Platelet activation near point-like source of agonist: Experimental insights and computational model.

Disorders of hemostasis resulting in bleeding or thrombosis are leading cause of mortality in the world. Blood platelets are main players in hemostasis, providing the primary response to the vessel wall injury. In this case, they rapidly switch to the activated state in reaction to the exposed chemical substances such as ADP, collagen and thrombin. Molecular mechanisms of platelet activation are known, and detailed computational models are available. However, they are too complicated for large-scale problems (e.g. simulation of the thrombus growth) where less detailed models are required, which still should take into account the variation of agonist concentration and heterogeneity of platelets. In this paper, we present a simple model of the platelet population response to a spatially inhomogeneous stimulus. First, computational nodes modeling platelets are placed randomly in space. Each platelet is assigned the specific threshold for agonist, which determines whether it becomes activated at a given time. The distribution of the threshold value in a population is assumed to be log-normal. The model was validated against experimental data in a specially designed system, where the photorelease of ADP was caused by localized laser stimulus. In this system, a concentration of ADP obeys 2-dimensional Gaussian distribution which broadens due to the diffusion. The response of platelets to the point-like source of ADP is successfully described by the presented model. Our results advance the understanding of platelet function during hemostatic response. The simulation approach can be incorporated into larger computational models of thrombus formation.

Nitric Oxide Photorelease from Silicone Films Doped with N-Nitroso BODIPY.

Nitric oxide (NO) is a unique biochemical mediator involved in the regulation of vital processes. Light-controllable NO releasers show promise in the development of smart therapies. Here, we present a novel biocompatible material based on polydimethylsiloxane (PDMS) doped with BODIPY derivatives containing an N-nitroso moiety that is capable of the photoinduced generation of NO. We study the green-light-induced NO-release properties with the following three methods: electrochemical gas-phase sensor, liquid-phase sensor, and the Griess assay. Prolonged release of NO from the polymer films after short irradiation by narrow-band LED light sources and a laser beam is demonstrated. Importantly, this was accompanied by no or little release of the parent compound (BODIPY-based photodonor). Silicone films with the capability of controllable and clean NO release can potentially be used as a highly portable NO delivery system for different therapeutic applications.

Wireless monitoring of cell cultures based on light scattering: A novel optical scheme and portable prototype.

Cell cultures are widely used in scientific research, biomedicine, and industry. When culturing, it is important to maintain certain conditions, including the concentration of cells. Monitoring of the culture growth and cell counting is an urgent task for the optimization of technological processes. Most existing methods require sampling from a culture flask. This procedure is time-consuming and associated with the risks of contamination. We present a device able to monitor the growth of cells number in a suspension noninvasively. The device uses a laser beam that pass through the culture flask and measures the intensity of scattered light as a function of coordinate along the beam. This optical scheme allows one to obtain accurate results for both high- and low-scattering samples. We constructed the wireless portable prototype for monitoring of cell culture growth directly in the incubator and demonstrated the applicability of the device for Jurkat cells and Escherichia coli bacteria.

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.

Singlet oxygen luminescence detector based on low-cost InGaAs avalanche photodiode.

Molecular oxygen excited to singlet state (Singlet oxygen, 1O2) becomes highly reactive and cytotoxic chemical. 1O2 is commonly generated by photoexcitation of dyes (photosensitizers), including the photodynamic therapy and diagnostics of cancer. However, the formation of singlet oxygen is often unwanted for various light-sensitive compounds, e.g. it causes the photobleaching of fluorescent probes. In either case, during a development of new photosensitive chemicals and drugs there is a need to evaluate the amount of 1O2 formed during photoexcitation. The direct approach in measuring the amount of singlet oxygen is based on the detection of its luminescence at 1270 nm. However, this luminescence is usually weak, which implies the use of highly sensitive single-photon detectors. Thus the existing instruments are commonly complicated and expensive. Here we suggest an approach and report a device to measure the 1O2 luminescence using low-cost InGaAs avalanche photodiode and simple electronics. The measurements can be performed in stationary (not time-resolved) mode in organic solvents such as tetrachloromethane (CCl4), ethanol and DMSO. In particular, we performed spectral-resolved measurements of the singlet oxygen luminescence in CCl4 with the device and demonstrated high complementarity to literature data. The simple setup allows to evaluate the efficiency (or speed) of singlet oxygen generation and hence facilitates the development and characterization of new photosensitizers and other photosensitive chemicals.

Diaminorhodamine and Light-Activatable NO Donors: Photorelease Quantification and Potential Pitfalls.

Light-activatable nitric oxide (NO) donors have become of interest in the recent years. They produce NO when illuminated by light, which enables the control of its local concentration and is promising for biomedical applications. Several successful prototypes of photodonors have been published, but further research is needed to improve their properties such as water-solubility, activation wavelength, biocompatibility etc. One of major challenges on this way is to evaluate the efficiency of NO generation. Several methods may be used to track NO, including spin traps, specific electrodes and fluorescence-based probes. We have studied the applicability of well-known fluorescent reporter, diaminorhodamine (DAR-2), for the evaluation of NO production by photodonors. Our results indicate that DAR-2 can be used for the quantification of NO photorelease if this process is not accompanied by the singlet oxygen formation. Otherwise the oxidation of probe results in huge fluorescence increase, which interferes with signal due to reaction with NO. This issue should be taken into account when studying hybrids releasing both NO and 1O2, which are promising for photodynamic therapy.

