Bornfleth H, Aldinger K, Hausmann M, Jauch A, Cremer C
Institute of Applied Physics, University of Heidelberg, Germany.
Cytometry. 1996 May 1;24(1):1-13. doi: 10.1002/(SICI)1097-0320(19960501)24:1<1::AID-CYTO1>3.0.CO;2-O.
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.
在比较基因组杂交（CGH）中，对中期染色体上荧光强度比率进行定量测量是检测测试DNA中拷贝数变化的基本方法。显微镜图像通常由高分辨率、高灵敏度的黑白（B&W）CCD相机采集。这需要随后使用适当的激发和发射滤光片组合记录不同的彩色图像。我们描述了一种使用单芯片真彩色CCD相机Kappa CF 15 MC和Omega三带通滤光片同时记录三种染料德克萨斯红、异硫氰酸荧光素（FITC）和4',6-二脒基-2-苯基吲哚（DAPI）的替代方法。对该系统的成像特性进行了详细检查。评估了相机在红（R）、绿（G）和蓝（B）三个颜色平面中的响应，并计算了图像校正的校准因子。给出了误差估计。以商用软件包Optimas作为图像记录的基础，为80486个人计算机开发了一个用于CGH实验图像分析的完整计算机程序。给出了证实该系统适用于CGH比率成像的实例。将结果与使用由黑白CCD相机（Photometrics）和基于TCL软件包并在Macintosh Quadra 950上运行的软件程序组成的既定装置从相同显微镜载玻片获得的其他结果进行了比较。发现使用两种不同系统获得的结果非常吻合。
