Quantitative fluorescence in confocal microscopy

SUMMARY The relationship between integrated fluorescence intensity and integrated absorbance was measured in Feulgen‐stained pigeon erythrocyte nuclei hydrolysed for different periods of time and stained at different dye concentrations. In conventional as well as confocal quantitative fluorescence microscopy the relationship between the integrated fluorescence intensity and the integrated absorbance shows a maximum. This is due to inner filtering and re‐absorption of the excitation light and emission light respectively. In conventional quantitative fluorescence microscopy the relationship is influenced by the numerical aperture of the objective lens. Under confocal observation, as measured with the BIO‐RAD MRC‐500 Confocal Imaging System, no influence of the numerical aperture of the objective lens on the relationship between the integrated fluorescence intensity and the integrated absorbance could be observed.