Light-scattering Profiles Research Articles

Determining blood platelet morphology modelled by a superellipsoid from the solution of the inverse light-scattering problem

In this work, we propose a new optical model to describe the morphology of a platelet in the form of a special axisymmetric superellipsoid. This model is more general than the widely used classical oblate spheroid model of the platelet and includes the oblate spheroid as a special case. The inverse light scattering (ILS) problem within the developed optical model was solved using the light scattering profile (LSP) of individual platelets obtained by the scanning flow cytometer. Solving the ILS problem we used a hybrid method that combines the DIRECT algorithm of global optimization with the linear interpolation between the nodes of the pre-calculated regular database of superellipsoid LSPs. The statistical F-test shows that the new optical model is better than the conventional oblate spheroid model in more than 60 percent of cases. This made it possible to obtain the refined shape index distributions for platelets consisting of three fractions, namely resting, partially and fully activated platelets.

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.

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.

Revisiting the Molar Mass and Conformation of Derivatized Fractionated Softwood Kraft Lignin.

The limited utilization of reliable tools and standards for determination of the softwood kraft lignin molar mass and the corresponding molecular conformation hampers elucidation of the structure-property relationships of lignin. At issue, conventional size exclusion chromatography (SEC) is unable to robustly measure the molar mass because of a lack of calibration standards with a similar structure to lignin. In the present work, the determination of the absolute molar mass of acetylated technical lignin was revisited utilizing SEC combined with multi-angle light scattering with a band pass filter to suppress the fluorescence. Fractionated lignin isolated using sequential techniques of solvent and membrane methods was used to enhance the clarity of light-scattering profiles by narrowing the molar mass distribution of lignin fractions. Further information on the molecular conformation of derivatized samples was studied utilizing a differential viscometer, and chemical structures were identified by NMR spectroscopy analysis. Through the help of fractionation, intrinsic viscosity values were determined for the different fractions as a function of molecular weight cut-off membranes. The derivatized acetone-soluble lignin was found to possess a lower molecular weight and an extremely compact structure relative to the derivatized acetone-insoluble fraction based on a significantly lower "α" value in the Mark-Houwink-Sakurada plot (0.15 acetone-soluble vs 0.33 acetone-insoluble). The differences in geometry were supported by the linkage analysis from NMR showing the acetone-soluble part containing fewer native linkages. In both of these examples, kraft lignin behaved like a solid sphere, limiting the ability to provide entanglements between molecular chains. From this standpoint, macroscopic properties of lignin are justified with this knowledge of a dense and extremely compact structure.

Retrieving the refractive index of a sphere from the phase spectrum of its light-scattering profile

We studied the Fourier spectrum of the light-scattering profiles of single particles in the Rayleigh-Gans-Debye (RGD) and Wentzel–Kramers–Brillouin (WKB) approximations. In the case of a homogeneous sphere, we found the relationship between the key parameters of the spectrum (including its phase) and the sphere characteristics – both analytically and numerically in the framework of the approximations and the rigorous Lorentz–Mie theory, respectively. Based on these results, we have improved the existing spectral characterization method for spheres extending the applicability range to particles with a higher refractive index.

Dual-wavelength angle-resolved light scattering used in the analysis of particles by scanning flow cytometry

For the first time, a scanning flow cytometer (SFC) was applied for the analysis of individual particles using angle-resolved light scattering at two wavelengths simultaneously. The SFC was equipped with three lasers with wavelengths of 405, 444 and 660 nm. The performance of dual-wavelength angle-resolved light-scattering (DWARLS) flow cytometry was demonstrated by the analysis of polymer beads, spherized red blood cells (RBCs) and blood platelets. The main advantage of dual-wavelength flow cytometry relates to an increment in the precision of the inverse light-scattering problem solution. The solution allows one to retrieve the characteristics of individual particles by measuring the light-scattering profiles of these particles. DWARLS has allowed us to measure diameters and refractive indices (RIs) of polystyrene beads with record precision: a median error of 12 nm and a few thousandths for diameter and RI, respectively. Analysis of spherized RBCs with DWARLS flow cytometry has provided record precision in the sizing of spherized RBCs, with a median error of 16 nm for diameter, and an adequate value of the specific refraction increment for hemoglobin. By means of DWARLS, we have formed a numerical criterion for shape separation of blood platelets, which can be described by an oblate spheroid model to increase the precision of the distribution parameters of the platelet fractions.

