Automated Analysis of Telomere Intensity from Quantitative Fluorescence In-Situ Hybridization using CellProfiler

Abstract

Telomeres are noncoding DNA sequences that protect chromosome ends from degradation. Quantitative fluorescence in situ hybridization (Q-FISH) is the favored technique for measuring telomere length. It can be applied to individual chromosomes in metaphase cells. Much of the currently available software to analyze fluorescence signal intensity as a proxy for telomere length is only commercially available. This study sought to develop a pipeline for the automated analysis of telomere fluorescence intensities in metaphase Q-FISH using modules from the open-source, freely available CellProfiler software. Intensities telomere and centromere were stained with fluorophore-conjugated peptide nucleic acid probes then evaluated using our pipeline. Telomere and centromere intensities were measured in a total of 30 images, giving an average relative telomere intensity of 22.17 %. Overall, our study showed that the pipeline can automatically identify and measure fluorescence signals from telomeres and centromeres in approximately 36 sec/image. However, the pipeline requires high-quality metaphase chromosome images with minimal touching and overlapping of chromosomes. Nevertheless, CellProfiler provides a free and flexible platform for the automated analysis of Q-FISH images that can be easily optimized and shared between researchers