We describe in detail a simple technique to construct the size distribution of liposome formulations from single-object fluorescence measurements. Liposomes that are fluorescently labeled in their membrane are first immobilized on a surface at dilute densities and then imaged individually using epi-fluorescence microscopy. The integrated intensities of several thousand single liposomes are collected and evaluated within minutes by automated image processing, using the user-friendly freeware ImageJ. The mean intensity of the liposome population is then calculated and scaled in units of length (nm) by relating the intensity data to the mean diameter obtained from a reference measurement with dynamic light scattering. We explain the process of constructing the size distributions in a step-by-step manner, starting with the preparation of liposomes through the final acquisition of size histograms. Detailed advice is given concerning critical parameters of image acquisition and processing. Size histograms constructed from single-particle measurements provide detailed information on complex distributions that may be easily averaged out in ensemble measurements (e.g., light scattering). In addition, the technique allows accurate measurements of polydisperse samples (e.g., nonextruded liposome preparations).
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