Digital image color compensation with unequal integration periods

Abstract

In the preparation of fluorescence in situ hybridization (FISH) specimens, when multiple DNA probes, each tagged with a different fluorophore, are used in combination, several different cellular structures can be visualized simultaneously. When such specimens are digitized, the different cellular components appear in separate color channels of the multi-channel digital image. Spectral overlap among the emission spectra of the fluorophores, the sensitivity spectra of the image sensor and the passbands of the optical filter set, however, spread the image of each fluorophore across the color channels. The result can be poor isolation of each fluorophore to a separate color channel. Each probe image is thus contaminated by 'crosstalk' from the other channels, to a greater or lesser degree. In an earlier paper we presented a processing method that effectively removes the spreading of fluorophore brightness among the color channels, thereby isolating each fluorophol e to a single (monochrome) image. That technique assumes the black level is zero, and the integration time is the same for each channel. This paper extends the technique to account for non-zero black level and for unequal integration periods for the different fluorophores.