Optical characterization and selective addressing of the resonant modes of a micropillar cavity with a white light beam

We have performed white-light reflectivity measurements on GaAs/AlAs micropillar cavities with diameters ranging from 1 {\mu}m up to 20 {\mu}m. We are able to resolve the spatial field distribution of each cavity mode in real space by scanning a small-sized beam across the top facet of each micropillar. We spectrally resolve distinct transverse optical cavity modes in reflectivity. Using this procedure we can selectively address a single mode in the multimode micropillar cavity. Calculations for the coupling efficiency of a small-diameter beam to each mode are in very good agreement with our reflectivity measurements.Comment: 7 pages, 8 figure

Innovation in detection of microparticles and exosomes

Cell-derived or extracellular vesicles, including microparticles and exosomes, are abundantly present in body fluids such as blood. Although such vesicles have gained strong clinical and scientific interest, their detection is difficult because many vesicles are extremely small with a diameter of less than 100 nm, and, moreover, these vesicles have a low refractive index and are heterogeneous in both size and composition. In this review, we focus on the relatively high throughput detection of vesicles in suspension by flow cytometry, resistive pulse sensing, and nanoparticle tracking analysis, and we will discuss their applicability and limitations. Finally, we discuss four methods that are not commercially available: Raman microspectroscopy, micro nuclear magnetic resonance, small-angle X-ray scattering (SAXS), and anomalous SAXS. These methods are currently being explored to study vesicles and are likely to offer novel information for future developments

Size and fluorescence calibrated imaging flow cytometry:From arbitrary to standard units

Imaging flow cytometry (IFCM) is a technique that can detect, size, and phenotype extracellular vesicles (EVs) at high throughput (thousands/minute) in complex biofluids without prior EV isolation. However, the generated signals are expressed in arbitrary units, which hinders data interpretation and comparison of measurement results between instruments and institutes. While fluorescence calibration can be readily achieved, calibration of side scatter (SSC) signals presents an ongoing challenge for IFCM. Here, we present an approach to relate the SSC signals to particle size for IFCM, and perform a comparability study between three different IFCMs using a plasma EV test sample (PEVTES). SSC signals for different sizes of polystyrene (PS) and hollow organosilica beads (HOBs) were acquired with a 405 nm 120 mW laser without a notch filter before detection. Mie theory was applied to relate scatter signals to particle size. Fluorescence calibration was accomplished with 2 μm phycoerythrin (PE) and allophycocyanin (APC) MESF beads. Size and fluorescence calibration was performed for three IFCMs in two laboratories. CD235a-PE and CD61-APC stained PEVTES were used as EV-containing samples. EV concentrations were compared between instruments within a size range of 100–1000 nm and a fluorescence intensity range of 3–10,000 MESF. 81 nm PS beads could be readily discerned from background based on their SSC signals. Fitting of the obtained PS bead SSC signals with Mie theory resulted in a coefficient of determination &gt;0.99 between theory and data for all three IFCMs. 216 nm HOBs were detected with all instruments, and confirmed the sensitivity to detect EVs by SSC. The lower limit of detection regarding EV-size for this study was determined to be ~100 nm for all instruments. Size and fluorescence calibration of IFCM data increased cross-instrument data comparability with the coefficient of variation decreasing from 33% to 21%. Here we demonstrate – for the first time – scatter calibration of an IFCM using the 405 nm laser. The quality of the scatter-to-diameter relation and scatter sensitivity of the IFCMs are similar to the most sensitive commercially available flow cytometers. This development will support the reliability of EV research with IFCM by providing robust standardization and reproducibility, which are pre-requisites for understanding the biological significance of EVs.</p

