Characterizing vesicle leakage by fluorescence lifetime measurements

The membrane leakage assay based on vesicles loaded with self-quenching dyes has been widely used for quantifying the activity of antibiotic peptides and other compounds that induce membrane pores or leaks. Here we show that this assay can be substantially improved if it is based on time-resolved decay curves rather than steady-state intensities only. A bi-or triexponential fit of calcein fluorescence decays allows for a parallel quantification of the free and (one or two) entrapped dye fractions and their effective local concentrations (governing the lifetime). The advantages of this technique are that it (i) allows the distinguishing of all-or-none from graded leakage for each sample, (ii) reveals the heterogeneity of graded leakage, (iii) truly quantifies the released dye rather than providing an empiric dequenching value, and (iv) is independent of many errors that may affect the intensity. We introduce the method by studying graded leakage caused by the detergent C12EO8.