Fluorescent dot counting in interphase cell nuclei

Fluorescence in situ hybridization allows the enumeration of chromosomal abnormalities in interphase cell nuclei. This process is called dot counting. To estimate the distribution of chromosomes per cell, a large number of cells have to be analysed, particularly when the frequency of aberrant cells is low. Automation of dot counting is desirable because manual counting is tedious, fatiguing, and time consuming. We have developed a completely automated fluorescence microscope system that counts fluorescent hybridization dots for one probe in interphase cell nuclei. This system works with two fluorescent dyes—one for the DNA hybridization dots and one for the cell nucleus. A fully automated scanning procedure has been used for the image acquisition. After an image is acquired it has to be analysed in order to find the nuclei and to detect the dots. This article focuses upon the dot detection procedure. Three different algorithms are presented. The problems of 'overlapping' dots and split dots are discussed. The automated dot counter has been tested on a number of normal specimens where DAPI was used for the nucleus counter stain and a centromeric probe was used to mark the chromosome 12. The slides contained lymphocytes from cultured blood. The performance of the different algorithms has been evaluated and compared with manually obtained results. The automated counting results approximate the results of manual counting.

[1]  Keinosuke Fukunaga,et al.  Introduction to Statistical Pattern Recognition , 1972 .

[2]  G. Zack,et al.  Automatic measurement of sister chromatid exchange frequency. , 1977, The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society.

[3]  T. W. Ridler,et al.  Picture thresholding using an iterative selection method. , 1978 .

[4]  F. Meyer.  Iterative image transformations for an automatic screening of cervical smears. , 1979, The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society.

[5]  Xinhua Zhuang,et al.  Image Analysis Using Mathematical Morphology , 1987, IEEE Transactions on Pattern Analysis and Machine Intelligence.

[6]  Ian T. Young,et al.  Acuity: Image Analysis for the Personal Computer , 1988 .

[7]  R. Aikens,et al.  Chapter 16 Solid-State Imagers for Microscopy , 1988 .

[8]  I T Young,et al.  Sampling density and quantitative microscopy. , 1988, Analytical and quantitative cytology and histology.

[9]  R. Aikens,et al.  Solid-state imagers for microscopy. , 1989, Methods in cell biology.

[10]  D. Pinkel,et al.  Aneuploidy detection by analysis of interphase nuclei using fluorescence in situ hybridization with chromosome-specific probes. , 1989, Progress in clinical and biological research.

[11]  H. Tanke,et al.  Detection of chromosome aberrations in interphase tumor nuclei by nonradioactive in situ hybridization. , 1989, Cancer genetics and cytogenetics.

[12]  F. Ramaekers,et al.  Numerical chromosome 1, 7, 9, and 11 aberrations in bladder cancer detected by in situ hybridization. , 1991, Cancer research.

[13]  P. Nederlof,et al.  Quantification of inter- and intra-nuclear variation of fluorescence in situ hybridization signals. , 1992, Cytometry.

[14]  L. J. van Vliet,et al.  Grey-Scale Measurements in Multi-Dimensional Digitized Images , 1993 .

[15]  A. Raap,et al.  Statistical methods in interphase cytogenetics: an experimental approach. , 1993, Cytometry.

[16]  Ian T. Young,et al.  Methods for CCD camera characterization , 1994, Electronic Imaging.

[17]  H. Netten,et al.  Autofocusing in microscopy based on the OTF and sampling , 1994 .

[18]  Lucas J. van Vliet,et al.  A fast scanner for fluorescence microscopy using a 2-D CCD and time delayed integration , 1994 .

[19]  A D Carothers,et al.  Counting, measuring, and mapping in FISH-labelled cells: sample size considerations and implications for automation. , 1994, Cytometry.

[20]  A fast scanner for fluorescence microscopy using a 2‐D CCD and time delayed integration. Bioimaging 2:184–192 1994 , 1995 .

[21]  K. Castleman,et al.  Dot count proportion estimation in FISH specimens , 1995 .

[22]  Roland Eils,et al.  Three‐dimensional distribution of centromeric or paracentromeric heterochromatin of chromosomes 1, 7, 15 and 17 in human lymphocyte nuclei studied with light microscopic axial tomography , 1995 .