Applicability of a noncooled video-rated CCD camera for detection of fluorescence in situ hybridization signals.

Cooled CCD cameras provide good sensitivity and linearity with a high dynamic range and are therefore well suited for quantification of fluorescence in situ hybridization signals. However, for a fraction of the cost, conventional noncooled, video-rated CCD cameras can also be applied for most applications in the field of fluorescence in situ hybridization, provided that they allow for longer integration times. This paper describes the use of the Sony camera, model XC-77RR-CE, for this purpose. Tests were carried out to compare the dark current, linearity, and signal-to-noise ratio of this camera with a Photometrics cooled CCD camera model KAF 1400, and the suitability for quantitative measurements was investigated on a model system of fluorescent beads. It is shown that if the dark current of the video-rated camera is internally corrected, integration times of up to 30 s can be used; under such conditions good linearity is maintained. The camera was found suitable for the detection of in situ hybridization spots in interphase nuclei using centromere-specific probes. The fast readout rate of the camera offers interesting facilities for quickly locating objects with relatively strong fluorescence, such as counterstained metaphases. The less intense probe signals may then be analyzed at higher magnification.