Ultraviolet light scattering scanning flow cytometry in the characterization of submicron microparticles.

Ultraviolet lasers are commonly used in flow cytometry to excite fluorochrome molecules with subsequent measurement of the specific fluorescence of individual cells. In this study, the performance of the ultraviolet light scattering (UVLS) in the analysis of individual particles with flow cytometry has been demonstrated for the first time. The main advantage of the UVLS relates to the improvement of the analysis of submicron particles due to the strong dependence of the scattering efficiency on the wavelength of the incident light. In this work, submicron particles were analyzed using a scanning flow cytometer (SFC) that allows measurements of light scattering in an angle-resolved regime. The measured light-scattering profiles of individual particles were utilized in solution of the inverse light-scattering problem to retrieve the particle characteristics using a global optimization. The standard polystyrene microspheres were successfully characterized from the analysis of UVLS which provided the size and refractive index (RI) of individual beads. We believe that the main application of UVLS relates to the analysis of microparticles in a serum, in particular in the analysis of chylomicrons (CMs). We have demonstrated the performance of the UVLS SFC in the analysis of CMs of a donor. The RI versus size scatterplot of CMs was successfully retrieved from the analysis. The current set-up of the SFC has allowed us to characterize individual CMs starting from the size of 160 nm that provides determination of the CM concentration in a serum with flow cytometry. This feature of the UVLS should help with the analysis of lipid metabolism measuring RI and size map evolution after lipase action.

A light scatter based model relating erythrocyte vesiculation to lifetime in circulation.

Methods for measuring erythrocyte age distribution are not available as a simple analytical tool. Most of them utilize the fluorescence or radioactive isotopes labeling to construct the age distribution and support physicians with aging indices of donor's erythrocytes. The age distribution of erythrocyte may be a useful snapshot of patient state over 120-days period of life. Previously, we introduced the enhanced assay of erythrocytes with measurement of 48 indices in four categories: concentration/content, morphology, aging and function (10.1002/cyto.a.24554). The aging category was formed by the indices based on the evaluation of the derived age of individual cells. The derived age does not exactly mean the real age of erythrocytes and its evaluation utilizes changes of cellular morphology during a lifespan. In this study, we are introducing the improved methodological approach that allows us to retrieve the derived age of individual erythrocytes, to construct the aging distribution, and to reform the aging category consisting of eight indices. The approach is based on the analysis of the erythrocyte vesiculation. The erythrocyte morphology is analyzed by scanning flow cytometry that measures the primary characteristics (diameter, thickness, and waist) of individual cells. The surface area (S) and sphericity index (SI) are calculated from the primary characteristics and the scattering diagram SI versus S is used in the evaluation of the derived age of each erythrocyte in a sample. We developed the algorithm to evaluate the derived age that provides eight indices in the aging category based on a model using light scatter features. The novel erythrocyte indices were measured for simulated cells and blood samples of 50 donors. We determined the first-ever reference intervals for these indices.

Erythrocyte lysis and angle-resolved light scattering measured by scanning flow cytometry result to 48 indices quantifying a gas exchange function of the human organism.

Molecular/cell level of gas exchange function assumes the accurate measurement of erythrocyte characteristics and rate constants concerning to molecules involved into the CO2 /O2 transport. Unfortunately, common hematology analyzers provide the measurement of eight indices of erythrocytes only and say little about erythrocyte morphology and nothing about rate constants of cellular function. The aim of this study is to demonstrate the ability of the Scanning Flow Cytometer (SFC) in the complete morphological analysis of mature erythrocytes and characterization of erythrocyte function via measurement of lysing kinetics. With this study we are introducing 48 erythrocyte indices. To provide the usability of application of the SFC in clinical diagnosis, we formed four categories of indices which are as follows: content/concentration (9 indices), morphology (26 indices), age (5 indices), and function (8 indices). The erythrocytes of 39 healthy volunteers were analyzed with the SFC to fix the first-ever reference intervals for the new indices introduced. The essential measurable reliability of the presented method is expressed in terms of errors of characteristics of single erythrocytes retrieved from the solution of the inverse light-scattering problem and errors of parameters retrieved from the fitting of the experimental kinetics by molecular-kinetics model of erythrocyte lysis.