Single-step isolation of extracellular vesicles by size-exclusion chromatography

Background: Isolation of extracellular vesicles from plasma is a challenge due to the presence of proteins and lipoproteins. Isolation of vesicles using differential centrifugation or density-gradient ultracentrifugation results in co-isolation of contaminants such as protein aggregates and incomplete separation of vesicles from lipoproteins, respectively. Aim: To develop a single-step protocol to isolate vesicles from human body fluids. Methods: Platelet-free supernatant, derived from platelet concentrates, was loaded on a sepharose CL-2B column to perform size-exclusion chromatography (SEC; n=3). Fractions were collected and analysed by nanoparticle tracking analysis, resistive pulse sensing, flow cytometry and transmission electron microscopy. The concentrations of high-density lipoprotein cholesterol (HDL) and protein were measured in each fraction. Results: Fractions 9–12 contained the highest concentrations of particles larger than 70 nm and platelet-derived vesicles (46%±6 and 61%±2 of totals present in all collected fractions, respectively), but less than 5% of HDL and less than 1% of protein (4.8%±1 and 0.65%±0.3, respectively). HDL was present mainly in fractions 18–20 (32%±2 of total), and protein in fractions 19–21 (36%±2 of total). Compared to the starting material, recovery of platelet-derived vesicles was 43%±23 in fractions 9–12, with an 8-fold and 70-fold enrichment compared to HDL and protein. Conclusions: SEC efficiently isolates extracellular vesicles with a diameter larger than 70 nm from platelet-free supernatant of platelet concentrates. Application SEC will improve studies on the dimensional, structural and functional properties of extracellular vesicles

GANGs: Generative Adversarial Network Games

Generative Adversarial Networks (GAN) have become one of the most successful frameworks for unsupervised generative modeling. As GANs are difficult to train much research has focused on this. However, very little of this research has directly exploited game-theoretic techniques. We introduce Generative Adversarial Network Games (GANGs), which explicitly model a finite zero-sum game between a generator ($G$) and classifier ($C$) that use mixed strategies. The size of these games precludes exact solution methods, therefore we define resource-bounded best responses (RBBRs), and a resource-bounded Nash Equilibrium (RB-NE) as a pair of mixed strategies such that neither $G$ or $C$ can find a better RBBR. The RB-NE solution concept is richer than the notion of `local Nash equilibria' in that it captures not only failures of escaping local optima of gradient descent, but applies to any approximate best response computations, including methods with random restarts. To validate our approach, we solve GANGs with the Parallel Nash Memory algorithm, which provably monotonically converges to an RB-NE. We compare our results to standard GAN setups, and demonstrate that our method deals well with typical GAN problems such as mode collapse, partial mode coverage and forgetting

P2Y12 antagonist ticagrelor inhibits the release of procoagulant extracellular vesicles from activated platelets

Background: Activated platelets release platelet extracellular vesicles (PEVs). Adenosine diphosphate(ADP) receptors P2Y1 and P2Y12 both play a role in platelet activation, The present hypothesis hereinis that the inhibition of these receptors may affect the release of PEVs.Methods: Platelet-rich plasma from 10 healthy subjects was incubated with saline, P2Y1 antagonistMRS2179 (100 μM), P2Y12 antagonist ticagrelor (1 μM), and a combination of both antagonists.Platelets were activated by ADP (10 μM) under stirring conditions at 37°C. Platelet reactivity wasassessed by impedance aggregometry. Concentrations of PEVs– (positive for CD61 but negative forP-selectin and phosphatidylserine) and PEVs+ (positive for all) were determined by a state-of-the-artflow cytometer. Procoagulant activity of PEVs was measured by a fibrin generation test.Results: ADP-induced aggregation (57 ± 13 area under curve {AUC] units) was inhibited 73%by the P2Y1 antagonist, 86% by the P2Y12 antagonist, and 95% when combined (p &lt; 0.001 for all).The release of PEVs– (2.9 E ± 0.8 × 108/mL) was inhibited 48% in the presence of both antagonists(p = 0.015), whereas antagonists alone were ineffective. The release of PEVs+ (2.4 ± 1.6 × 107/mL)was unaffected by the P2Y1 antagonist, but was 62% inhibited by the P2Y12 antagonist (p = 0.035),and 72% by both antagonists (p = 0.022). PEVs promoted coagulation in presence of tissue factor.Conclusions: Inhibition of P2Y1 and P2Y12 receptors reduces platelet aggregation and affects therelease of distinct subpopulations of PEVs. Ticagrelor decreases the release of procoagulant PEVs fromactivated platelets, which may contribute to the observed clinical benefits in patients treated with ticagrelor

