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Measurement of refractive index by nanoparticle tracking analysis reveals heterogeneity in extracellular vesicles

Gardiner, C; Shaw, M; Hole, P; Smith, J; Tannetta, D; Redman, CW; Sargent, IL; (2014) Measurement of refractive index by nanoparticle tracking analysis reveals heterogeneity in extracellular vesicles. Journal of Extracellular Vesicles , 3 (1) , Article 25361. 10.3402/jev.v3.25361. Green open access

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Abstract

INTRODUCTION: Optical techniques are routinely used to size and count extracellular vesicles (EV). For comparison of data from different methods and laboratories, suitable calibrators are essential. A suitable calibrator must have a refractive index (RI) as close to that of EV as possible but the RI of EV is currently unknown. To measure EV, RI requires accurate knowledge of size and light scattering. These are difficult to measure as most EVs cannot be resolved by light microscopy and their diameter is smaller than the wavelength of visible light. However, nanoparticle tracking analysis (NTA) provides both size and relative light scattering intensity (rLSI) values. We therefore sought to determine whether it was possible to use NTA to measure the RI of individual EVs. METHODS: NTA was used to measure the rLSI and size of polystyrene and silica microspheres of known size and RI (1.470 and 1.633, respectively) and of EV isolated from a wide range of cells. We developed software, based on Mie scattering code, to calculate particle RI from the rLSI data. This modelled theoretical scattering intensities for polystyrene and silica microspheres of known size (100 and 200 nm) and RI. The model was verified using data from the polystyrene and silica microspheres. Size and rLSI data for each vesicle were processed by the software to generate RI values. RESULTS: The following modal RI measurements were obtained: fresh urinary EV 1.374, lyophilised urinary EV 1.367, neuroblastoma EV 1.393, blood EV 1.398, EV from activated platelets 1.390, small placental EV 1.364–1.375 and 1.398–1.414 for large placental EV (>200 nm). Large placental EV had a significantly higher RI than small placental EV (p<0.0001). The spread of RI values was narrower for small EV than for the more heterogeneous large EV. DISCUSSION: Using NTA and Mie scattering theory, we have demonstrated that it is possible to estimate the RI of sub-micron EV using NTA data. EV typically had a modal RI of 1.37–1.39, whereas values of >1.40 were observed for some large (>200 nm) microvesicles. CONCLUSION: This method for measuring EV RI will be useful for developing appropriate calibrators for EV measurement.

Type: Article
Title: Measurement of refractive index by nanoparticle tracking analysis reveals heterogeneity in extracellular vesicles
Open access status: An open access version is available from UCL Discovery
DOI: 10.3402/jev.v3.25361
Publisher version: http://dx.doi.org/10.3402/jev.v3.25361
Language: English
Additional information: Journal of Extracellular Vesicles 2014. # 2014 Chris Gardiner et al. This is an Open Access article distributed under the terms of the Creative Commons Attribution-Noncommercial 3.0 Unported License (http://creativecommons.org/licenses/by-nc/3.0/), permitting all non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Keywords: Extracellular vesicles; refractive index; nanoparticle tracking analysis; light scattering; standardisation
UCL classification: UCL
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Medical Sciences
URI: https://discovery.ucl.ac.uk/id/eprint/1468498
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