I knew collagen can be intensely birefringent but in res. papers and in my previous slides never got much feeling for it. They were mostly rather dull. Then by chance got a new (but not recently made) tendon etc. slide and it blew the lid off. It was also surprise since from the outset it seemed prepared much the standard lab way, probably just H&E staining.
Investigating a bit learned that the birefringence can be both amplifed or surpressed by details of staining procedure and choice of mountant. Can't get more info on my slide prep. anymore. I'm curious if anyone else has encountered wild pol differences due to preparation in their birefingent bio slides and figured out the root cause?
Anyways here are some views. Initially got into this since I modded a rotating stage to my scope. Used to think it was a somewhat redundant piece of hardware but not anymore! Even if you don't measure any angles, it is just a wonderfully natural thing when these birefringent things come up.
All with Zetopan & Reichert Apo 20x, 15-25 pic stacks, some contrast adj. in PP.
Cheers, Karl
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Collagen
Moderators: rjlittlefield, ChrisR, Chris S., Pau
Re: Collagen
Thanks Pau.
Indeed there is much else than tendon in these slides, ligaments, muscle, adipose stuff. Often a bit ripped - I can imagine cutting these parts must be tricky given such varying toughness in tissues. Don't have access to good microtome nor expertise to operate one, but would be nice to see it done close up. And then see, what's done afterwards...
Indeed there is much else than tendon in these slides, ligaments, muscle, adipose stuff. Often a bit ripped - I can imagine cutting these parts must be tricky given such varying toughness in tissues. Don't have access to good microtome nor expertise to operate one, but would be nice to see it done close up. And then see, what's done afterwards...
Re: Collagen
Thanks Chris R.
Cheers, Karl
Cheers, Karl
Re: Collagen
Really excellent photos, ModelZ. Not sure why you have such spectacular colours, but this might be of interest: http://lists.utsouthwestern.edu/mailman ... 68827.html . It is about Picro Sirius Red, well known to change the birefringence of collagen to which it bonds. Don't know how this fits in with a similarity to H&E, but PSR can be used with (at least) Alcian Blue, Haemalum, and Tartrazine. Also your muscle fibres are birefringent, and I haven't seen that before, at least to the extent you have here, but the document mentioned above touches upon the possible effects of fixation on birefringence so maybe that's an issue. Collodion embedding makes everything birefringent, but your background appears isotropic (?), and I see no reason why it should enhance the birefringence of whatever is embedded. Anyway, what a nice post.
Re: Collagen
Interesting reading & excellent info, thanks john w!
Yes, indeed uniform background in my sample. Its a shame I can't anymore get info on its making. Just comparing the color in BF to e.g. www.hindawi.com/journals/scientifica/2015/802980 pics makes me think it likely isn't pure Picro Sirius Red. Much more magenta shade. However your link notes that there is some color variability in PSR, too, procedure dependent. Or combination with other dyes as you suggest - my H&E was just a layman¨s guess.
PSR is all new to me. Seems to be related to Congo Red but outcomewise a lot depends on the details of the treatment. Each histowizard has his/her own reciepe...
Learning some of this biobirefringence stuff is fascinating. Opens up your mind thinking beyond usual lattice structures. Why birefringence becomes almost inevitable when Nature starts making complex molecules by stacking up from a finite set of building blocks. Never knew that other triple helices (apart from collagen) include versions of DNA & RNA, too.
Yes, indeed uniform background in my sample. Its a shame I can't anymore get info on its making. Just comparing the color in BF to e.g. www.hindawi.com/journals/scientifica/2015/802980 pics makes me think it likely isn't pure Picro Sirius Red. Much more magenta shade. However your link notes that there is some color variability in PSR, too, procedure dependent. Or combination with other dyes as you suggest - my H&E was just a layman¨s guess.
PSR is all new to me. Seems to be related to Congo Red but outcomewise a lot depends on the details of the treatment. Each histowizard has his/her own reciepe...
Learning some of this biobirefringence stuff is fascinating. Opens up your mind thinking beyond usual lattice structures. Why birefringence becomes almost inevitable when Nature starts making complex molecules by stacking up from a finite set of building blocks. Never knew that other triple helices (apart from collagen) include versions of DNA & RNA, too.
Re: Collagen
I see Picrosirius Red was already mentioned. Tartrazine yellow is a common counterstain used for Gram stains which polarizes beautifully in shades of green and yellow. Very easy procedure, just stain for a few seconds and rinse in water. I believe you may lose the stain in alcohol, typically a universal solvent is used to bring it to xylene in the lab, but you could just air dry before clearing instead.
You should also try polarizing some cortical bone if its available to you, gives an incredible view of the concentric rings of separate osteons, especially when stained with Tartrazine.
You should also try polarizing some cortical bone if its available to you, gives an incredible view of the concentric rings of separate osteons, especially when stained with Tartrazine.