A parameter for the distribution of fluorophores in cells derived from measurements of inner filter effect and reabsorption phenomenon

AbstractThe linear relationship between the emitted fluorescence and the amount of fluorophore at relatively high local absorbance values is significantly disturbed by two major phenomena, inner filter effect and reabsorption of fluorescence emission light. For a theoretical model in which excitation and emission rays were considered parallel and the fluorophore distribution was assumed to be homogeneous, Rigler (Acta Physiol Scand (Suppl.) 267:1, 1966) showed that these errors become significant at local absorbance >0.1 and may become more important for heterogeneously stained cells measured with conical illumination.In this study we demonstrate theoretically and experimentally that the inner filter effect and reabsorption phenomenon depend not only on the average local absorption but also on the distribution of the fluorophore in the measuring field. The ratio of the fluorescence intensities in the longer and the shorter emission wavelength range is introduced as a new parameter called the “heterogeneity index.” This index is not affected by the total amount of fluorophore, but depends on the fluorophore distribution within the measuring field.The theory has been tested on Sephadex beads and glycoprotein films stained with periodic acid acriflavine‐SO2 or acrolein acriflavine‐SO2. The results of the measurements on the model systems were found to be qualitatively in agreement with the theoretical predictions.Heterogeneity index measurements on Feulgen acriflavine‐SO2 stained cells using both microscope fluorometry and flow cytometry showed significant differences for cell nuclei with the same total amount of fluorophore but with different fluorophore distributions. The avoidance of inner filter effect and reabsorption phenomenon or their use to study changes in DNA compactness such as occur during cell differentiation and degeneration are discussed.