Towards defining reference materials for extracellular vesicle size, concentration, refractive index and epitope abundance

Accurate characterization of extracellular vesicles (EVs) is critical to explore their diagnostic and therapeutic applications. As the EV research field has developed, so too have the techniques used to characterize them. The development of reference materials is required for the standardization of these techniques. This work, initiated from the ISEV 2017 Biomarker Workshop in Birmingham, UK, and with further discussion during the ISEV 2019 Standardization Workshop in Ghent, Belgium, sets out to elucidate which reference materials are required and which are currently available to standardize commonly used analysis platforms for characterizing EV size, concentration, refractive index, and epitope expression. Due to their predominant use, a particular focus is placed on the optical methods nanoparticle tracking analysis and flow cytometry.Comment: 30 pages, 6 figures, 2 table

Ticagrelor attenuates the increase of extracellular vesicle concentrations in plasma after acute myocardial infarction compared to clopidogrel

Background Platelet P2Y12 antagonist ticagrelor reduces mortality after acute myocardial infarction (AMI) compared to clopidogrel, but the underlying mechanism is unknown. Because activated platelets, leukocytes, and endothelial cells release proinflammatory and prothrombotic extracellular vesicles (EVs), we hypothesized that the release of EVs is more efficiently inhibited by ticagrelor compared to clopidogrel. Objectives We compared EV concentrations and EV procoagulant activity in plasma of patients after AMI treated with ticagrelor or clopidogrel. Methods After percutaneous coronary intervention, 60 patients with first AMI were randomized to ticagrelor or clopidogrel. Flow cytometry was used to determine concentrations of EVs from activated platelets (CD61(+), CD62p(+)), fibrinogen(+), phosphatidylserine (PS+), leukocytes (CD45(+)), endothelial cells (CD31(+), 146(+)), and erythrocytes (CD235a(+)) in plasma at randomization, after 72 hours and 6 months of treatment. A fibrin generation test was used to determine EV procoagulant activity. Results Concentrations of platelet, fibrinogen(+), PS+, leukocyte, and erythrocyte EVs increased 6 months after AMI compared to the acute phase of AMI (P = .17). Conclusions Ticagrelor attenuates the increase of EV concentrations in plasma after acute myocardial infarction compared to clopidogrel. The ongoing release of EVs despite antiplatelet therapy might explain recurrent thrombotic events after AMI and worse clinical outcomes on clopidogrel compared to ticagrelor.Peer reviewe

Hollow organosilica beads as reference particles for optical detection of extracellular vesicles

Background: The concentration of extracellular vesicles (EVs) in body fluids is a promising biomarker for disease, and flow cytometry remains the clinically most applicable method to identify the cellular origin of single EVs in suspension. To compare concentration measurements of EVs between flow cytometers, solid polystyrene reference beads and EVs were distributed in the first ISTH organized inter-laboratory comparison studies. The beads were used to set size gates based on light scatter, and the concentration of EVs was measured within the size gates. However, polystyrene beads lead to false size determination of EVs due to the mismatch in refractive index between beads and EVs. Moreover, polystyrene beads gate different EV sizes on different flow cytometers. Objective: To prepare, characterize and test hollow organosilica beads (HOBs) as reference beads to set EV size gates in flow cytometry investigations. Methods: HOBs were prepared by a hard template sol-gel method and extensively characterized for morphology, size and colloidal stability. The applicability of HOBs as reference particles was investigated by flow cytometry using HOBs and platelet-derived EVs. Results: HOBs proved monodisperse with homogeneous shell thickness. Two angle light scattering measurements by flow cytometry confirmed that HOBs have light scattering properties similar to platelet-derived EVs. Conclusions: Because HOBs resemble the structure and light scattering properties of EVs, HOBs with a given size will gate EVs of the same size. Therefore, HOBs are ideal reference beads to standardize optical measurements of the EV concentration within a predefined size range