Rj. Florijn et al., EFFECT OF CHROMATIC ERRORS IN MICROSCOPY ON THE VISUALIZATION OF MULTICOLOR FLUORESCENCE IN-SITU HYBRIDIZATION, Cytometry, 23(1), 1996, pp. 8-14
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.