Spectral approach to recognize spherical particles among non-spherical ones by angle-resolved light scattering

Most of known light-scattering technologies, which allow one to separate spherical from non-spherical single particles, utilize either analysis of 2D light-scattering pattern or depolarization of light scattered. Both approaches force one to use high-sensitive detectors to provide a suitable signal to noise ratio for two-dimensionless photo matrix or for optical system with crossed polarizers. In this study, we introduce the method for discrimination of spherical and non-spherical single particles. The approach is based on measurement of leading, most intensive, element S11 of light-scattering matrix. To provide maximal signal to noise ratio we specified the light-scattering profile (LSP) in terms of integrated over azimuthal angle S11 as a function of polar scattering angle. The shape-sensitive vector-invariant for individual spherical particles was constructed from the parameters of LSP spectrum. The vector-invariant plays a role of the numerical criterion to identify spherical particles from LSPs. It can be applied to find a sphere with characteristics ranging from 16.5 to 70 and from 0.5 to 7.0 for size and phase-shift parameters respectively (size parameter α = πdn0/λ, where d – sphere diameter, λ – wavelength of the incident light, and n0 – medium refractive index, RI, phase-shift parameter ρ = 2α(m − 1), where relative RI m = n/n0 and n is the sphere RI). These ranges cover all possible characteristics of blood cells within the visible region of wavelengths. The ability of the vector-invariant to recognize spherical cells among non-spherical ones was tested theoretically by LSP databases of optical models of platelets and mature red blood cells. Moreover, experimentally the vector-invariant demonstrated good performance in searching of near-perfect spheres among milk fat globules, isolated nuclei of mononuclear cells, and completely spherized cells in a course of red blood cell lysis.

Sensitive detection and estimation of particle non-sphericity from the complex Fourier spectrum of its light-scattering profile

We develop a fast method to estimate the non-sphericity of arbitrary-shaped particles from the complex Fourier spectrum of its light-scattering profile (LSP), measured with the scanning flow cytometer (SFC). We show that previously used amplitude spectrum is not sufficiently sensitive to the non-sphericity and extensively study the phase of the spectral peak for spheroids in the framework of the Rayleigh–Gans–Debye (RGD) approximation. Based on this analysis we construct a new spectral parameter P – the weighted deviation of the complex spectrum around the peak from that for an equivalent sphere determined by the previously published spectral characterization method for spheres (SCMS). We also propose a geometric indicator of non-sphericity η as the relative volume difference from that of the best-fit sphere. These two new parameters apply to particles of arbitrary shape and strongly correlate with each other for rigorously simulated LSPs for spheroids and biconcave disks in a wide range of sizes, refractive indices, and orientations. This correlation is the core of the new method, allowing one to provide both the estimate and the confidence range of η from the experimental value of P. The method is both sensitive and specific to small non-sphericity. For instance, the median error of estimated aspect ratio for simulated LSPs of spheroids is 0.024. We test the resulting algorithm on the real experimental measurements of milk fat globules and red blood cells (RBCs) during the spherization process. These results raise a question about the actual shape of a spherized RBC in the flow inside the SFC. The applicability domain of the method is determined mainly by that of the SCMS and includes biological objects with sizes larger than 7 wavelengths in the liquid host medium. Moreover, we briefly discuss the potential extension of the method to larger refractive indices.

