Quantification of fluorescent molecules in heterogeneous media by use of the fluorescence decay amplitude analysis

In heterogeneous media, including biological objects, fluorescent molecules of one kind often exist as a mixture of species with different fluorescence parameters. Fractional concentrations of these species can be measured by analyzing their fluorescence decay amplitudes. The amplitudes are linear functions of concentrations of actually fluorescent molecules, i.e., molecules whose fluorescence decay can be measured. Other (quenched) molecules do not influence these amplitudes. The other parameter that has to be measured to calculate these concentrations is the radiative rate constant. The parameter can be excluded by comparison of decay amplitudes of the sample studied and a standard. The comparison should be made taking into account the dependence of the radiation rates on emision wavelength. The method has been tested in experiments with the fluorescent probe 3-methoxybenzanthrone (MBA) bound with phosphatidylcholine bilayer membranes. The probe has a complex fluorescence decay in these membranes. The decay can be described as two exponentials, with decay times of 2 and 12 ns and a blue-shifted fluorescence spectrum of the short-life component as compared with long-life one. The shift was used to correct calculated radiative rate values. After this, about 100% of the MBA molecules were found to be fluorescent in these membranes. Thus, this approach can be used to measure absolute concentrations of subpopulations of fluorescent molecules in heterogeneous biological objects.