Abstract
Telomere length and dynamics are central to understanding cell aging, genomic instability and cancer. Currently, there are limited guidelines for analyzing telomeric features in 3D using different cellular models. Image processing for telomere analysis is of increasing interest in many fields, however a lack of standardization can make comparisons and reproducibility an issue. Here we provide a user's guide for quantitative immunofluorescence microscopy of telomeres in interphase cells that covers image acquisition, processing and analysis. Strategies for determining telomere size and number are identified using normal human diploid Hs68 fibroblasts. We demonstrate how to accurately determine telomere number, length, volume, and degree of clustering using quantitative immunofluorescence. Using this workflow, we make the unexpected observation that hTERT-immortalized Hs68 cells with longer telomeres have fewer resolvable telomeres in interphase. Rigorous quantification indicates that this is due to telomeric clustering, leading to systematic underestimation of telomere number and overestimation of telomere size.
Highlights
Telomere length and dynamics are central to understanding cell aging, genomic instability and cancer
The probe generates a fluorescence signal that is proportional in intensity to the length of the telomere and can be used to estimate the relative lengths within the same cell. quantitativefluorescence in situ hybridization (qFISH) is often used to examine telomeres in metaphase spreads, which allows for the staining of individual chromosomes and their identification if they are labelled with chromosome-specific probes
Wide-field microscopy, when combined with current powerful digital deconvolution methods, yields optical sections comparable to the optical sectioning inherent in confocal techniques. Another advantage is that the camera technologies, such as scientific complementary metal–oxide–semiconductor cameras, allow researchers to acquire larger fieldsof-view, reducing experimental time at the wide-field microscope[29]
Summary
Telomere length and dynamics are central to understanding cell aging, genomic instability and cancer. It is thought that when telomere sequences shorten to a critical length, a DNA damage response is triggered which leads to activation of ATM4, p535 and downstream molecules such as p21 to block further cell replication. This results in a permanent cell cycle arrest called replicative senescence. One standard method to measure the telomere length of individual chromosomes is quantitativefluorescence in situ hybridization (qFISH)[18] In this procedure, a peptide nucleic acid (PNA) probe conjugated to a fluorophore is used to label telomeric DNA. Detailed observations of telomere intensities using this technique revealed that the telomeres of subsets of chromosomes can be quite short in some strains of normal cells and that telomeres begin to fuse upon depletion of members of the shelterin complex
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