Proposed Dynamics of CDB3 Activation in Human Erythrocytes by Nifedipine Studied with Scanning Flow Cytometry.

Nifedipine is calcium channels and pumps blocker widely used in medicine. However, mechanisms of nifedipine action in blood are not clear. In particular, the influence of nifedipine on erythrocytes is far from completely understood. In this work, applying scanning flow cytometry, we observed experimentally for the first time the dynamics behind a significant increase of HCO3- /Cl- transmembrane exchange rate of CDB3 (main anion exchanger, AE1, Band 3, SLC4A1) of human erythrocytes in the presence of nifedipine in blood. It was found that the rate of CDB3 activation is not limited by the rate of nifedipine binding and/or Ca2+ transport. In order to explain the experimental data, we suggested a kinetic model assuming that the rate of CDB3 activation is limited by the dynamics of the balance between two intracellular processes (1) the activation of CDB3 limited by its interaction with intracellular Ca2+ , and (2) the spontaneous deactivation of CDB3. Thus the use of scanning flow cytometry allowed to clarify quantitatively the molecular kinetic mechanism of nifedipine action on human erythrocytes. In particular, the efficiency (~30) and rates of activation (~0.3 min-1 ) and deactivation (~10-3 min-1 ) of CDB3 in human erythrocytes was evaluated for two donors. © 2019 International Society for Advancement of Cytometry.

Interface-Assisted Synthesis of the Mn3-xFexO4 Gradient Film with Multifunctional Properties.

Anisotropic gradient materials are considered as promising and novel in that they have numerous functional properties and are able to transform into hierarchical microstructures. We report a facile method of gradient inorganic thin film synthesis through diffusion-controlled deposition at the gas-solution interface. To investigate the reaction of interfacial phase boundary controllable hydrolysis by gaseous ammonium, an aqueous solution of FeCl3 and MnCl2 was chosen, as the precipitation pH values for the hydroxides of these metals differ gradually. As a result of synthesis using the gas-solution interface technique (GSIT), a thin film is formed on the surface of the solution that consists of Mn2+(Fe,Mn)23+O4 nanoparticles with hausmannite crystal structure. The ratio between iron and manganese in the film can be adjusted over a wide range by varying the synthetic procedure. Specific conditions are determined that allow the formation of a Mn-Fe mixed oxide film with a gradient of composition, morphology, and properties, as well as its further transformation into microscrolls with a diameter of 10-20 µm and a length of up to 300 µm, showing weak superparamagnetic properties. The technique reported provides a new interfacial route for the development of functional gradient materials with tubular morphology.

Method for the simulation of blood platelet shape and its evolution during activation.

We present a simple physically based quantitative model of blood platelet shape and its evolution during agonist-induced activation. The model is based on the consideration of two major cytoskeletal elements: the marginal band of microtubules and the submembrane cortex. Mathematically, we consider the problem of minimization of surface area constrained to confine the marginal band and a certain cellular volume. For resting platelets, the marginal band appears as a peripheral ring, allowing for the analytical solution of the minimization problem. Upon activation, the marginal band coils out of plane and forms 3D convoluted structure. We show that its shape is well approximated by an overcurved circle, a mathematical concept of closed curve with constant excessive curvature. Possible mechanisms leading to such marginal band coiling are discussed, resulting in simple parametric expression for the marginal band shape during platelet activation. The excessive curvature of marginal band is a convenient state variable which tracks the progress of activation. The cell surface is determined using numerical optimization. The shapes are strictly mathematically defined by only three parameters and show good agreement with literature data. They can be utilized in simulation of platelets interaction with different physical fields, e.g. for the description of hydrodynamic and mechanical properties of platelets, leading to better understanding of platelets margination and adhesion and thrombus formation in blood flow. It would also facilitate precise characterization of platelets in clinical diagnosis, where a novel optical model is needed for the correct solution of inverse light-scattering problem.

Calibration-free quantitative immunoassay by flow cytometry: Theoretical consideration and practical implementation for IgG antibody binding to CD14 receptors on human leukocytes.

