首页 | 本学科首页   官方微博 | 高级检索  
   检索      


Quantification of fluorescence in situ hybridization signals by image cytometry.
Authors:P M Nederlof  S van der Flier  N P Verwoerd  J Vrolijk  A K Raap  H J Tanke
Institution:Sylvius Laboratory, Department of Cytochemistry and Cytometry, University of Leiden, The Netherlands.
Abstract:In this study we aimed at the development of a cytometric system for quantification of specific DNA sequences using fluorescence in situ hybridization (ISH) and digital imaging microscopy. The cytochemical and cytometric aspects of a quantitative ISH procedure were investigated, using human peripheral blood lymphocyte interphase nuclei and probes detecting high copy number target sequences as a model system. These chromosome-specific probes were labeled with biotin, digoxigenin, or fluorescein. The instrumentation requirements are evaluated. Quantification of the fluorescence ISH signals was performed using an epi-fluorescence microscope with a multi-wavelength illuminator, equipped with a cooled charge couple device (CCD) camera. The performance of the system was evaluated using fluorescing beads and a homogeneously fluorescing specimen. Specific image analysis programs were developed for the automated segmentation and analysis of the images provided by ISH. Non-uniform background fluorescence of the nuclei introduces problems in the image analysis segmentation procedures. Different procedures were tested. Up to 95% of the hybridization signals could be correctly segmented using digital filtering techniques (min-max filter) to estimate local background intensities. The choice of the objective lens used for the collection of images was found to be extremely important. High magnification objectives with high numerical aperture, which are frequently used for visualization of fluorescence, are not optimal, since they do not have a sufficient depth of field. The system described was used for quantification of ISH signals and allowed accurate measurement of fluorescence spot intensities, as well as of fluorescence ratios obtained with double-labeled probes.
Keywords:
设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号