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

Gardiner, Chris; Shaw, Michael; Hole, Patrick; Smith, J.; Tannetta, Dionne; Redman, Christopher W.G.; Sargent, Ian L.

doi:10.3402/jev.v3.25361

Cited by 148 publications

(160 citation statements)

References 18 publications

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“…RIs vary between types of particles based on their composition [8]. Calculations from NTA studies utilizing Mie theory reveal that smaller EVs have RIs in the range of 1.37–1.39, whereas larger EVs (>200 nm) were seen to have RIs >1.4 [6]. Beads typically have higher RIs (1.47–1.63) which more closely resemble lipoproteins, various types of viruses or protein aggregates than biological EVs [6,8].…”

Section: Discussionmentioning

confidence: 99%

“…Calculations from NTA studies utilizing Mie theory reveal that smaller EVs have RIs in the range of 1.37–1.39, whereas larger EVs (>200 nm) were seen to have RIs >1.4 [6]. Beads typically have higher RIs (1.47–1.63) which more closely resemble lipoproteins, various types of viruses or protein aggregates than biological EVs [6,8]. With this in mind, we assessed RIs ranging from 1.34 to 1.46 to encompass typical ranges for EVs and reference materials.…”

Section: Discussionmentioning

confidence: 99%

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Improved resolution in extracellular vesicle populations using 405 instead of 488 nm side scatter

McVey

Spring

Kuebler

2018

J of Extracellular Vesicle

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Improvements in identification and assessment of extracellular vesicles (EVs) have fuelled a recent surge in EV publications investigating their roles as biomarkers and mediators of disease. Meaningful scientific comparisons are, however, hampered by difficulties in accurate, reproducible enumeration and characterization of EVs in biological fluids. High-sensitivity flow cytometry (FCM) is presently the most commonly applied strategy to assess EVs, yet its utility is limited by variant ability to resolve smaller EVs. Here, we propose the use of 405 nm (violet) wavelength lasers in place of 488 nm (blue) for side scatter (SSC) detection to obtain greater resolution of EVs using high-sensitivity FCM. To test this hypothesis, we modelled EV resolution by violet versus blue SSC in silico and compared resolution of reference beads and biological EVs from plasma and bronchoalveolar lavage (BAL) fluid using either violet or blue wavelength SSC EV detection. Mie scatter modelling predicted that violet as compared to blue SSC increases resolution of small (100–500 nm) spherical particles with refractive indices (1.34–1.46) similar to EVs by approximately twofold in terms of light intensity and by nearly 20% in SSC signal quantum efficiency. Resolution of reference beads was improved by violet instead of blue SSC with two- and fivefold decreases in coefficients of variation for particles of 300–500 nm and 180–240 nm size, respectively. Resolution was similarly improved for detection of EVs from plasma or BAL fluid. Violet SSC detection for high-sensitivity FCM allows for significantly greater resolution of EVs in plasma and BAL compared to conventional blue SSC and particularly improves resolution of smaller EVs. Notably, the proposed strategy is readily implementable and inexpensive for machines already equipped with 405 nm SSC or the ability to accommodate 405/10 nm bandpass filters in their violet detector arrays.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Improved resolution in extracellular vesicle populations using 405 instead of 488 nm side scatter

McVey

Spring

Kuebler

2018

J of Extracellular Vesicle

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“…A 5 mm obscuration bar was placed in front of the FSC collection lens. FSC was measured with a collection angle of [15][16][17][18][19][20][21][22][23][24][25] (reduced wide-angle FSC; rw-FSC). We previously optimized the detection of FSC induced by nano-sized particles by using a larger (5 mm) obscuration bar, high numerical aperture and long working distance lens, and by reconstruction of the BD small particle detector (11).…”

Section: High-resolution Flow Cytometric Analysis Of Submicron-sized mentioning

confidence: 99%

Prerequisites for the analysis and sorting of extracellular vesicle subpopulations by high‐resolution flow cytometry

