This special issue of Cytometry marks the transition of spectral flow cytometry from an emerging technology into a transformative force that will shape the fields of cytometry and single‐cell ...analysis for some time to come. Tracing its roots to the earliest years of flow cytometry, spectral flow cytometry has evolved from the domain of individual researchers pushing the limits of hardware, reagents, and software to the mainstream, where it is being harnessed and adapted to meet the analytical challenges presented by modern biomedical research. In particular, the current form of spectral flow technology has arisen to address the needs of multiparameter immunophenotyping of immune cells in basic and translational research, and much of the current instrumentation and software reflects the needs of those applications. Yet, the possibilities enabled by high‐resolution analysis of the spectral properties of optical absorbance, scatter, and emission have only begun to be exploited. In this brief review, the author highlights the origins and early milestones of single‐cell spectral analysis, assesses the current state of instrumentation and software, and speculates as to future directions of spectral flow cytometry technology and applications.
Extracellular vesicles (EVs) are released by cells and can be found in cell culture supernatants and biofluids. EVs carry proteins, nucleic acids, and other cellular components and can deliver these ...to nearby or distant cells, making EVs of interest as both disease biomarkers and therapeutic targets. EVs in biofluids are heterogeneous, coming from different cell types and from different sources with the cell, which limits the usefulness of bulk EV analysis methods that report the average features of all EVs present. Single-particle measurements such as flow cytometry would be preferred, but the small size and low abundance of surface antigens challenges conventional flow cytometry approaches, leading to the development of vesicle-specific assays and experimental design. Among the key issues that have emerged are: (a) judicious choice of detection (triggering) approach; (b) appropriate control experiments to confirm the vesicular nature of the detected events and the contribution of coincidence (aka swarm detection); and (c) the importance of fluorescence calibration to allow data to be compared over time and between laboratories. We illustrate these issues in the context of fluorescence-triggered Vesicle Flow Cytometry (VFC), a general approach to the quantitative measurement of EV number, size, and surface marker expression.
Although popular discussion of testosterone’s influence on males often centers on aggression and antisocial behavior, contemporary theorists have proposed that it instead enhances behaviors involved ...in obtaining and maintaining a high social status. Two central distinguishing but untested predictions of this theory are that testosterone selectively increases status-relevant aggressive behaviors, such as responses to provocation, but that it also promotes nonaggressive behaviors, such as generosity toward others, when they are appropriate for increasing status. Here, we tested these hypotheses in healthy young males by injecting testosterone enanthate or a placebo in a double-blind, between-subjects, randomized design (n = 40). Participants played a version of the Ultimatum Game that was modified so that, having accepted or rejected an offer from the proposer, participants then had the opportunity to punish or reward the proposer at a proportionate cost to themselves. We found that participants treated with testosterone were more likely to punish the proposer and that higher testosterone levels were specifically associated with increased punishment of proposers who made unfair offers, indicating that testosterone indeed potentiates aggressive responses to provocation. Furthermore, when participants administered testosterone received large offers, they were more likely to reward the proposer and also chose rewards of greater magnitude. This increased generosity in the absence of provocation indicates that testosterone can also cause prosocial behaviors that are appropriate for increasing status. These findings are inconsistent with a simple relationship between testosterone and aggression and provide causal evidence for a more complex role for testosterone in driving status-enhancing behaviors in males.
Stable distributions with elliptical contours are a class of distributions that are useful for modeling heavy tailed multivariate data. This paper describes the theory of such distributions, presents ...formulas for calculating their densities, and methods for fitting the data and assessing the fit. Efficient numerical routines are implemented and evaluated in simulations. Applications to data sets of a financial portfolio with 30 assets and to a bivariate radar clutter data set are presented.
Traditional compressive sensing (CS) primarily assumes light-tailed models for the underlying signal and/or noise statistics. Nevertheless, this assumption is not met in the case of highly impulsive ...environments, where non-Gaussian infinite-variance processes arise for the signal and/or noise components. This drives the traditional sparse reconstruction methods to failure, since they are incapable of suppressing the effects of heavy-tailed sampling noise. The family of symmetric alpha-stable (SαS) distributions, as a powerful tool for modeling heavy-tailed behaviors, is adopted in this paper to design a robust algorithm for sparse signal reconstruction from linear random measurements corrupted by infinite-variance additive noise. Specifically, a novel greedy reconstruction method is developed, which achieves increased robustness to impulsive sampling noise by solving a minimum dispersion (MD) optimization problem based on fractional lower-order moments. The MD criterion emerges naturally in the case of additive sampling noise modeled by SαS distributions, as an effective measure of the spread of reconstruction errors around zero, due to the lack of second-order moments. The experimental evaluation demonstrates the improved reconstruction performance of the proposed algorithm when compared against state-of-the-art CS techniques for a broad range of impulsive environments.
