Posted by
Ekaterina PAPUSHEVA-2 on
URL: http://confocal-microscopy-list.275.s1.nabble.com/imaging-zebrafish-tp7582876p7582877.html
Hi Megha,
There can be three things:
- tubulin is accumulated on the surface (improbable at 512 cells stage)
-your staining does not go into deep layers (you can check this by sectioning stained embryo)
- you use a lens that has strong spherical aberration with this sample (like, oil immersion lens with an agarose mounted sample). Do you have an access to an upright confocal? Try dipping lens then, otherwise try water immersion lens to match refraction indices of the sample and the immersion medium.
Best,
Katja
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From: Confocal Microscopy List [mailto:
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Sent: Tuesday, November 11, 2014 8:59 AM
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Subject: imaging zebrafish
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Hello everyone!
I am imaging the 256 and 512 cell stage of zebrafish using confocal (Leica TCS SP5 II). The embryos are stained with alpha and gamma tubulin antibody.
I take z stacks and reconstruct the entire embryo using a 3D software. I am unable to visualize the deeper cells below although I catch the first top most layer pretty well, which gives me a hollow half dome shape in 3D. Any ideas why I dont see the deeper cells at 512 cell stage? Is this a microscope issue or penetration is not deep enough or something else?
--
Megha Kumar, Ph.D.
Young Investigator
Regional Center for Biotechnology
180 Udyog Vihar phase I
Gurgaon 122016
India
ph: 8826422770