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

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.

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