We propose a calibration-free method to determine the number of receptors per cell, as well as the direct and the reverse reaction rate constants for a single receptor. The method is based on the analysis of the temporal evolution of the cells mean fluorescent intensity measured by a flow cytometer during the ligand-receptor (antigen-antibody) binding under the conditions of their comparable concentrations. We developed the kinetic approach accounting both for the delay between the dilution and the measurement and for the practical duration of the measurement itself. The method was applied to determine thenumber of CD14 receptors on human blood mononuclear (granulocytes, monocytes, lymphocytes) cells of several donors. We also obtained the direct ( k+= (5.6 ± 0.2) × 107 M-1 min-1 ) and reverse ( k-= (1.3 ± 0.2) × 10-2 min-1 ) rate constants of ligand-receptor interaction, and estimated the size of the binding site as b = 0.5 nm. The latter allows one to recalculate the rate constants for a different ligand, fluorescent label, medium viscosity, and/or temperature. The knowledge of the rate constants is essential for the calibration-free determination of the number of receptors per cell from a single kinetic curve of the cells mean fluorescence intensity.

Massive Vector Fields in Rotating Black-Hole Spacetimes: Separability and Quasinormal Modes.

We demonstrate the separability of the massive vector (Proca) field equation in general Kerr-NUT-AdS black-hole spacetimes in any number of dimensions, filling a long-standing gap in the literature. The obtained separated equations are studied in more detail for the four-dimensional Kerr geometry and the corresponding quasinormal modes are calculated. Two of the three independent polarizations of the Proca field are shown to emerge from the separation ansatz and the results are found in an excellent agreement with those of the recent numerical study where the full coupled partial differential equations were tackled without using the separability property.

Nuclear apoptotic volume decrease in individual cells: Confocal microscopy imaging and kinetic modeling.

The dynamics of nuclear morphology changes during apoptosis remains poorly investigated and understood. Using 3D time-lapse confocal microscopy we performed a study of early-stage apoptotic nuclear morphological changes induced by etoposide in single living HepG2 cells. These observations provide a definitive evidence that nuclear apoptotic volume decrease (AVD) is occurring simultaneously with peripheral chromatin condensation (so called "apoptotic ring"). In order to describe quantitatively the dynamics of nuclear morphological changes in the early stage of apoptosis we suggest a general molecular kinetic model, which fits well the obtained experimental data in our study. Results of this work may clarify molecular mechanisms of nuclear morphology changes during apoptosis.

Interface-Assisted Synthesis of Single-Crystalline ScF3 Microtubes.

Scandium fluoride (ScF3) microtubes with nanoscale wall thickness were for the first time successfully synthesized by an interface-assisted technique at the surface of a scandium nitrate aqueous solution without the addition of any surfactant as a result of interaction with hydrofluoric acid from the gaseous phase in only 30 min. X-ray diffraction analysis, scanning electron microscopy, helium ionic microscopy, transmission electron microscopy (TEM), and high-resolution TEM (HRTEM) were used to examine the morphology and crystal structure of ScF3 microtubes. The results show that the ScF3 microtube is single-crystalline and has a hexagonal structure. A hypothetical model of thin-walled microtube formation is proposed.

Black holes, hidden symmetries, and complete integrability.

The study of higher-dimensional black holes is a subject which has recently attracted vast interest. Perhaps one of the most surprising discoveries is a realization that the properties of higher-dimensional black holes with the spherical horizon topology and described by the Kerr-NUT-(A)dS metrics are very similar to the properties of the well known four-dimensional Kerr metric. This remarkable result stems from the existence of a single object called the principal tensor. In our review we discuss explicit and hidden symmetries of higher-dimensional Kerr-NUT-(A)dS black hole spacetimes. We start with discussion of the Killing and Killing-Yano objects representing explicit and hidden symmetries. We demonstrate that the principal tensor can be used as a "seed object" which generates all these symmetries. It determines the form of the geometry, as well as guarantees its remarkable properties, such as special algebraic type of the spacetime, complete integrability of geodesic motion, and separability of the Hamilton-Jacobi, Klein-Gordon, and Dirac equations. The review also contains a discussion of different applications of the developed formalism and its possible generalizations.

Super-resolved calibration-free flow cytometric characterization of platelets and cell-derived microparticles in platelet-rich plasma.

