Determination of the refractive index of vesicles using nanoparticle tracking analysis
||E. van der Pol|
||Scientic and Standardization Committee meeting of the International Society on Thrombosis and Haemostasis, Amsterdam, The Netherlands|
||June 29, 2013|
||Amsterdam 2013 Refractive index of vesicles.pdf (504 kB)|
Background: Microparticles and exosomes are often studied by optical detection methods based on light scattering, such as flow cytometry. Light scattering is a complex process involving the size, shape, and refractive index of these vesicles. Consequently, the refractive index affects the detection limit of optical techniques and it is essential to relate the detected light scattering signal to the diameter of vesicles. The refractive index may also provide a new label-free parameter. However, knowledge on the refractive index of vesicles is limited, since no methods are available to measure this physical property.
Methods: We have applied nanoparticle tracking analysis (NS500, Nanosight Ltd) to detect light scattering from single beads of known size and refractive index. We analytically described the relation between the diameter, refractive index, and light scattering of beads by Mie theory to determine the refractive index of vesicles from cell-free urine and platelet poor plasma.
Results: We obtained a median refractive index of urine vesicles of 1.36, with 95% of the vesicles having a refractive index between 1.35 and 1.45. The refractive index distribution of plasma vesicles was much broader and had a median refractive index of 1.49.
Conclusions: Nanoparticle tracking analysis can be used to assess the refractive index of vesicles. Urine vesicles had a different median refractive index (n=1.36) than plasma vesicles (n=1.49). We hypothesize that the relatively high refractive index of plasma vesicles is due to the presence of lipoproteins. These results imply that currently used calibration standards are insufficient and new reference materials have to be developed to calibrate optical instruments for vesicle detection.