4π light scattering flow cytometry: enhancing the identification and characterization of individual cells.

The analysis of individual particles with complex morphologies from light scattering is crucial in disperse systems studies, such as blood cells. Characterization, which assumes determining particle characteristics, has a higher likelihood of succeeding in solving the inverse light-scattering problem if an instrument provides enough light-scattering data. In this study, we demonstrate how we extend the operating angular interval for the 4π Scanning Flow Cytometer (4πSFC), which measures angle-resolved light-scattering profiles (LSPs) of individual particles. The angular interval is extended by additionally measuring light scattering for the backward hemisphere. Currently, the 4πSFC setup uses three lasers, a single optical cell, and three photomultipliers. It enables the measurement of the LSP of individual particles within the angular interval of 10 to 170° for polar angles with integration over azimuth angles, which covers the spatial angle of 98.5% of the 4π angle. We demonstrate the 4πSFC's performance in measuring LSPs from the analysis of polymer beads, mature and spherized erythrocytes, and platelets. The 4πSFC has the potential to be very useful in identifying platelet dimers and granulocytes without labels, characterizing lymphocytes, monocytes, and abnormal erythrocytes.

Kinetic titration method in flow cytometry for quantification of cell receptors.

For the quantitative determination of cell receptors by fluorescence flow cytometry, we proposed a new method, which takes into account the reaction kinetics. The binding reaction of the ligand with receptors begins after placing the cells in the ligand solution. In the proposed method, there are several samples with the same concentration of cells and different initial concentrations of fluorescently labeled ligand, and each sample is measured by a flow cytometer once at the time when the following condition is met: the product of the incubation time (cells with ligand) and the initial concentration of ligand is the same for all samples. The proposed approach eliminates disadvantages and combines advantages of both kinetic and titration methods for quantification of receptors on single cells without the use of traditional calibration fluorescent beads. Practical application of the method was demonstrated in quantification of CD8 and CD14 on peripheral blood human leukocytes. Particularly, we found decreased (by a factor of two) mean number of CD14 on monocytes and granulocytes in patients with atherosclerosis (treated in the hospital) compared to conditionally healthy donors, whereas no difference was found in the mean CD8 expression on leukocytes between the same patient and donor groups.

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.

Blood platelet quantification by light scattering: from morphology to activation.

Analysis of blood platelets encounters a number of different preanalytical issues, which greatly decrease the reliability and accuracy of routine clinical analysis. Modern hematology analyzers determine only four parameters relating to platelets. Platelet shape and dose-dependent activation parameters are outside the scope of commercial instruments. We used the original scanning flow cytometer for measurement of angle-resolved light scattering and the discrete dipole approximation for simulation of light scattering from a platelet optical model, as an oblate spheroid, and global optimization with two algorithms: the DATABASE algorithm to retrieve platelet characteristics from light scattering and the DIRECT algorithm to retrieve dose-dependent activation parameters. We developed the original sampling protocol to decrease spontaneous platelet activation. The new protocol allows us to keep most of the platelets in resting and partially activated states before analysis. The analysis delivers 13 content and morphological parameters of the platelets. To analyze platelet shape change during ADP activation we developed a phenomenological model. This model was applied to the analysis of ADP activation of platelets to give 8 dose-dependent activation parameters. To demonstrate the applicability of the developed protocol and analytical method, we analyzed platelets from five donors. This novel approach to the analysis of platelets allows the determination of 21 parameters relating to their content, morphology and dose-dependent activation.

Non-uniform sampling in pulse dipolar spectroscopy by EPR: the redistribution of noise and the optimization of data acquisition.

Pulse dipolar spectroscopy (PDS) in Electron Paramagnetic Resonance (EPR) is the method of choice for determining the distance distribution function for mono-, bi- or multi- spin-labeled macromolecules and nanostructures. PDS acquisition schemes conventionally use uniform sampling of the dipolar trace, but non-uniform sampling (NUS) schemes can decrease the total measurement time or increase the accuracy of the resulting distance distributions. NUS requires optimization of the data acquisition scheme, as well as changes in data processing algorithms to accommodate the non-uniformly sampled data. We investigate in silico the applicability of the NUS approach in PDS, considering its effect on random, truncation and sampling noise in the experimental data. Each type of noise in the time-domain data propagates differently and non-uniformly into the distance spectrum as errors in the distance distribution. NUS schemes seem to be a valid approach for increasing sensitivity and/or throughput in PDS by decreasing and redistributing noise in the distance spectrum so that it has less impact on the distance spectrum.

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.

Analytical solution of the PELDOR inverse problem using the integral Mellin transform.

