CGH
Images were acquired through a Zeiss Axiophot fluorescence microscope using a Plan NEOFLUAR oil objective x63, N.A. 1.25 (Zeiss, Oberkochen, Germany) equipped with filter sets appropriate for DAPI (Zeiss filter set 02, excitation: G365, beamsplitter: FT 395, emission: LP 420), FITC (Zeiss filter set 10, excitation: BP 450-490, beamsplitter: FT 510, emission: BP 515-565) and TRITC (Chroma filter set HQ Cy3+excitation filter from Zeiss filter set 15, excitation: BP 546/12, beamsplitter: FT 565, emission: BP 570-650) with a cooled CCD camera (Photometrics, Tucson, Arizona, U.S.A.) connected to a Macintosh Quadra 950 (Apple, Cupertino, California, U.S.A.). The resolution of this apparatus configuration is 0.108 m/pixel. The maximum image size is 1320x1035x12 bit.
The 100 W mercury lamp and the diaphragms of the microscope were precisely adjusted to get a homogeneous illumination of the optical field.
For each metaphase spread three monochrome images were digitized, one image for each fluorochrome. An image size of 512x512 or 768x768 pixels was chosen, according to catch the whole metaphase with one image. The images were inverted in order to make it possible to use a standard segmentation process and then transferred as 8 bit TIFF-files to a server PC via a local area network.