imaging zebrafish

[megha kumar]

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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

[Mark Cannell-2]

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What objective lens are you using? What is the refractive index of the mountant? If there is a mismatch the signal can drop quite quickly due to spherical aberration. Is the embryo mounted close to the coverslip?

Cheers

On 11/11/2014, at 7:58 am, megha kumar <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> 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

Mark  B. Cannell Ph.D. FRSNZ
Professor of Cardiac Cell Biology
School of Physiology &  Pharmacology
Medical Sciences Building
University of Bristol
Bristol
BS8 1TD UK

[hidden email]

[Ekaterina PAPUSHEVA-2]

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



-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of megha kumar
Sent: Tuesday, November 11, 2014 8:59 AM
To: [hidden email]
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

[Martin Wessendorf-2]

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Dear Dr. Kumar--

What wavelength fluorophore are you using?  I would expect that you'd
have best results with far-red--there'd be less scattering.

Good luck!

Martin Wessendorf


On 11/11/2014 1:58 AM, megha kumar wrote:
> 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?
>

--
Martin Wessendorf, Ph.D.                   office: [phone]
Assoc Prof, Dept Neuroscience                 lab: [phone]
University of Minnesota             Preferred FAX: [phone]
6-145 Jackson Hall, 321 Church St. SE    Dept Fax: [phone]
Minneapolis, MN  55455                    e-mail: [hidden email]

[weber]

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You can use a water lens with a long distance but we will have to use a clamp to hold water.
The collars are available from Zeiss. I have already made such an arrangement with a 20 X
water immersion 2mm WD. It's better to use an inverted microscope to do that. You can add
drugg and nutriments during your acquisition.