Flow cytometry as a novel tool to evaluate and separate vesicles using characteristic scatter signatures

Vesicles are usually characterized for their structure by microscopy or, less often, by the addition of fluorescent dyes in a flow cytometer. We present a new method of studying these structures and associated forms by forward and side scatter analysis on a flow cytometer which has the advantage of simultaneous handling of large population of vesicles to identify their shapes and lamellarities. The technique is suitable for several types of vesicular structures like Multivesicular vesicles (MVV), multilamellar and unilamellar vesicles. Characteristic signatures are given by tubular structures and fine features thereon allow detection of complex structures such as fused and ellipsoidal forms. Coexistence of tubular and spherical structures, such as those known to form when surfactants/salt solutions are diluted, can clearly be detected by the signature pattern, which separates into two distinctly identifiable populations. The population can be sorted or separated easily based on these signatures and such sorting has allowed us to confirm our findings by microscopic observations. This novel method can thus be used for concurrent observations of vesicle populations with dye or more advantageously without employing any fluorescent tag.