Title: Fluorescent Dot Counting in Interphase Cell Nuclei
H. Netten, L.J. van Vliet, H.
Vrolijk, W.C.R. Sloos, H.J. Tanke, and Ian T. Young
in: BioImaging, vol. 4, no. 2, 1996, 93-106.
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 analyzed, 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 analyzed in order to find the nuclei and to detect
the dots. This manuscript focuses upon the dot detection procedure. Three
different algorithms are presented. The problem 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.
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