Ultimate peculiarity in angular spectrum enhances the parametric solution of the inverse Mie problem

With this study, we are introducing a new parameter, ultimate spectral peculiarity Lu, to be used in solution of the inverse Mie problem. This parameter is evaluated from a power spectrum of an angle-resolved light-scattering profile (LSP). Direct correlation of single sphere size and Lu allows sizing a homogeneous sphere from 0.6 µm. In order to determine the refractive index (RI) of the sphere we utilized the integral of the LSP within the particular angular range providing the largest uniqueness domain where both size and RI can be unambiguously determined. The integral and Lu were used in the spectral solution of the inverse Mie problem providing the real-time analysis of milk fat globules, spherized red blood cells, polymer and silica beads with the scanning flow cytometer.

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.

Fluorescence-free flow cytometry for measurement of shape index distribution of resting, partially activated, and fully activated platelets.

Whereas commercially available hematological analyzers measure volume of individual platelets, angle-resolved light-scattering provides unique ability to additionally measure their shape index. We utilized the scanning flow cytometer to measure light-scattering profiles (LSPs) of individual platelets taken from 16 healthy donors and the solution of the inverse light-scattering problem to retrieve the volume and shape index of each platelet. In normal conditions, the platelet shape index distribution (PSID) demonstrates three peaks, which relate to resting, partially activated, and fully activated platelets. We developed an algorithm, based on fitting PSID by a sum of three peak functions, to determine the percentage, mean platelet shape index, and distribution width of each platelet fraction. In total, this method gives eight additional parameters of platelet morphology and function to be used in clinical hematological analysis. We also stimulated the platelets with adenosine diphosphate (ADP) and measured the dependence of equilibrium PSID, including the total percentage of activated platelets, on ADP concentration. © 2016 International Society for Advancement of Cytometry.

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.

L-Phenylalanine functionalized silver nanoparticles: Photocatalytic and nonlinear optical applications

An extensive study on the behavior of l-Phenylalanine capped silver nanoparticles (Phe-Ag NPs) in the aqueous phase and in a sol–gel thin film showed different UV/Vis, Transmission Electron Microscope (TEM), Dynamic Light Scattering and Zeta potential profiles. Scanning Electron Microscope (SEM) images of the samples in the sol gel film showed Ag embedded in the SiO2 matrix. Surface Enhanced Raman Spectra (SERS) confirmed that both in the aqueous media and in the sol gel film, the attachment of Phe to the Ag NP surface was through the benzene ring, with the sol–gel film showing a better enhancement. Photocatalytic degradation of crystal violet was measured spectrophotometrically using Phe-Ag NPs as a nanocatalyst under visible light illumination. Intensity-dependent nonlinear optical absorption of Phe-Ag measured using the open aperture Z-scan technique revealed that the material is an efficient optical limiter with potential applications.

Enhanced characterisation of milk fat globules by their size, shape and refractive index with scanning flow cytometry

We present a high-precision method for characterisation of a milk sample by the distributions over milk fat globules (MFG) size, shape, and refractive index (RI). We measured light-scattering profiles of individual MFGs and used global optimisation to retrieve their characteristics. We tested two optical models, a sphere and an oblate spheroid, and found that the latter is a more adequate model for part of the MFGs. We applied the developed method to samples of raw bovine milk and milk from two commercial manufacturers. Diameter and RI of individual MFGs were determined with median errors of 74 nm and 0.0094, respectively, which proves the method to be sensitive to small changes in the MFG properties. Moreover, the distributions over size, surface area, and RI showed a good sensitivity to the details of the milk treatment. In particular, the MFG specific surface area is significantly different for all three milk samples studied.

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.

