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In this paper we present methods for characterizing CCD cameras. Interesting properties are linearity of photometric response, signal-to-noise ratio (SNR), sensitivity, dark current and spatial frequency response (SFR). The techniques to characterize CCD cameras are carefully designed to assist one in selecting a camera to solve a certain problem. The methods described were applied to a variety of cameras: an Astromed TE3/A with P86000 chip, a Photometrics CC200 series with Thompson chip TH7882, a Photometrics CC200 series with Kodak chip KAF1400, a Xillix' Micro Imager 1400 with Kodak chip KAF1400, an HCS MXR CCD with a Philips chip and a Sony XC-77RRCE.

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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.

In situ hybridization techniques allow the enumeration of chromosomal abnormalities and form a great potential for many clinical applications. Although the use of fluorescent labels is preferable regarding sensitivity and colormultiplicity, chromogenic labels can provide an excellent alternative in relatively simple situations, e.g., where it is sufficient to use a centromere specific probe to detect abnormalities of one specific chromosome. When the frequency of chromosomal aberrations is low, several hundreds or even thousands of cells have to be evaluated to achieve sufficient statistical confidence. Since manual counting is tedious, fatiguing, and time consuming, automation can assist to process the slides more efficiently. Therefore, a system has been developed for automated spot counting using brightfield microscopy. This paper addresses both the hardware system aspects and the software image analysis algorithms for nuclei and spot detection. As a result of the automated slide analysis the system provides the frequency spot distribution of the selected cells. The automatic classification can, however, be overruled by human interaction, since each individual cell is stored in a gallery and can be relocated for visual inspection. With this system a thousand cells can be automatically analyzed in approximately 10 min, while an extra 5–10 min is necessary for visual evaluation. The performance of the system was analyzed using a model system for trisomy consisting of a mixture of male and female lymphocytes hybridized with probes for chromosomes 7 and Y. The sensitivity for trisomy detection in the seeding experiment was such that a frequency of 3% trisomic cells could be picked up automatically as being abnormal according to the multiple proportion test, while trisomy as low as 1.5% could be detected after interaction. © 1996 Wiley‐Liss, Inc.