Comparative genomic hybridization imaging by the one-chip true-color CCD camera kappa CF 15 MC

In comparative genomic hybridization (CGH), quantitative measurement of fluorescence intensity ratios on metaphase chromosomes is the basic method for detecting copy number changes in the test DNA. The microscope images are usually acquired by high-resolution, highly sensitive, black and white (B&W) CCD cameras. This requires subsequent recording of the different color images using appropriate filter combinations for excitation and emission. We describe an alternative approach using the one-chip true-color CCD camera Kappa CF 15 MC and an Omega triple-bandpass filter for simultaneous registration of the three dyes Texas red, FTTC, and DAPI. A detailed examination of the imaging properties of the system was performed. The camera response in the three color planes R, G, and B was evaluated, and calibration factors for image correction were calculated. An error estimate is given. A complete computer program for the image analysis of CGH experiments has been developed for an 80486 PC, using the commercially available software package Optimas as the basis for image recording. Examples that confirm the suitability of the system for ratio imaging in CGH are presented. The results were compared with others obtained from the same microscope slides using an established setup consisting of a B&W CCD camera (Photometrics) and a software program based on the TCL software package and run on a Macintosh Quadra 950. The results obtained using the two different systems were found to correspond closely.