Glycosylation of Skeletal Calsequestrin

Calsequestrin (CASQ) serves as a major Ca(2+) storage/buffer protein in the sarcoplasmic reticulum (SR). When purified from skeletal muscle, CASQ1 is obtained in its glycosylated form. Here, we have confirmed the specific site and degree of glycosylation of native rabbit CASQ1 and have investigated its effect on critical properties of CASQ by comparison with the non-glycosylated recombinant form. Based on our comparative approach utilizing crystal structures, Ca(2+) binding capacities, analytical ultracentrifugation, and light-scattering profiles of the native and recombinant rabbit CASQ1, we propose a novel and dynamic role for glycosylation in CASQ. CASQ undergoes a unique degree of mannose trimming as it is trafficked from the proximal endoplasmic reticulum to the SR. The major glycoform of CASQ (GlcNAc(2)Man(9)) found in the proximal endoplasmic reticulum can severely hinder formation of the back-to-back interface, potentially preventing premature Ca(2+)-dependent polymerization of CASQ and ensuring its continuous mobility to the SR. Only trimmed glycans can stabilize both front-to-front and the back-to-back interfaces of CASQ through extensive hydrogen bonding and electrostatic interactions. Therefore, the mature glycoform of CASQ (GlcNAc(2)Man(1-4)) within the SR can be retained upon establishing a functional high capacity Ca(2+) binding polymer. In addition, based on the high resolution structures, we propose a molecular mechanism for the catecholaminergic polymorphic ventricular tachycardia (CPVT2) mutation, K206N.

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.

Laser speckle analysis on correlation between gelation and phase separation in aqueous methyl cellulose solutions

AbstractStructure formation by coupling between formation of crosslinking points and liquid–liquid phase separation was investigated for aqueous methyl cellulose solution by small‐angle X‐ray scattering (SAXS) and light scattering (LS) techniques. The sol–gel phase diagram and the SAXS results suggested that the liquid–liquid phase separation occurred before gelation. By LS measurements, the structure due to the liquid–liquid phase separation was directly observed. By applying speckle analysis on the LS profiles, it was suggested that the gelation and the phase separation strongly coupled each other: the increase in the apparent molecular weight by crosslinking induced the liquid–liquid phase separation. © 2009 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 48: 168–174, 2010

Is there a difference between T- and B-lymphocyte morphology?

We characterize T- and B-lymphocytes from several donors, determining cell diameter, ratio of nucleus to cell diameter, and refractive index of the nucleus and cytoplasm for each individual cell. We measure light-scattering profiles with a scanning flow cytometer and invert the signals using a coated sphere as an optical model of the cell and by relying on a global optimization technique. The main difference in morphology of T- and B-lymphocytes is found to be the larger mean diameters of the latter. However, the difference is smaller than the natural biological variability of a single cell. We propose nuclear inhomogeneity as a possible reason for the deviation of measured light-scattering profiles from real lymphocytes from those obtained from the coated sphere model.

The impact of the concentration of casein micelles and whey protein-stabilized fat globules on the rennet-induced gelation of milk

The rennet-induced aggregation of skim milk recombined with whey protein-stabilized emulsion droplets was studied using diffusing wave spectroscopy (DSW) and small deformation rheology. The effect of different volume fractions of casein micelles and fat globules was investigated by observing changes in turbidity (1/ l*), apparent radius, elastic modulus and mean square displacement (MSD), in addition to confocal imaging of the gels. Skim milk containing different concentration of casein micelles showed comparable light-scattering profiles; a higher volume fraction of caseins led to the development of more elastic gels. By following the development of 1/ l* in recombined milks, it was possible to describe the behaviour of the fat globules during the initial stages of rennet coagulation. Increasing the volume fraction of fat globules showed a significant increase in gel elasticity, caused by flocculation of the oil droplets. The presence of flocculated oil globules within the gel structure was confirmed by confocal microscopy observations. Moreover, a lower degree of κ-casein hydrolysis was needed to initiate casein micelles aggregation in milk containing whey protein-stabilized oil droplets compared to skim milk. This study for the first time clearly describes the impact of a mixture of casein micelles and whey protein-stabilized fat globules on the pre-gelation stages of rennet coagulation, and further highlights the importance of the flocculation state of the emulsion droplets in affecting the structure formation of the gel.