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Telomere length measurements using fluorescence microscopy

University of British Columbia

We describe a system to estimate the lengths of telomere repeat DNA sequences in individual chromosomes of cells. Conventional systems based on gel electrophoresis of digested DNA an only determine the telomere length distribution of a population of cells but cannot provide estimation of telomere lengths of individual chromosomes using a limited number of cells. Our method is based on analyzing microscopy images of metaphase chromosomes prepared using fluorescence in-situ hybridization technology. In order to obtain reliable and reproducible images for measurements, we have developed and optimized an image acquisition system specifically for this purpose using commercially available components. In this study, two types of images are used. The first image highlights the chromosome regions. The second highlights only the telomere regions and consists of a set of multi-focus plane images. We first perform segmentation on the acquired multi-focus plane images in order to determine the region that each telomere occupies. For each telomere region determined, the total integrated fluorescence intensity (IFI) is obtained. This calculated IFI value is normalized for spatial unevenness in illumination in the field of view as well as the day to day variation in the illumination intensity. The acquired chromosome image is then segmented using novel and simple algorithms developed for such purpose. The location of the segmented telomere and chromosome boundaries are then overlaid onto the chromosome image. Different colour boundaries are used to highlight and identify different telomeres within a chromosome as well as the number of telomeres detected within each chromosome. The resulting image and the calculated telomere IFI values for each chromosome are then presented on the computer screen for user verification and editing as required. The algorithms described i n this thesis are presently being used on a daily basis to collect data on telomeres and to study the role telomeres play in the biology of cells. Our method of analysis has made it possible to generate reliable estimates of the length of telomeres in individual chromosome arms using a limited number of cells. In addition, our automation of the analysis has significantly reduced the user interaction time for editing and verification of the results. To date, at least two different biological studies of telomeres have been completed on the system we developed.

Thesis/Dissertation

application/pdf

2009-06-02

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http://hdl.handle.net/2429/8569

Electrical and Computer Engineering

University of British Columbia

UBCV

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