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Automated 3D Gene Position Analysis Using a Customized Imaris Plugin: XTFISHInsideNucleus

  • Mariamawit S. Ashenafi
  • Célia BarouxEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1675)

Abstract

Fluorescence in situ hybridization (FISH) is commonly used to visualize chromosomal regions or genomic loci within the nucleus, and can largely contribute to unraveling the link between structure and function in the nucleus. Three-dimensional (3D) analyses are required to best capture the nucleus’ organizing principles, but the experimental setup and computational analyses are far from trivial. Here, we present a robust workflow for 3D FISH against repeats and single copy loci in embedded intact nuclei from Arabidopsis leaves. We then describe in detail the image acquisition, subsequent image deconvolution before 3D image processing, and the image reconstruction. We developed an automated batch image processing pipeline using a customized, open source plugin implemented in the Imaris environment.

Key words

Fluorescence in situ hybridization 3D FISH Confocal imaging Deconvolution Automated image processing 3D gene position Imaris 

Notes

Acknowledgments

The authors thank Dr. Jana Döhner (Microscopy Imaging Facility, University of Zürich) for technical assistance with image acquisition and deconvolution; Dr. Peter Majer, Sacha Guyer, (Bitplane CH) for advice on the customized plugin; Drs. Wenjing She, Kostas Kritsas (University of Zürich), Imen Mestiri, and Fredy Barneche (IBENS Paris) for sharing experimental protocols as described here. This work was supported by a SystemsX iPhD grant (2014/235), the University of Zürich and the Baugarten Stiftung.

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Copyright information

© Springer Science+Business Media LLC 2018

Authors and Affiliations

  1. 1.Department of Plant and Microbial Biology, Zürich-Basel Plant Science CenterUniversity of ZürichZürichSwitzerland

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