In in multi-color fluorescence, endogenous fluorophores have been considered more of a nuisance than a signal. Many of them co-exist (e.g., mitochondrial NADH and flavins) or, as ceroids and lipofuscins, have intrinsically broad fluorescence excitation and emission spectra. Thus, the presence of autofluorescence, along with cross-excitation and fluorescence bleed-through of one color channel into the neighboring one, bring up the question to which extent different color channels contain truly independent fluorophore information.Here, we use a rigorously defined spectral separability index that combines the absorption and emission characteristics of fluorophore j with the spectral properties of the light source, excitation and emission filter(s), dichroic mirror and detector into one figure of merit that quantifies the amount of cross-talk in both excitation (i) and emission (k) channels. We used Xijk to detect in mouse cortical astrocytes two exogenous fluorophores (EGFP and Texas Red) in front of a multi-component autofluorescent background comprising at least three different components. We believe that Xijk offers a valuable tool to experimenters and reviewers for choosing suitable recording conditions and for evaluating and comparing co-localization, FRET and photoswitching data across set-ups and publications.View Large Image | View Hi-Res Image | Download PowerPoint Slide