Effect of chromatic errors in microscopy on the visualization of multi-color fluorescence in situ hybridization

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. Köhler 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 Köhler 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.