Size and refractive index determination of extracellular vesicles smaller than 70 nm in a nano-fluidic optical fiber

Oral presentation
E. van der Pol, F.A. Coumans, S. Weidlich, Y. Lahini, M.A. Schmidt, T.G. van Leeuwen, A. Sturk, R. Nieuwland, and S. Faez
International Society for Extracellular Vesicles 2016, Rotterdam, The Netherlands
May 6, 2016


Introduction: State-of-the-art detection methods show that typically ~70% of the extracellular vesicles (EV) have a diameter <70 nm. Thus far, EV <70 nm have only been studied in frozen state or with methods requiring EV to be adhered to a surface. Thus, the majority of EV have never been studied in their physiological environment. Here we present a novel label-free optical method to track single EV <70 nm in suspension. We use the method to determine the diameter and refractive index (RI) of EV.

Methods: EV from human cell-free urine (n=5) were contained within a single-mode light-guiding silica fiber with a 450 nm or 660 nm nano-fluidic channel. Laser light (660 nm wavelength) was coupled to the fiber, resulting in a confined optical mode in the nano-fluidic channel, which continuously illuminated the freely diffusing EV inside the channel. Elastic light scattering from the EV, in the direction orthogonal to the fiber axis, was collected using a microscope objective (NA=0.95) and imaged with a home-built microscope. Light scattering was calibrated with 35 nm and 50 nm polystyrene beads. To derive the diameter and RI of EV, we described the measured thermal diffusion by the Stokes-Einstein equation and light scattering by the Rayleigh approximation.

Results: Using a nano-fluidic optical fiber, we could track single EV as small as 50 nm for >60 seconds by elastic light scattering. Furthermore, by combining data on thermal diffusion and light scattering, we confirmed that most EV have an RI <1.4.

Summary/conclusion: For the first time, we have tracked single EV <70 nm freely diffusing in suspension to determine their diameter and RI. Knowledge on the diameter and RI of EV is essential to data interpretation and standardization. Potential applications of the nano-fluidic optical fiber are dimensional characterization, zeta potential analyses and particle-particle interaction analyses of the smallest EV present.