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.

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.

Chylomicrons against light scattering: The battle for characterization.

Chylomicrons (CMs) are lipoprotein particles circulating in blood and transporting dietary lipids. Optically speaking, CMs are small compared to the wavelength of visible light and widely distributed by the size and refractive index (RI). Consequently, intensity of light scattered by the CMs scales with up to the sixth power of their size, hampering simultaneous analysis of 60 and 600 nm CMs. We present an accurate method for quantitative characterization of large-size CM subpopulation by the distributions over size and RI. For the first time the CM characteristics have been determined at a single particle level based on angle-resolved light-scattering measurements. We applied the developed method to 2 key processes relating to CM metabolism, namely in vivo dynamics of CMs in blood plasma after a meal and in vitro lipolysis of CMs by the lipoprotein lipase in postheparin plasma. We have observed the substantial variations in CM concentration, size and RI distributions. This opens the way for a multitude of medical applications involving screening of CM metabolism, which we exemplified by revealing large differences in CM characteristics after a 12-hour fast between a healthy volunteer and a patient with atherosclerosis.

Light-scattering gating and characterization of plasma microparticles.

Flow cytometry method (FCM) is widely used for analysis of cell-derived microparticles (MPs). Numerous efforts are currently aimed to standardize these measurements among different instruments. We push the FCM characterization of MPs to the limit based on rigorous simulation of measured signals. We measured forward- and side-scatter (FSC/SSC) signals and angle-resolved light-scattering profiles (LSPs) of polystyrene microspheres and MPs, including their aggregates, using a scanning flow cytometer (SFC). We used the Mie theory to (1) accurately evaluate instrument detection limits; (2) construct FSC/SSC gates for MPs in absolute scales of size and refractive index (RI); and (3) determine size and RI of individual spherical MPs. LSPs were used for advanced characterization, including differentiation of spherical and nonspherical particles. The proposed absolute FSC/SSC gating is naturally standardized for any FCM instrument, given the knowledge of its optical system and leads to instrument-independent analysis of MPs. The inverse Mie problem has a unique solution only for some regions of size and RI and uncertainties rapidly increase with decreasing size and RI. The developed methods are applicable to any flow cytometer, but are limited by assumption of particle sphericity. The latter can be relaxed only if additional signals, such as LSP, are measured.

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.

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.

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.

Light-scattering flow cytometry for identification and characterization of blood microparticles.

We describe a novel approach to study blood microparticles using the scanning flow cytometer, which measures light scattering patterns (LSPs) of individual particles. Starting from platelet-rich plasma, we separated spherical microparticles from non-spherical plasma constituents, such as platelets and cell debris, based on similarity of their LSP to that of sphere. This provides a label-free method for identification (detection) of microparticles, including those larger than 1 µm. Next, we rigorously characterized each measured particle, determining its size and refractive index including errors of these estimates. Finally, we employed a deconvolution algorithm to determine size and refractive index distributions of the whole population of microparticles, accounting for largely different reliability of individual measurements. Developed methods were tested on a blood sample of a healthy donor, resulting in good agreement with literature data. The only limitation of this approach is size detection limit, which is currently about 0.5 µm due to used laser wavelength of 0.66 µm.