Optical uncaging of ADP reveals the early calcium dynamics in single, freely moving platelets.

Platelet activation is considered to be a cornerstone in pathogenesis of cardiovascular disease. The assessment of platelet activation at the single-cell level is a promising approach for the research of platelet function in physiological and pathological conditions. Previous studies used the immobilization of platelets on the surface, which significantly alters the activation signaling. Here we show that the use of photolabile "caged" analog of ADP allows one to track the very early stage of platelet activation in single, freely moving cells. In this approach, the diffusion step and ADP receptor ligation are separated in time, and a millisecond-timescale optical pulse may trigger the activation. The technique allows us to measure the delay (lag time) between the stimulus and calcium response in platelets. We also propose a simple model function for calcium peaks, which is in good agreement with the measured data. The proposed technique and model function can be used for in-depth studies of platelet physiology.

Long-wavelength photoremovable protecting groups: On the way to in vivo application.

Photoremovable protective groups (PPGs) and related "caged" compounds have been recognized as a powerful tool in an arsenal of life science methods. The present review is focused on recent advances in design of "caged" compounds which function in red or near-infrared region. The naive comparison of photon energy with that of organic bond leads to the illusion that long-wavelength activation is possible only for weak chemical bonds like N-N. However, there are different means to overcome this threshold and shift the uncaging functionality into red or near-infrared regions for general organic bonds. We overview these strategies, including the novel photochemical and photophysical mechanisms used in newly developed PPGs, singlet-oxygen-mediated photolysis, and two-photon absorption. Recent advances in science places the infrared-sensitive PPGs to the same usability level as traditional ones, facilitating in vivo application of caged compounds.

Photolysis of dimethoxynitrobenzyl-"caged" acids yields fluorescent products.

Carboxylic acids conjugated with 4,5-dimethoxy-2-nitrobenzyl photoremovable protecting group are well known and widely used for biological studies. In this paper, we study the photolysis of likewise "caged" acetic, caprylic and arachidonic acids. Unexpectedly, we observed huge growth of fluorescence emission at ~430 nm during photolysis. Following further UV irradiation, a product with fluorescence at longer wavelength was formed (470 nm excitation / ~500-600 nm emission). While it may be used to monitor the "uncaging", these fluorescent products may interfere with widespread dyes such as fluorescein in biomedical experiments. This effect might be negligible if the photolysis products dissolve in the medium. On the other hand, we observed that arachidonic and caprylic acids derivatives self-organize in emulsion droplets in water environment due to long lipophilic chains. Illumination of droplets by UV rapidly induces orange fluorescence excited by 488 nm light. This fluorescence turn-on was fast (~0.1 s) and apparently caused by the accumulation of water-insoluble fluorescent residuals inside droplets. These self-organized lipophilic structures with fluorescence turn-on capability may be of interest for biomedical and other application. We have identified and hypothesized some compounds which may be responsible for the observed fluorescense.

The platelet shape change: biophysical basis and physiological consequences.

The well-known platelet shape change is the universal hallmark of activation. This review uncovers the biophysics underlying this rapid and dramatic transformation. We aim to give a broad vision of the interplay between different cytoskeletal subsystems, which is based on physical considerations and recent advances in mathematics and computational biology. These novel findings lead to the understanding that the ring of microtubules counterbalances cortical tension in the resting platelet, making it a "mechanically charged" system. Platelet activation breaks the balance via several mechanisms, triggering rapid ring buckling and cell rounding. Based on the review of known data concerning the relations between platelet shape and function, we hypothesize that disk-to-sphere transformation facilitates platelet adhesion under flow. Conclusions of the paper may be useful for the development of novel, cytoskeletal-based strategies of antiplatelet therapy.

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.

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.

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.

Additivity of light-scattering patterns of aggregated biological particles.

The paper is focused on light scattering by aggregates of optically soft particles with a size larger than the wavelength, in particular, blood platelets. We conducted a systematic simulation of light scattering by dimers and larger aggregates of blood platelets, each modeled as oblate spheroids, using the discrete dipole approximation. Two-dimensional (2-D) light scattering patterns (LSPs) and internal fields showed that the multiple scattering between constituent particles can be neglected. Additionally, we derived conditions of the scattering angle and orientation of the dimer, under which the averaging of the 2-D LSPs over the azimuthal scattering angle washes out interference in the far field, resulting in averaged LSPs of the aggregate being equal to the sum of that for its constituents. We verified theoretical conclusions using the averaged LSPs of blood platelets measured with the scanning flow cytometer (SFC). Moreover, we obtained similar results for a model system of aggregates of polystyrene beads, studied both experimentally and theoretically. Finally, we discussed the potential of discriminating platelet aggregates from monomers using the SFC.

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.

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.