Kormelink

Arkesteijn

Nauwelaers³

et al. 2015

Cytometry Pt A

175

183

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Submicron-sized vesicles released by cells are increasingly recognized for their role in intercellular communication and as biomarkers of disease. Methods for highthroughput, multi-parameter analysis of such extracellular vesicles (EVs) are crucial to further investigate their diversity and function. We recently developed a highresolution flow cytometry-based method (using a modified BD Influx) for quantitative and qualitative analysis of EVs. The fact that the majority of EVs is <200 nm in size requires special attention with relation to specific conditions of the flow cytometer, as well as sample concentration and event rate. In this study, we investigated how (too) high particle concentrations affect high-resolution flow cytometry-based particle quantification and characterization. Increasing concentrations of submicron-sized particles (beads, liposomes, and EVs) were measured to identify coincidence and swarm effects, caused by the concurrent presence of multiple particles in the measuring spot. As a result, we demonstrate that analysis of highly concentrated samples resulted in an underestimation of the number of particles and an interdependent overestimation of light scattering and fluorescence signals. On the basis of this knowledge, and by varying nozzle size and sheath pressure, we developed a strategy for high-resolution flow cytometric sorting of submicron-sized particles. Using the adapted sort settings, subsets of EVs differentially labeled with two fluorescent antibodies could be sorted to high purity. Moreover, sufficient numbers of EVs could be sorted for subsequent analysis by western blotting. In conclusion, swarm effects that occur when measuring high particle concentrations severely hamper EV quantification and characterization. These effects can be easily overlooked without including proper controls (e.g., sample dilution series) or tools (e.g., oscilloscope). Providing that the event rate is well controlled, the sorting strategy we propose here indicates that high-resolution flow cytometric sorting of different EV subsets is feasible. V C 2015 International Society for Advancement of Cytometry Key terms extracellular vesicle; exosome; microvesicle; microparticle; high-resolution flow cytometry; characterization; sorting; coincidence; swarm; liposome EXTRACELLULAR vesicles (EVs) are small membrane-enclosed vesicles released by cells either by outward budding from the plasma membrane or by the fusion of multivesicular bodies with the plasma membrane resulting in the release of intracellular stored vesicles (1). The release of EVs and their content, i.e., proteins, lipids and RNAs, is tightly regulated and varies not only between different cell types but also depends on the physiological state of the producing cell (2-4). Consequently, EV release is very dynamic and the EV population is very heterogeneous. EVs can function in an autocrine or paracrine fashion, but can also enter the circulatory system and act at distant sites. Hence EVs are present in body fluids like blood, milk, urin...

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“…In addition to standard diameter and concentration measurements, NanoSight R scattering intensity data has been used to identify several extracellular vesicle subpopulations with different refractive index [47]. To the authors' knowledge, our method is the first to use exported position data from NanoSight R , which allowed investigation of different particle tracking analysis methods without constructing a custom video microscopy suite or developing particle tracking algorithms.…”

Section: Discussionmentioning

confidence: 99%

Decorrelation correction for nanoparticle tracking analysis of dilute polydisperse suspensions in bulk flow

Hartman

Kirby

2017

Phys. Rev. E

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Nanoparticle tracking analysis, a multi-probe single particle tracking technique, is a widely used method to quickly determine the concentration and size distribution of colloidal particle suspensions. Many popular tools remove non-Brownian components of particle motion by subtracting the ensemble-averaged displacement at each time step, which is termed 'de-drifting'. Though critical for accurate size measurements, de-drifting is shown here to introduce significant biasing error and can fundamentally limit the dynamic range of particle size that can be measured for dilute, heterogeneous suspensions, such as biological extracellular vesicles. We report a more accurate estimate of particle mean-squared displacement, which we call Decorrelation Analysis, that accounts for correlations between individual and ensemble particle motion, which are spuriously introduced by de-drifting. Particle tracking simulation and experimental results show that this approach more accurately determines particle diameters for low concentration, polydisperse suspensions when compared with standard de-drifting techniques.

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

Cited by 148 publications

References 18 publications

Improved resolution in extracellular vesicle populations using 405 instead of 488 nm side scatter

Improved resolution in extracellular vesicle populations using 405 instead of 488 nm side scatter

Prerequisites for the analysis and sorting of extracellular vesicle subpopulations by high‐resolution flow cytometry

Decorrelation correction for nanoparticle tracking analysis of dilute polydisperse suspensions in bulk flow

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