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

Mariamawit S Ashenafi1, Célia Baroux2

  • 1Department of Plant and Microbial Biology, Zürich-Basel Plant Science Center, University of Zürich, Zürich, Switzerland.

Methods in Molecular Biology (Clifton, N.J.)
|October 21, 2017
PubMed
Summary

We present a robust workflow for 3D fluorescence in situ hybridization (FISH) in Arabidopsis nuclei. This method enables detailed visualization of genomic structures, aiding in understanding nuclear organization and function.

Keywords:
3D FISH3D gene positionAutomated image processingConfocal imagingDeconvolutionFluorescence in situ hybridizationImaris

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Area of Science:

  • Plant biology
  • Genomics
  • Cell biology

Background:

  • Fluorescence in situ hybridization (FISH) is crucial for visualizing nuclear genomic structures.
  • Understanding nuclear organization requires advanced 3D analysis, which presents experimental and computational challenges.

Purpose of the Study:

  • To develop and present a robust workflow for 3D FISH in intact Arabidopsis leaf nuclei.
  • To detail the process from image acquisition to 3D reconstruction for genomic loci analysis.

Main Methods:

  • Established a 3D FISH protocol for repeats and single-copy loci in embedded Arabidopsis nuclei.
  • Detailed image acquisition, deconvolution, and 3D image processing steps.
  • Developed an automated batch image processing pipeline using a customized open-source Imaris plugin.

Main Results:

  • Successfully applied 3D FISH to visualize chromosomal regions and genomic loci within intact nuclei.
  • The workflow allows for detailed 3D analysis of nuclear organization.
  • An automated pipeline streamlines the complex image processing steps.

Conclusions:

  • The presented workflow provides a reliable method for 3D FISH in Arabidopsis.
  • This approach facilitates the study of the relationship between nuclear structure and function.
  • Automated image processing enhances the efficiency and reproducibility of 3D genomic analyses.