Utricularia - inside the bladder of doom

Images made through a microscope. All subject types.

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Pau
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Post by Pau »

Absolutely outstanding!!! :shock:
Pau

hkv
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Post by hkv »

Amazing!

Can you explain how you manage to rotate the specimen under the scope? You said you put the specimen between two cover glasses like a sandwhich, but how do you then manage the rotation, or do you extract the 3d data from the confocal data set without rotation?

blepharopsis
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Post by blepharopsis »

hkv wrote:Amazing!

Can you explain how you manage to rotate the specimen under the scope? You said you put the specimen between two cover glasses like a sandwhich, but how do you then manage the rotation, or do you extract the 3d data from the confocal data set without rotation?
Yes, the 3D information is in the confocal stack; open source freeware, like ImageJ or Icy, comes with plugins that can reconstruct the 3D object from a set of optical slices. Because of the point-spread function characteristics the resolution in the Z-dimension is much worse then in the XY plane (by a factor of 2 for the best high-aperture objectives) and so the image of a point light source is effectively a rotational ellipsoid - you can see how the image degrades when rotated ~ 90 degrees. We actually do have technology here at Janelia Research Campus that allows for rotation of the specimen embedded in a cylinder of agarose extruded from a tip of capillary; it uses two sources of light in the form of a sheet (hence the name, light sheet microscopy). This way several problems, including lack of isomorphic resolution in 3 dimensions, can be avoided. The technique gives wonderful data such as 3D movies of developing fly, mouse, fish or crustacean embryos. I don't have an access to it tho :)
Check out those links to learn more about it: http://www.andor.com/learning-academy/l ... ous-system
http://www.digital-embryo.org/
Cheers!
Igor

vasselle
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Post by vasselle »

Hello.
Just fantastic.
Your images are breathtaking.
Thank you for sharing.
Sincerely seb ..
Microscope Leitz Laborlux K
Boitier EOS 1200d

discomorphella
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Post by discomorphella »

Brilliant sectioning and imaging. I am assuming the agar immobilized the desmids inside the bladder? You mounted it in Tris-HCl buffer or PBS?

David

blepharopsis
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Post by blepharopsis »

discomorphella wrote:Brilliant sectioning and imaging. I am assuming the agar immobilized the desmids inside the bladder? You mounted it in Tris-HCl buffer or PBS?

David
Thanks, David! Yes, I decided to use agarose on the second approach - the fast and dirty method of simply cutting the traps with a razor yielded damaged specimens and the spillage of desmids (I wasn't familiar with those algae at the time, imagine my surprise when I "discovered" them inside bladderwort! One thing led to another and I ended up imaging lots of desmids). I remember using very diluted PBS with a dash of baking soda (slightly alkaline conditions increase fluorescence of Calcofluor).

discomorphella
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Post by discomorphella »

Sounds good. Must have been really cool to find the desmids inside...
Although my confocal at work is too subscribed to run my pond life through it (and its for semiconductor research, so it has metallurgical objectives on it so no coverglass) I can try agar embedding on my home setup, using regular agarose embedding and then double embedding the agar block into Steadman's wax and sectioning it like a regular ester wax block on my ancient microtome. The Calcofluar and AO wiill stain dewaxed sections too.
Thanks again for posting, this is great to see.

David

pwnell
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Post by pwnell »

Yep these images are fantastic. I just love the resolution you get with confocal. And naturally your technique - I tried to cut open a bladderwort once and it looked kind of pathetic ;)

discomorphella
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Post by discomorphella »

Still curious about one aspect of this. Doesn't Calcofluor White MR2 stain agarose polysaccharides too? Did you melt the agarose out of the sections or I am recalling something incorrectly? I would have thought the agarose would have contributed a lot of background. If it doesn't I am in luck for using Calcofluor for fungi.
Thanks, again spectacular, just spectacular shots.

David

blepharopsis
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Post by blepharopsis »

discomorphella wrote:Still curious about one aspect of this. Doesn't Calcofluor White MR2 stain agarose polysaccharides too? Did you melt the agarose out of the sections or I am recalling something incorrectly? I would have thought the agarose would have contributed a lot of background. If it doesn't I am in luck for using Calcofluor for fungi.
Thanks, again spectacular, just spectacular shots.

David
Nope, never observed background fluorescence from agarose when using Calcofluor - it is eluted very fast, few washes are usually needed :)

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