Scanning Flow Cytometer Research Articles

ИССЛЕДОВАНИЕ ВОЗМОЖНОСТИ ИЗМЕРЕНИЯ ГЛИКИРОВАННОГО ГЕМОГЛОБИНА МЕТОДОМ СКАНИРУЮЩЕЙ ПРОТОЧНОЙ ЦИТОМЕТРИИ

The article explores the possibility of measuring glycated hemoglobin in single erythrocytes using scanning flow cytometry. Calculations of the intensity of scattered radiation from an erythrocyte in a wide angular range (light scattering indicatrix) were carried out for wavelengths at the maximum of the absorption band of glycated hemoglobin. The maximum sensitivity for the concentration of glycated hemoglobin was set at a wavelength of 415 nm. As a result, it is possible to reliably separate the concentrations of glycated hemoglobin in donors and patients with diagnosed diabetes with a difference in HbA1c concentrations of more than 5% on the existing practical implementation of a scanning flow cytometer, which includes lasers: 405 nm (30 mW, Radius, Coherent Inc., Santa Clara, USA) and 660 nm (LM–660–20–S, 40 mW). A theoretical calculation for a wavelength of 415 nm showed that a noticeable difference in the value of the scattering intensity (more than 10%) is observed only in the case of a difference in the concentrations of glycated hemoglobin of at least 3%. Thus, if a laser with a wavelength of 415 nm is installed on the existing practical implementation of the device, then the determination of glycated hemoglobin will become possible with an accuracy of no more than 3%.

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.

A microfluidic scanning flow cytometer with superior signal-to-noise-ratio for label-free characterization of small particles

Single-cell analysis without immune-specific labelling is essential across research fields, but conventional flow cytometers (FCMs) struggle with label-free analysis. We introduce a novel microfluidic scanning flow cytometer (μSFC) designed for label-free analysis within a simple microfluidic chip. Our system outperforms traditional FCMs for label-free analysis but its signal-to-noise ratio (SNR) limits the minimum detectable size. We present three modifications to enhance SNR and improve the smallest detectable particle size: additional neutral optical density filtering, a lower noise-equivalent-power photoreceiver, and laser spot size reduction. These improvements enable reliable characterization of particles as small as 3 μm. Experimental results validate the correlation between angular profile oscillations and particle size. While reliable detection down to 1 μm is achieved, further refinement is needed. The simplicity and low setup of the μSFC make it promising for integration into multi-parametric single-cell analysis systems, facilitating comprehensive cellular characterization for diagnostic and point-of-care applications.

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.

Retrieving refractive index of single spheres using the phase spectrum of light-scattering pattern

We analyzed the behavior of the complex Fourier spectrum of the angle-resolved light scattering pattern (LSP) of a sphere in the framework of the Wentzel–Kramers–Brillouin (WKB) approximation. Specifically, we showed that the phase value at the main peak of the amplitude spectrum almost quadratically depends on the particle refractive index, which was confirmed by numerical simulations using both the WKB approximation and the rigorous Lorenz–Mie theory. Based on these results, we constructed a method for characterizing polystyrene beads using the main peak position and the phase value at this point. We tested the method both on noisy synthetic LSPs and on the real data measured with the scanning flow cytometer. In both cases, the spectral method was consistent with the reference non-linear regression one. The former method leads to comparable errors in retrieved particle characteristics but is 300 times faster than the latter one. The only drawback of the spectral method is a limited operational range of particle characteristics that need to be set a priori due to phase wrapping. Thus, its main application niche is fast and precise characterization of spheres with small variation range of characteristics.

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.

A low-cost, label-free microfluidic scanning flow cytometer for high-accuracy quantification of size and refractive index of particles.

Flow cytometers and fluorescence activated cells sorters (FCM/FACS) represent the gold standard for high-throughput single-cell analysis, but their usefulness for label-free applications is limited by the unreliability of forward and side scatter measurements. Scanning flow cytometers represent an appealing alternative, as they exploit measurements of the angle-resolved scattered light to provide accurate and quantitative estimates of cellular properties, but the requirements of current setups are unsuitable for integration with other lab-on-chip technologies or for point-of-care applications. Here we present the first microfluidic scanning flow cytometer (μSFC), able to achieve accurate angle-resolved scattering measurements within a standard polydimethylsiloxane microfluidic chip. The system exploits a low cost linearly variable optical density (OD) filter to reduce the dynamic range of the signal and to increase its signal-to-noise ratio. We present a performance comparison between the μSFC and commercial machines for the label free characterization of polymeric beads with different diameters and refractive indices. In contrast to FCM and FACS, the μSFC yields size estimates linearly correlated with nominal particle sizes (R2 = 0.99) and quantitative estimates of particle refractive indices. The feasibility of using the μSFC for the characterization of biological samples is demonstrated by analyzing a population of monocytes identified based on the morphology of a peripheral blood mononuclear cells sample, which yields values in agreement with the literature. The proposed μSFC combines low setup requirements with high performance, and has great potential for integration within other lab-on-chip systems for multi-parametric cell analysis and for next-generation point-of-care diagnostic applications.

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.

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.

Antibacterial Activity of Positively and Negatively Charged Hematite (α-Fe2O3) Nanoparticles to Escherichia coli, Staphylococcus aureus and Vibrio fischeri.

