Comparing microrheological methods for measuring lipid membrane viscosity

The fluidity of lipid membranes governs the motions of bound proteins and macromolecules. Despite this, measurements of the viscosity of a lipid bilayer remain challenging to perform and interpret. Two different microrheological methods for measuring bilayer viscosity have been developed in recent years, one involving tracking phase-separated domains in giant unilamellar vesicles, and the other involving tracking elliptical lipid-anchored tracer particles. The latter approach has so far been applied only to pore-spanning black lipid membranes. The membrane viscosity values obtained by these two methods differ by an order of magnitude, however, and it is unclear whether this indicates inaccuracy of one or both techniques, or whether it is due to the difference in membrane systems examined. To resolve this discrepancy, we applied both methods simultaneously to the same lipid vesicles, featuring both phase separated domains and bound elliptical beads. We show that when applied to identical systems these methods are in agreement. The elliptical tracer method is generally applicable to vesicles of arbitrary composition, and we use it to quantify the viscosity of bilayers composed of phosphatidylcholine lipids of different chain lengths.