Determination of the encapsulation efficiency of individual vesicles using single-vesicle photolysis and confocal single-molecule detection

This paper describes a new method to measure the encapsulation efficiency of individual lipid vesicles. Single vesicles were first optically trapped (with a CW Nd:YAG laser at 1064 nm) and then photolyzed with a single 3-ns UV laser pulse (from a N-2 laser at 337 nm) to release the molecules encapsulated within the vesicle; confocal detection with single-molecule sensitivity (laser excitation at 488 nm from a CW Ar+ laser) was used to measure the number of released molecules. By placing the confocal probe volume a few micrometers from the vesicle and by monitoring the transit times and the number of released molecules that crossed this probe volume, we could calculate the total number of molecules released from the vesicle using a three-dimensional diffusion equation. Unlike traditional definitions of encapsulation efficiencies based on bulk assays, because we can measure the contents of and release from individual vesicles, we can define the encapsulation efficiency by dividing the concentration of molecules encapsulated in each vesicle over the original concentration of the molecules present in the loading solution. We characterized the encapsulation efficiency of carboxyfluorescein for vesicles prepared by rotary evaporation and found oligolamellar vesicles have an encapsulation efficiency of 36.3 +/- 18.9%, while multilamellar vesicles have an encapsulation efficiency of 17.5 +/- 8.9%.