Ethidium bromide fluorescence photo-enhancement in glycerol

Frog erythrocyte nuclei were stained with ethidium bromide at concentrations of 10(-6) M to 10(-4) M and mounted in phosphate buffer or in glycerol. Ethidium fluorescence was excited with different irradiation powers at 514 nm from an argon laser, and the variation of fluorescence was measured with a microfluorimeter over the first twenty seconds of illumination. The following differences between glycerol and buffer mounting were observed: 1) fading was more delayed in glycerol at all dye concentrations and light powers; 2) fluorescence yield was significantly lower in glycerol compared to buffer at high dye concentration; 3) whereas from the start of illumination fluorescence exponentially declined in buffer, there was an initial fluorescence photo-enhancement in glycerol followed by exponential fading. Both retardation of fading and fluorescence photo-enhancement were more prominent with increasing dye concentration. It is suggested that the light induced fluorescence increase was caused by concentration dequenching and was observed in glycerol, not in buffer solution, through one or both of the following glycerol effects: 1) aggravation of self-quenching of the secondary type of binding of ethidium; 2) delayed fading. The concomitant attenuation of the inner filter effect through the photo-destruction of the non-fluorescent ethidium dimer, which absorbs within the excitation spectrum of the ethidium monomer, could also contribute to an apparent amplification of the fluorescence build-up.