Telomere length measurements using digital fluorescence microscopy

Background: The ends of chromosomes (telomeres) are important to maintain c hromosome stability, and the loss of telomere repeat sequences has been imp licated in cellular senescence and genomic instability of canter cells. The traditional method for measuring the length of telomeres (Southern analysi s) requires a large number of cells (>10(5)) and does not provide informati on on the telomere length of individual chromosomes. Here, we describe a di gital image microscopy system for measurements of the fluorescence intensit y derived from telomere repeat sequences in metaphase cells following quant itative fluorescence in situ hybridization (Q-FISH). Methods: Samples are prepared for microscopy using Q-FISH with Cy3 labeled peptide nucleic acid probes specific for (T(2)AG(3))(n) sequences and the D NA dye DAPI. Separate images of Cy3 and DAPI fluorescence are acquired and processed with a dedicated computer program (TFL-TELO). With the program, t he integrated fluorescence intensity value for each telomere, which is prop ortional to the number of hybridized probes, is calculated and presented to the user. Results: Indirect tests of our method were performed using simulated as wel l as defined tests objects. The precision and consistency of human telomere length measurements was then analyzed in a number of experiments. It was f ound that by averaging the results of less than 30 cells, a good indication of the telomere length (SD of 10-15%) can be obtained. Conclusions: We demonstrate that accurate and repeatable fluorescence inten sity measurements can be made from Q-FISH images that provide information o n the length of telomere repeats at individual chromosomes from limited num ber of cells. (C) 1999 Wiley-Liss, Inc.