Characterizing the three-dimensional organization of telomeres

Abstract

Background: Quantitative analysis can be used in combination with fluorescence microscopy. Although the human eye is able to obtain good qualitative results, when analyzing the spatial organization of telomeres in interphase nuclei, there is a need for quantitative results based on image analysis. Methods: We developed a tool for analyzing three-dimensional images of telomeres stained by fluorescence in situ hybridization in interphase nuclei with DNA counterstained with 4′,6-diamidino-2-phenylindole. After deconvolution of the image, we segmented individual telomeres. From the location of the telomeres we derived a distribution parameter ρT, which indicated whether the telomeres were in a disk (ρT ≫ 1) or not (ρT ≈ 1). We sorted mouse lymphocyte nuclei and measured ρT. We also performed a bromodeoxyuridine synchronous cell sorting experiment on live cells and measured ρT at several instances. Results: Measuring ρT for nuclei in G0/G1, S, and G2 produced 1.4 ± 0.1, 1.5 ± 0.2, and 14 ± 2, respectively, showing a significant difference between G2 and G0/G1 or S. For the bromodeoxyuridine synchronous cell sorting experiment, we found a cell cycle dependency of ρT and a correlation between ρT and an observer. Conclusions: In this study we present a quantitative method to characterize the organization of telomeres using three-dimensional imaging, image processing, and image analysis. © 2005 International Society for Analytical Cytology.