EFFECT OF CHROMATIC ERRORS IN MICROSCOPY ON THE VISUALIZATION OF MULTICOLOR FLUORESCENCE IN-SITU HYBRIDIZATION

The relevant microscopical conditions for the optimal visualization of ratio-color FISH stained cells were investigated. Special attention w as given to the influence of the type of illumination, (semi)-critical vs. Kohler type illumination, in combination with the use of multi-ba nd excitation and emission fitters, on the registration of the colors of ratio labelled probes. Due to chromatic errors, many collecting len ses were found to cause a wavelength dependent excitation pattern with critical illumination. This resulted in a change of the observed colo r of microscopic objects when stained with a mixture of two dyes and e xcited with a dual band pass filter. A quantitative study of this effe ct for semi-critical illumination of FISH ratio-labelled chromosomes r evealed a difference of 20% between highest and lowest ratio values de pending on the position of the object in the microscopic held vs. only 2.5% for Kohler type of illumination, The impact of these errors on t he identification of ratio-labelled probes and on the sensitivity of c omparative genomic hybridization (CGH) to detect gene amplifications o r losses is discussed. Standard preparations consisting of solutions o f defined mixtures of fluorescent dyes or objects stained with defined ratios of fluorophores, are proposed to correct for the errors observ ed. (C) 1996 Wiley-Liss, Inc.