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Method Article
Wolfgang Driever
Driever Lab, University of Freiburg
Corresponding Author
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In this protocol we describe a method to produce multi-view confocal 3D datasets suitable to be processed by the Virtual Brain Explorer (ViBE-Z) software. The method is optimized for Zebrafish (Danio rerio) embryos and larvae from one to five days post fertilization, but may be used also for other small biological objects. Zebrafish larvae are stained using either fluorescent in situ hybridization or immunostaining. In addition, all samples are counterstained with a nuclear stain to generate information to be used for anatomical reference. Stained larval brains are imaged using standard laser scanning confocal microscopes. To properly represent regions of very high as well as very low signal intensity we generate image stacks at different laser intensities and merge them to high dynamic range datasets. Further, multiple views are recorded and merged into high resolution combined datasets. To reduce the loss of information by restricted optical depth as a result of absorption and light scattering occurring in thick samples, image stacks are recorded both from the dorsal and ventral side of larvae. Both dorsal and ventral recordings are fused using attenuation correction of the ViBE-Z software, leading to a data representation that significantly reduces absorption and diffraction artifacts typical for microscopy of tissues deep inside biological samples.
Keywords
confocal microscopy, zebrafish
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The authors declare no competing financial interests.
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Associated Publications
ViBE-Z: a framework for 3D virtual colocalization analysis in zebrafish larval brains
by Olaf Ronneberger, Kun Liu, Meta Rath, +10
Nature Methods (04 June, 2012)
An open repository of community-contributed protocols sponsored by Nature Research.
Learn more about Protocol Exchange
Method Article
Wolfgang Driever
Driever Lab, University of Freiburg
Corresponding Author
Version History
CURRENT STATUS: Posted
Version 1
Posted 22 Jun, 2012
No community comments so far
Metrics
Comments: 0
HTML Views: ...
License:
This work is licensed under a CC BY-NC 3.0 License. Read Full License
In this protocol we describe a method to produce multi-view confocal 3D datasets suitable to be processed by the Virtual Brain Explorer (ViBE-Z) software. The method is optimized for Zebrafish (Danio rerio) embryos and larvae from one to five days post fertilization, but may be used also for other small biological objects. Zebrafish larvae are stained using either fluorescent in situ hybridization or immunostaining. In addition, all samples are counterstained with a nuclear stain to generate information to be used for anatomical reference. Stained larval brains are imaged using standard laser scanning confocal microscopes. To properly represent regions of very high as well as very low signal intensity we generate image stacks at different laser intensities and merge them to high dynamic range datasets. Further, multiple views are recorded and merged into high resolution combined datasets. To reduce the loss of information by restricted optical depth as a result of absorption and light scattering occurring in thick samples, image stacks are recorded both from the dorsal and ventral side of larvae. Both dorsal and ventral recordings are fused using attenuation correction of the ViBE-Z software, leading to a data representation that significantly reduces absorption and diffraction artifacts typical for microscopy of tissues deep inside biological samples.
Figure 1
Figure 2
Figure 3
The authors declare no competing financial interests.
Loading...
Associated Publications
ViBE-Z: a framework for 3D virtual colocalization analysis in zebrafish larval brains
by Olaf Ronneberger, Kun Liu, Meta Rath, +10
Nature Methods (04 June, 2012)