COMPARATIVE GENOMIC HYBRIDIZATION IMAGING BY THE ONE-CHIP TRUE-COLOR CCD CAMERA KAPPA-CF-15-MC

In comparative genomic hybridization (CGH), quantitative measurement o f fluorescence intensity ratios on metaphase chromosomes is the basic method for detecting copy number changes in the test DNA. The microsco pe images are usually acquired by high-resolution, highly sensitive, b lack and white (B&W) CCD cameras, This requires subsequent recording o f the different color images using appropriate filter combinations for excitation and emission, We describe an alternative approach using th e one-chip true-color CCD camera Kappa CF 15 MC and an Omega triple-ba ndpass filter for simultaneous registration of the three dyes Texas re d, FITC, and DAPI, A detailed examination of the imaging properties of the system was performed, The camera response in the three color plan es R, G, and B was evaluated, and calibration factors for image correc tion were calculated, An error estimate is given. A complete computer program for the image analysis of CGH experiments has been developed f or an 80486 PC, using the commercially available software package Opti mas as the basis for image recording, Examples that confirm the suitab ility of the system for ratio imaging in CGH are presented, The result s were compared with others obtained from the same microscope slides u sing 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 s ystems were found to correspond closely. (C) 1996 Wiley-Liss, Inc.