Refractive index of extracellular vesicles by nanoparticle tracking analysis
||E. van der Pol, F.A.W. Coumans, A.N. Böing, A. Sturk, T.G. van Leeuwen, R. Nieuwland|
||International Society for Extracellular Vesicles 2014, Rotterdam, The Netherlands|
||April 30, 2014|
||Rotterdam 2014 Refractive index NTA.pdf (873 kB)
Introduction: Vesicles are often studied by methods that detect light scattering, such as flow cytometry. The amount of light scattered by a vesicle depends on its size and refractive index. Consequently, knowledge of the vesicle refractive index is required to determine the size distribution of vesicles from the flow cytometry scatter signal. The refractive index may also become a new label-free parameter, for example to distinguish vesicles from protein aggregates. At present, however, no method is available to determine the refractive index of single vesicles.
Methods: We have measured the diameter and light scattering of vesicles and beads by tracking their Brownian motion with nanoparticle tracking analysis (NTA; NS500, Nanosight Ltd). We analytically described the relation between the diameter, refractive index, and light scattering of beads using Mie theory to determine the refractive index of vesicles from cell-free human urine. Urine was used because it contains a high concentration of vesicles with low contamination from lipoproteins and protein aggregates compared to blood plasma.
Results: The median refractive index of urine vesicles was 1.36 with 90% of the vesicles between 1.35 and 1.41.
Conclusion: The refractive index of single vesicles in suspension can be determined by NTA. Urine vesicles have a median refractive index of 1.36. Taking into account that vesicles have a phospholipid membrane with a refractive index of 1.48, our findings indicate that the lumen of most urine vesicles have a refractive index of 1.34, which is equal to water. Since the cytosol typically has a refractive index of 1.38±0.02 due to the abundant presence of proteins, our findings suggest a relatively low protein concentration in most urine vesicles.
Funding: This work was funded by the European Metrology Research Programme (EMRP) under the joint research project HLT02 (Metves). The EMRP is jointly funded by the EMRP participating countries within EURAMET and the European Union.