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The relevant microscopical conditions for the optimal visualization of ratio-color FISH stained cells were investigated. Special attention was given to the influence of the type of illumination, (semi)-critical vs. Kohler type illumination, in combination with the use of multi-band excitation and emission filters, on the registration of the colors of ratio labelled probes. Due to chromatic errors, many collecting lenses were found to cause a wavelength dependent excitation pattern with critical illumination. This resulted in a change of the observed color of microscopic objects when stained with a mixture of two dyes and excited with a dual band pass filter. A quantitative study of this effect for semi-critical illumination of FISH ratio-labelled chromosomes revealed a difference of 20% between highest and lowest ratio values depending on the position of the object in the microscopic field vs. only 2.5% for Kohler type of illumination. The impact of these errors on the identification of ratio-labelled probes and on the sensitivity of comparative genomic hybridization (CGH) to detect gene amplifications or losses is discussed. Standard preparations consisting of solutions of defined mixtures of fluorescent dyes or objects stained with defined ratios of fluorophores, are proposed to correct for the errors observed. © 1996 Wiley-Liss, Inc.

The utility of p-phenylenediamine, 1,4-di-azobicyclo-(2.2.2.)-octane, and the commercial products Citifluor, Slowfade, and Vectashield, antifading agents frequently used as mounting media for fluorescence in situ hybridization, was investigated. Fading curves for bound fluorochromes were recorded with digital microscopy, and relative fluorescence intensities of fluorochromes in solution were measured with an aperture defined measurement system. The three commonly used fluorochromes, fluorescein, tetramethyl rhodamine, and coumarin, were studied. Vectashield offered the best antifading properties for all three fluorochromes, although their relative fluorescence intensity was slightly less in Vectashield in comparison with other antifading agents. In Vectashield, fluorescein, tetramethyl rhodamine, and coumarin showed half-life times of 96, 330, and 106 s, respectively, whereas in 90% glycerol in PBS (pH 8.5), these half-life time values were 9, 7, and 25 s, respectively. Vectashield is particularly recommended as a mounting medium for quantitative digital imaging microscopy and for multicolor applications, where it is easy to have errors due to differences in fading rates of the fluorochromes. © 1995 Wiley-Liss, Inc.