Importance of microparticles (MPs), also regarded as extracellular vesicles, in many physiological processes and clinical conditions motivates one to use the most informative and precise methods for their characterization. Methods based on individual particle analysis provide statistically reliable distributions of MP population over characteristics. Although flow cytometry is one of the most powerful technologies of this type, the standard forward-versus-side-scattering plots of MPs and platelets (PLTs) overlap considerably because of similarity of their morphological characteristics. Moreover, ordinary flow cytometry is not capable of measurement of size and refractive index (RI) of MPs. In this study, we 1) employed the potential of the scanning flow cytometer (SFC) for identification and characterization of MPs from light scattering; 2) suggested the reference method to characterize MP morphology (size and RI) with high precision; and 3) determined the lowest size of a MP that can be characterized from light scattering with the SFC. We equipped the SFC with 405 and 488 nm lasers to measure the light-scattering profiles and side scattering from MPs, respectively. The developed two-stage method allowed accurate separation of PLTs and MPs in platelet-rich plasma. We used two optical models for MPs, a sphere and a bisphere, in the solution of the inverse light-scattering problem. This solution provides unprecedented precision in determination of size and RI of individual spherical MPs-median uncertainties (standard deviations) were 6 nm and 0.003, respectively. The developed method provides instrument-independent quantitative information on MPs, which can be used in studies of various factors affecting MP population.

Influence of magnesium sulfate on HCO3/Cl transmembrane exchange rate in human erythrocytes.

Magnesium sulfate (MgSO4) is widely used in medicine but molecular mechanisms of its protection through influence on erythrocytes are not fully understood and are considerably controversial. Using scanning flow cytometry, in this work for the first time we observed experimentally (both in situ and in vitro) a significant increase of HCO3(-)/Cl(-) transmembrane exchange rate of human erythrocytes in the presence of MgSO4 in blood. For a quantitative analysis of the obtained experimental data, we introduced and verified a molecular kinetic model, which describes activation of major anion exchanger Band 3 (or AE1) by its complexation with free intracellular Mg(2+) (taking into account Mg(2+) membrane transport and intracellular buffering). Fitting the model to our in vitro experimental data, we observed a good correspondence between theoretical and experimental kinetic curves that allowed us to evaluate the model parameters and to estimate for the first time the association constant of Mg(2+) with Band 3 as KB~0.07mM, which is in agreement with known values of the apparent Mg(2+) dissociation constant (from 0.01 to 0.1mM) that reflects experiments on enrichment of Mg(2+) at the inner erythrocyte membrane (Gunther, 2007). Results of this work partly clarify the molecular mechanisms of MgSO4 action in human erythrocytes. The method developed allows one to estimate quantitatively a perspective of MgSO4 treatment for a patient. It should be particularly helpful in prenatal medicine for early detection of pathologies associated with the risk of fetal hypoxia.

Mass Gap for Black-Hole Formation in Higher-Derivative and Ghost-Free Gravity.

We study a spherical gravitational collapse of a small mass in higher-derivative and ghost-free theories of gravity. By boosting a solution of linearized equations for a static point mass in such theories we obtain in the Penrose limit the gravitational field of an ultrarelativistic particle. Taking a superposition of such solutions we construct a metric of a collapsing null shell in the linearized higher-derivative and ghost-free gravity. The latter allows one to find the gravitational field of a thick null shell. By analyzing these solutions we demonstrate that in a wide class of the higher dimensional theories of gravity as well as for the ghost-free gravity there exists a mass gap for mini-black-hole production. We also found conditions when the curvature invariants remain finite at r=0 for the collapse of the thick null shell.

Convergence of the discrete dipole approximation. I. Theoretical analysis: erratum.

We report and address the errors in the analysis of the weighted discretization in Section 2.F of our published paper [J. Opt. Soc. Am. A23, 2578 (2006)JOAOD61084-752910.1364/JOSAA.23.002578].

Synthesis of birnessite structure layers at the solution-air interface and the formation of microtubules from them.

H(x)MnO2·nH2O layers have been successfully produced through a facile low-temperature process at the solution-air interface without using any templates. The crystalline structures of layers can be tuned by the compositions and the pH of the growth solutions. The analysis of birnessite-like layers indicates that they are formed by nanosheets approximately 4-6 nm thick that are oriented for the most part normally to the interface. Our results demonstrated that 1-3-µm-thick layers can roll up into microtubules 20 to 100 µm in diameter and up to 10 mm long. The hypothesis explaining the formation of the microtubular structures is assumed.

