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 fluo rescence in situ hybridization signals. However, for a fraction of the cost, conventional noncooled, video-rated CCD cameras can also be app lied for most applications in the field of fluorescence in situ hybrid ization, provided that they allow for longer integration times. This p aper 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 measur ements 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 co nditions good linearity is maintained. The camera was found suitable f or the detection of in situ hybridization spots in interphase nuclei u sing centromere-specific probes. The fast readout rate of the camera o ffers interesting facilities for quickly locating objects with relativ ely strong fluorescence, such as counterstained metaphases. The less i ntense probe signals may then be analyzed at higher magnification. (C) 1994 Wiley-Liss, Inc.