blade, and same wax block criteria...how is a 'slide microtome/ sledge microtome' generating different forces to the botanical tissue than John B's Shandon (?rotary?) microtome?
I enjoy your You-Tube video, Yvan , on performance of your: 'sledge microtome'...quite a few You-Tube tutorials depict this sectioning equipment as a:'slide microtome'...I/m curious why you call your's: 'sledge microtome'?
In ancient times I used an A/O rotary microtome with waxed blocked botanical tissues for 7-9 micron meter sections.
So I keenly desire to have your comments on how a sledge/slide microtome would generate different (potentially distorting forces) distortion forces than a rotary, or a rocking microtome...all settings and block and specimen conditions being equal?
I always want to ask you, Mr.Sonchas...do you ever have an esthetic and statistical goal in your produced botanical sections of capture of most tissues with majority containing cells with nuclei and other organelles...vrs sections so thin that often enough...the plane of section misses the nucleus and other intracellular structures?
Is there a sweet spot section thickness range for a given botanical specimen to depict a large number of cells with pleasing cytoplasm stain reaction...and cells with nuclei? Or is it always the case that ( like the MRI section planes through a persons head say!)..is it always the case that you get tangental swaths of tissue with no cell nuclei...and cell walls with no cytoplasm stain reaction. etc. realities?
I enjoy your Safranin current trials, Joh B., thank you! charlie guevara
Thank you Yvan for your keen eye and shared thoughts on this posted set of botanical tissues...intresting for me indeed.
1) I must ask you, Yvan, specifically where in this sequence of sections you sense tissue distortions to an extent that 'rotary sectioning' might be at play...vrs: 'sledge microtome sectioning' as a possible less distorting section method? To my untrained eyes..I sense only 'missed cytoplasm'; inside cell walls due section plane/section thickness...missed cell nuclei inside cell walls in areas due section thickness/section plane. Please offer comments on the distortion areas you notice, Yvan..it greatly enhances this threads botanical images for me and perhaps many others.
I am not hijacking Mr.Sonchas'es thread here.., my coments here are a part of John B's current thread..I have sincere and burning querry on how: when the blade meets the tissue block..with settings of bevel and attack angle of blade, and same wax block criteria...how is a 'slide microtome/ sledge microtome' generating different forces to the botanical tissue than John B's Shandon (?rotary?) microtome?
I enjoy your You-Tube video, Yvan , on performance of your: 'sledge microtome'...quite a few You-Tube tutorials depict this sectioning equipment as a:'slide microtome'...I/m curious why you call your's: 'sledge microtome'?
In ancient times I used an A/O rotary microtome with waxed blocked botanical tissues for 7-9 micron meter sections.
So I keenly desire to have your comments on how a sledge/slide microtome would generate different (potentially distorting forces) distortion forces than a rotary, or a rocking microtome...all settings and block and specimen conditions being equal?
I always want to ask you, Mr.Sonchas...do you ever have an esthetic and statistical goal in your produced botanical sections of capture of most tissues with majority containing cells with nuclei and other organelles...vrs sections so thin that often enough...the plane of section misses the nucleus and other intracellular structures?
Is there a sweet spot section thickness range for a given botanical specimen to depict a large number of cells with pleasing cytoplasm stain reaction...and cells with nuclei? Or is it always the case that ( like the MRI section planes through a persons head say!)..is it always the case that you get tangental swaths of tissue with no cell nuclei...and cell walls with no cytoplasm stain reaction. etc. realities?
I enjoy your Safranin current trials, Joh B., thank you! charlie guevara
Re: A few pictures from this evening's Safranin trials
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Last edited by mrsonchus on Mon Nov 28, 2016 12:24 am, edited 1 time in total.
John B
Re: A few pictures from this evening's Safranin trials
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Last edited by Yvan on Sun Dec 04, 2016 4:56 pm, edited 6 times in total.
Re: A few pictures from this evening's Safranin trials
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Last edited by mrsonchus on Mon Nov 28, 2016 12:19 am, edited 1 time in total.
John B
Re: A few pictures from this evening's Safranin trials
Thanks John B. for this constant botanical microtechnique work you share in postings..yes,yes it's terrific...err I may be giving myself more of your slides for this years Christmas! You root tip cell mitotic depicting slide in this thread suggests for me the type of slides I wil err..request from you in the name of Santa for me.
Thank you again, Yvan for your thoughtful comments on microtechnique as we forum share,hear,hear!
For me the 'ripping or drag forces' of a sharp steak knife being 'drawn through' and sectioning steak muscle cells, arteries,nerves and fascia...for me this 'drag force' differs from the 'compressive force' of a sharp clever 'chop sectioning' a similar parcel of steak flesh. Yes I sense,Yvan, that without vector algebra , yes I sense 'drag forces' differ from compressive forces as a properly processed botanical tissue is sectioned.
I just thought there are trade offs with all sectioning...especially of hard or brittle tissues...I did not understand that compressive forces of botanical sectioning are often more distortion causing than the drag forces of a tangentially advancing microtome blade...a blade/tissue contact through equal area of the tissue specimen as in case of rotary ...but with the tangentally advancing slide microtome blade...so much more blade area drags through the tissue sample!
Yvan I love your mention of the germanic term for the microtechnique processed specimens...the term to always remind the observer that the reality far exceeds what the processed specimen captures...thank you,Yvan!
Sigh...it's Platonic appreciation of this botanical microtechnique John B. shares here. In processed specimens we strive for elegance, beauty, and stable depictions of the tissue specimens. We strive for an essence of a tissues ideal whole, we always understand that in microtechnique many aspects of the tissue are missed...yet incredible realities are suggested!
Slide microtome drag forces vrs rotary microtome compressive forces...humm...imbed this finger lakes thread hijacker in hot wax! charlie guevara
Thank you again, Yvan for your thoughtful comments on microtechnique as we forum share,hear,hear!
For me the 'ripping or drag forces' of a sharp steak knife being 'drawn through' and sectioning steak muscle cells, arteries,nerves and fascia...for me this 'drag force' differs from the 'compressive force' of a sharp clever 'chop sectioning' a similar parcel of steak flesh. Yes I sense,Yvan, that without vector algebra , yes I sense 'drag forces' differ from compressive forces as a properly processed botanical tissue is sectioned.
I just thought there are trade offs with all sectioning...especially of hard or brittle tissues...I did not understand that compressive forces of botanical sectioning are often more distortion causing than the drag forces of a tangentially advancing microtome blade...a blade/tissue contact through equal area of the tissue specimen as in case of rotary ...but with the tangentally advancing slide microtome blade...so much more blade area drags through the tissue sample!
Yvan I love your mention of the germanic term for the microtechnique processed specimens...the term to always remind the observer that the reality far exceeds what the processed specimen captures...thank you,Yvan!
Sigh...it's Platonic appreciation of this botanical microtechnique John B. shares here. In processed specimens we strive for elegance, beauty, and stable depictions of the tissue specimens. We strive for an essence of a tissues ideal whole, we always understand that in microtechnique many aspects of the tissue are missed...yet incredible realities are suggested!
Slide microtome drag forces vrs rotary microtome compressive forces...humm...imbed this finger lakes thread hijacker in hot wax! charlie guevara