Extracellular vesicles (EVs) have emerged as a promising biomarker platform for glioblastoma patients. However, the optimal method for quantitative assessment of EVs in clinical bio-fluid remains a ...point of contention. Multiple high-resolution platforms for quantitative EV analysis have emerged, including methods grounded in diffraction measurement of Brownian motion (NTA), tunable resistive pulse sensing (TRPS), vesicle flow cytometry (VFC), and transmission electron microscopy (TEM). Here we compared quantitative EV assessment using cerebrospinal fluids derived from glioblastoma patients using these methods. For EVs <150 nm in diameter, NTA detected more EVs than TRPS in three of the four samples tested. VFC particle counts are consistently 2-3 fold lower than NTA and TRPS, suggesting contribution of protein aggregates or other non-lipid particles to particle count by these platforms. While TEM yield meaningful data in terms of the morphology, its particle count are consistently two orders of magnitude lower relative to counts generated by NTA and TRPS. For larger particles (>150 nm in diameter), NTA consistently detected lower number of EVs relative to TRPS. These results unveil the strength and pitfalls of each quantitative method alone for assessing EVs derived from clinical cerebrospinal fluids and suggest that thoughtful synthesis of multi-platform quantitation will be required to guide meaningful clinical investigations.
Extracellular vesicles (EVs) are small, heterogeneous and difficult to measure. Flow cytometry (FC) is a key technology for the measurement of individual particles, but its application to the ...analysis of EVs and other submicron particles has presented many challenges and has produced a number of controversial results, in part due to limitations of instrument detection, lack of robust methods and ambiguities in how data should be interpreted. These complications are exacerbated by the field's lack of a robust reporting framework, and many EV-FC manuscripts include incomplete descriptions of methods and results, contain artefacts stemming from an insufficient instrument sensitivity and inappropriate experimental design and lack appropriate calibration and standardization. To address these issues, a working group (WG) of EV-FC researchers from ISEV, ISAC and ISTH, worked together as an EV-FC WG and developed a consensus framework for the minimum information that should be provided regarding EV-FC. This framework incorporates the existing Minimum Information for Studies of EVs (MISEV) guidelines and Minimum Information about a FC experiment (MIFlowCyt) standard in an EV-FC-specific reporting framework (MIFlowCyt-EV) that supports reporting of critical information related to sample staining, EV detection and measurement and experimental design in manuscripts that report EV-FC data. MIFlowCyt-EV provides a structure for sharing EV-FC results, but it does not prescribe specific protocols, as there will continue to be rapid evolution of instruments and methods for the foreseeable future. MIFlowCyt-EV accommodates this evolution, while providing information needed to evaluate and compare different approaches. Because MIFlowCyt-EV will ensure consistency in the manner of reporting of EV-FC studies, over time we expect that adoption of MIFlowCyt-EV as a standard for reporting EV- FC studies will improve the ability to quantitatively compare results from different laboratories and to support the development of new instruments and assays for improved measurement of EVs.
The composition and function of platelet-derived extracellular vesicles (EVs) in health and in disease are a major topic of investigation in biomedical research. However, efforts to delineate ...specific molecular repertoires and roles for different types of EVs in the circulation are limited not only by the lack of flow cytometers capable of analyzing submicron- and nano-materials across the full size spectrum of plasma EVs, but also by the lack of standardized methods and reference materials that would permit inter-laboratory reproducibility for these analyses. In this review, we summarize the flow cytometry of EVs, with a focus on platelet vesicles in plasma. In addition to delineating the basic principles that govern what precautions must be considered when using flow cytometry for the analysis of platelet vesicles, we provide an overview for how to standardize, control, annotate, and report EV flow cytometry data reproducibly, while looking forward to a next generation of high sensitivity instruments for the analysis of EVs and other submicron biomaterials in the circulation.
Ultrasensitive detection and characterization of single nanoparticles (<100 nm) is important in nanotechnology and life sciences. Direct measurement of the elastically scattered light from individual ...nanoparticles represents the simplest and the most direct method for particle detection. However, the sixth-power dependence of scattering intensity on particle size renders very small particles indistinguishable from the background. Adopting strategies for single-molecule fluorescence detection in a sheathed flow, here we report the development of high sensitivity flow cytometry (HSFCM) that achieves real-time light-scattering detection of single silica and gold nanoparticles as small as 24 and 7 nm in diameter, respectively. This unprecedented sensitivity enables high-resolution sizing of single nanoparticles directly based on their scattered intensity. With a resolution comparable to that of TEM and the ease and speed of flow cytometric analysis, HSFCM is particularly suitable for nanoparticle size distribution analysis of polydisperse/heterogeneous/mixed samples. Through concurrent fluorescence detection, simultaneous insights into the size and payload variations of engineered nanoparticles are demonstrated with two forms of clinical nanomedicine. By offering quantitative multiparameter analysis of single nanoparticles in liquid suspensions at a throughput of up to 10 000 particles per minute, HSFCM represents a major advance both in light-scattering detection technology and in nanoparticle characterization.
Optimal sampling period selection for high-frequency data is at the core of financial instruments based on algorithmic trading. The unique features of such data, absent in data measured at lower ...frequencies, raise significant challenges to their statistical analysis and econometric modelling, especially in the case of heavy-tailed data exhibiting outliers and rare events much more frequently. To address this problem, this paper proposes a new methodology for optimal sampling period selection, which better adapts to heavy-tailed statistics of high-frequency financial data. In particular, the novel concept of the degree of impulsiveness (DoI) is introduced first based on alpha-stable distributions, as an alternative source of information for characterising a broad range of impulsive behaviours. Then, a DoI-based generalised volatility signature plot is defined, which is further employed for determining the optimal sampling period. The performance of our method is evaluated in the case of risk quantification for high-frequency indexes, demonstrating a significantly improved accuracy when compared against the well-established volatility-based approach.
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