We describe a new model-free approach to solve the inverse problem in pulsed double electron-electron resonance (PELDOR, also known as DEER) spectroscopy and obtain the distance distribution function between two radicals from time-domain PELDOR data. The approach is based on analytical solutions of the Fredholm integral equations of the first kind using integral Mellin transforms to provide the distance distribution function directly. The approach appears to confine the noise in the computed distance distribution to short distances and does not introduce systematic distortions. Thus, the proposed analysis method can be a useful supplement to current methods to determine complicated distance distributions.

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.

Accurate measurement of volume and shape of resting and activated blood platelets from light scattering.

We introduce a novel approach for determination of volume and shape of individual blood platelets modeled as an oblate spheroid from angle-resolved light scattering with flow-cytometric technique. The light-scattering profiles (LSPs) of individual platelets were measured with the scanning flow cytometer and the platelet characteristics were determined from the solution of the inverse light-scattering problem using the precomputed database of theoretical LSPs. We revealed a phenomenon of parameter compensation, which is partly explained in the framework of anomalous diffraction approximation. To overcome this problem, additional a priori information on the platelet refractive index was used. It allowed us to determine the size of each platelet with subdiffraction precision and independent of the particular value of the platelet aspect ratio. The shape (spheroidal aspect ratio) distributions of platelets showed substantial differences between native and activated by 10 µM adenosine diphosphate samples. We expect that the new approach may find use in hematological analyzers for accurate measurement of platelet volume distribution and for determination of the platelet activation efficiency.

Polarized light-scattering profile-advanced characterization of nonspherical particles with scanning flow cytometry.

We instrumentally, theoretically, and experimentally demonstrate a new approach for characterization of nonspherical individual particles from light scattering. Unlike the original optical scheme of the scanning flow cytometer that measures an angle-resolved scattering corresponding in general to S11 element of the light-scattering matrix, the modernized instrument allows us to measure the polarized light-scattering profile of individual particles simultaneously. Theoretically, the polarized profile is expressed by the combination of a few light-scattering matrix elements. This approach supports us with additional independent data to characterize a particle with a complex shape and an internal structure. Applicability of the new method was demonstrated from analysis of polymer bispheres. The bisphere characteristics, sizes, and refractive indices of each sphere composing the bisphere were successfully retrieved from the solution of the inverse light-scattering problem. The solution provides determination of the Eulerian angles, which describe the orientation of the bispheres relative to the direction of the incident laser beam and detecting polarizer of the optical system. Both the ordinary and polarized profiles show a perfect agreement with T-matrix simulation resulting to 50-nm precision for sizing of bispheres.

Scanning flow cytometer modified to distinguish phytoplankton cells from their effective size, effective refractive index, depolarization, and fluorescence.

A laser flow cytometer based on scanning flow cytometry has been assembled. The unpolarized and linearly polarized light-scattering profiles, as well as the side emitted light in different spectral bands, were measured, allowing the simultaneous and real-time determination of the effective size and the effective refractive index of each spherelike particle. Additionally, each particle could be identified from depolarization and fluorescence measured simultaneously. The tests with aqueous samples of polystyrene spheres, fluorescent or nonfluorescent, and phytoplankton cells demonstrate that the system is able to retrieve size and refractive index with an accuracy of 1% and that the depolarization and fluorescence measurements allow the classification of particles otherwise indistinguishable.

Erythrocyte lysis in isotonic solution of ammonium chloride: theoretical modeling and experimental verification.

A mathematical model of erythrocyte lysis in isotonic solution of ammonium chloride is presented in frames of a statistical approach. The model is used to evaluate several parameters of mature erythrocytes (volume, surface area, hemoglobin concentration, number of anionic exchangers on membrane, elasticity and critical tension of membrane) through their sphering and lysis measured by a scanning flow cytometer (SFC). SFC allows measuring the light-scattering pattern (indicatrix) of an individual cell over the angular range from 10 degrees to 60 degrees . Comparison of the experimentally measured and theoretically calculated light scattering patterns allows discrimination of spherical from non-spherical erythrocytes and evaluation of volume and hemoglobin concentration for individual spherical cells. Three different processes were applied for erythrocytes sphering: (1) colloid osmotic lysis in isotonic solution of ammonium chloride, (2) isovolumetric sphering in the presence of sodium dodecyl sulphate and albumin in neutrally buffered isotonic saline, and (3) osmotic fragility test in hypotonic media. For the hemolysis in ammonium chloride, the evolution of distributions of sphered erythrocytes on volume and hemoglobin content was monitored in real-time experiments. The analysis of experimental data was performed in the context of a statistical approach, taking into account that parameters of erythrocytes vary from cell to cell.