Brownian aggregation rate of colloid particles with several active sites.

We theoretically analyze the aggregation kinetics of colloid particles with several active sites. Such particles (so-called "patchy particles") are well known as chemically anisotropic reactants, but the corresponding rate constant of their aggregation has not yet been established in a convenient analytical form. Using kinematic approximation for the diffusion problem, we derived an analytical formula for the diffusion-controlled reaction rate constant between two colloid particles (or clusters) with several small active sites under the following assumptions: the relative translational motion is Brownian diffusion, and the isotropic stochastic reorientation of each particle is Markovian and arbitrarily correlated. This formula was shown to produce accurate results in comparison with more sophisticated approaches. Also, to account for the case of a low number of active sites per particle we used Monte Carlo stochastic algorithm based on Gillespie method. Simulations showed that such discrete model is required when this number is less than 10. Finally, we applied the developed approach to the simulation of immunoagglutination, assuming that the formed clusters have fractal structure.

A feasible interlayer strategy for simultaneous light and heat management in photothermal catalysis.

Photothermal conversion represents one crucial approach for solar energy harvesting and its exploitation as a sustainable alternative to fossil fuels; however, an efficient, cost-effective, and generalized approach to enhance the photothermal conversion processes is still missing. Herein, we develop a feasible and efficient photothermal conversion strategy that achieves simultaneous light and heat management using supported metal clusters and WSe2 interlayer toward enhanced CO2 hydrogenation photothermal catalysis. The interlayer can simultaneously reduce heat loss in the catalytic layer and improve light absorption, leading to an 8-fold higher CO2 conversion rate than the controls. The optical and thermal performance of WSe2 interlayered catalysts on different substrates was quantified using Raman spectroscopy. This work demonstrates a feasible and generalized approach for effective light and heat management in solar harvesting. It also provides important design guidelines for efficient photothermal converters that facilitate the remediation of the energy and environmental crises faced by humans.

Modulation of the Structure-function Relationship of the "nano-greenhouse effect" towards Optimized Supra-photothermal Catalysis.

Photothermal catalytic CO2 hydrogenation holds great promise for relieving the global environment and energy crises. The "nano-greenhouse effect" has been recognized as a crucial strategy for improving the heat management capabilities of a photothermal catalyst by ameliorating the convective and radiative heat losses. Yet it remains unclear to what degree the respective heat transfer and mass transport efficiencies depend on the specific structures. Herein, the structure-function relationship of the "nano-greenhouse effect" was investigated and optimized in a prototypical Ni@SiO2 core-shell catalyst towards photothermal CO2 catalysis. Experimental and theoretical results indicate that modulation of the thickness and porosity of the SiO2 nanoshell leads to variations in both heat preservation and mass transport properties. This work deepens the understandings on the contributing factor of the "nano-greenhouse effect" towards enhanced photothermal conversion. It also provides insights on the design principles of an ideal photothermal catalyst in balancing heat management and mass transport processes.

High-precision characterization of individual E. coli cell morphology by scanning flow cytometry.

We demonstrate a flow-cytometric method to measure length and diameter of single Escherichia coli cells with sub-diffraction precision. The method is based on the original scanning flow cytometer that measures angle-resolved light-scattering patterns (LSPs) of individual particles. We modeled the shape of E. coli cells as a cylinder capped with hemispheres of the same radius, and simulated light scattering by the models using the discrete dipole approximation. We computed a database of the LSPs of individual bacteria in a wide range of model parameters and used it to solve the inverse light-scattering problem by the nearest-neighbor interpolation. The solution allows us to determine length and diameter of each individual bacterium, including uncertainties of these estimates. The developed method was tested on two strains of E. coli. The resulting precision of bacteria length and diameter measurements varied from 50 nm to 250 nm and from 5 nm to 25 nm, respectively. The measured distributions of samples over length and diameter were in good agreement with measurements performed by optical microscopy and literature data. The described approach can be applied for rapid morphological characterization of any rod-shaped bacteria.