AUTOFLUORESCENCE CORRECTION FOR FLUORESCENCE IN-SITU HYBRIDIZATION

Optimal sensitivity of fluorescence in situ hybridization (FISH) requi res bright signals and low background fluorescence. Use of locus-speci fic probes is especially dependent on high sensitivity. Some tissue pr eparations show high autofluorescence, masking small or dim signals. W e have developed a new method for subtracting autofluorescence from di gital images on a pixel-by-pixel basis. It is based on the observation that fluorescent labels for FISH have narrower excitation and emissio n spectra than the chemical components responsible for autofluorescenc e. Our new approach uses calculation of the ratio of autofluorescence between multiple color images for correction of autofluorescence in ea ch individual image. By subtracting autofluorescence components, we we re able to enhance centromeric signals and make previously indistingui shable cosmid signals clearly visible. This image-processing approach to autofluorescence correction may widen the applicability of gene-spe cific probes in FISH analysis of tumor material. (C) 1995 Wiley-Liss, Inc.