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Utricularia - inside the bladder of doom
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Pau
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Joined: 20 Jan 2010
Posts: 4807
Location: Valencia, Spain

PostPosted: Mon Dec 01, 2014 9:38 am    Post subject: Reply with quote

Absolutely outstanding!!! Shocked
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hkv



Joined: 02 Jan 2013
Posts: 443
Location: Sweden

PostPosted: Mon Dec 01, 2014 12:48 pm    Post subject: Reply with quote

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?
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blepharopsis



Joined: 28 Nov 2014
Posts: 143
Location: Virginia, USA

PostPosted: Mon Dec 01, 2014 1:06 pm    Post subject: Reply with quote

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 Smile
Check out those links to learn more about it: http://www.andor.com/learning-academy/light-sheet-microscopy-the-development-of-the-nervous-system
http://www.digital-embryo.org/
Cheers!
Igor
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vasselle



Joined: 05 Jan 2014
Posts: 1497
Location: France

PostPosted: Mon Dec 01, 2014 1:49 pm    Post subject: Reply with quote

Hello.
Just fantastic.
Your images are breathtaking.
Thank you for sharing.
Sincerely seb ..
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discomorphella



Joined: 01 Oct 2006
Posts: 605
Location: NW USA

PostPosted: Mon Dec 01, 2014 1:53 pm    Post subject: Reply with quote

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
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blepharopsis



Joined: 28 Nov 2014
Posts: 143
Location: Virginia, USA

PostPosted: Mon Dec 01, 2014 2:21 pm    Post subject: Reply with quote

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).
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discomorphella



Joined: 01 Oct 2006
Posts: 605
Location: NW USA

PostPosted: Mon Dec 01, 2014 2:34 pm    Post subject: Reply with quote

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
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pwnell



Joined: 18 Dec 2009
Posts: 2000
Location: Tsawwassen, Canada

PostPosted: Mon Dec 01, 2014 3:32 pm    Post subject: Reply with quote

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 Wink
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discomorphella



Joined: 01 Oct 2006
Posts: 605
Location: NW USA

PostPosted: Mon Dec 01, 2014 3:48 pm    Post subject: Reply with quote

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
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blepharopsis



Joined: 28 Nov 2014
Posts: 143
Location: Virginia, USA

PostPosted: Mon Dec 01, 2014 4:08 pm    Post subject: Reply with quote

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 Smile
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