In the current study, the antibacterial activity of positively and negatively charged spherical hematite (α-Fe2O3) nanoparticles (NPs) with primary size of 45 and 70 nm was evaluated against clinically relevant bacteria Escherichia coli (gram-negative) and Staphylococcus aureus (gram-positive) as well as against naturally bioluminescent bacteria Vibrio fischeri (an ecotoxicological model organism). α-Fe2O3 NPs were synthesized using a simple green hydrothermal method and the surface charge was altered via citrate coating. To minimize the interference of testing environment with NP’s physic-chemical properties, E. coli and S. aureus were exposed to NPs in deionized water for 30 min and 24 h, covering concentrations from 1 to 1000 mg/L. The growth inhibition was evaluated following the postexposure colony-forming ability of bacteria on toxicant-free agar plates. The positively charged α-Fe2O3 at concentrations from 100 mg/L upwards showed inhibitory activity towards E. coli already after 30 min of contact. Extending the exposure to 24 h caused total inhibition of growth at 100 mg/L. Bactericidal activity of positively charged hematite NPs against S. aureus was not observed up to 1000 mg/L. Differently from positively charged hematite NPs, negatively charged citrate-coated α-Fe2O3 NPs did not exhibit any antibacterial activity against E. coli and S. aureus even at 1000 mg/L. Confocal laser scanning microscopy and flow cytometer analysis showed that bacteria were more tightly associated with positively charged α-Fe2O3 NPs than with negatively charged citrate-coated α-Fe2O3 NPs. Moreover, the observed associations were more evident in the case of E. coli than S. aureus, being coherent with the toxicity results. Vibrio fischeri bioluminescence inhibition assays (exposure medium 2% NaCl) and colony forming ability on agar plates showed no (eco)toxicity of α-Fe2O3 (EC50 and MBC > 1000 mg/L).

PEGylated polylysine derived copolymers with reduction‐responsive side chains for anticancer drug delivery

AbstractReduction‐responsive drug delivery systems have recently gained intense attention in intracellular delivery of anticancer drugs. In this study, we developed a PEGylated polypeptide, poly(ethylene glycol)‐block‐poly(ϵ‐propargyloxycarbonyl‐l‐lysine) (PEG113‐b‐PPAL), as a novel clickable substrate for conjugation of reduction‐responsive side chains for antineoplastic drug delivery. PEG113‐b‐PPAL was synthesized through ring‐opening polymerization of alkyne‐containing N‐carboxyanhydride monomers. A designed disulfide‐containing side chain was introduced onto the PEGylated polypeptide by click reaction. The obtained copolymer PEG113‐b‐P(Lys‐DSA) formed micelles by self‐assembly, which exhibited reduction‐responsive behavior under the stimulus of 10 mmol L–1 glutathione (GSH) in water. A small molecule intermediate, compound 2, was used as a model to investigate the thiol reduction mechanism of PEG113‐b‐P(Lys‐DSA) copolymers. The anticancer drug doxorubicin (DOX) was then loaded into the micelles with a drug loading content of 6.73 wt% and a loading efficiency of 40.3%. Both the blank and the drug‐loaded micelles (DOX‐loaded polylysine derived polymeric micelles (LMs/DOX)) adopted a spherical morphology, with average diameters of 48.0 ± 13.1 and 63.8 ± 20.0 nm, respectively. The in vitro drug release results indicated that DOX could be released faster from the micelles by the trigger of GSH in phosphate buffered saline. Confocal laser scanning microscopy and flow cytometer analysis further proved the intracellular delivery of DOX by LMs/DOX and their GSH‐sensitive release behavior. A 3‐(4,5‐dimethyl‐thiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide assay showed that the polymers exhibited negligible cytotoxicity towards normal L929 cells or cancer MCF‐7 cells with a treated concentration up to 1.0 mg mL–1. In conclusion, our synthesized biocompatible and biodegradable PEGylated polypeptides hold great promise for intracellular antineoplastic drug delivery. © 2019 Society of Chemical Industry

Inhibition of proliferation and migration of tumor cells through phenylboronic acid-functionalized polyamidoamine-mediated delivery of a therapeutic DNAzyme Dz13.

BackgroundThe phenylboronic acid-functionalized polyamidoamine (PP) was employed as a gene carrier for Dz13 delivery, inducing an obvious anticancer response.Materials and methodsThe Dz13 condensation ability of PP was evaluated through gel retardation assay. The cellular uptake mechanism of PP/Dz13 nanoparticles was studied using confocal laser scanning microscope and flow cytometer. The inhibition ability of cell proliferation, migration and invasion was investigated through MTT assay, flow cytometry, wound healing and Transwell migration assays, using hepatocarcinoma cell line HepG2 as a model. Finally, Western blotting analysis was used to detect the signaling pathway associated with the inhibition of cell apoptosis and migration induced by Dz13 delivery.ResultsThe carrier PP could efficiently condense Dz13 into stable nanoparticles at mass ratios of >1.5. The hydrodynamic diameter and zeta potential of PP/Dz13 nanoparticles were measured to be 204.77 nm and +22.00 mV at a mass ratio of 10.0, respectively. The nanoparticles could realize an efficient cellular uptake in sialic acid-dependent endocytosis manner. Moreover, the nanoparticles exhibited an obvious antiproliferation effect through the induction of cell apoptosis and cell cycle arrest due to the cleavage of c-Jun mRNA. Besides, the suppression of cell migration and invasion could be achieved after the PP/Dz13 transfection, attributing to the decreased expression level of MMP-2 and MMP-9.ConclusionThe PP provided a potential delivery system to achieve the tumor-targeting gene therapy.

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.

Tailored surface silica nanoparticles for blood-brain barrier penetration: Preparation and in vivo investigation

Surface modified fluorescent silica nanoparticle derivatives (Ru@SNPs), namely, glucose (Glu) and glucose-poly (ethylene glycol) methyl ether amine (Glu-PEG) coated SNPs were designed and tested for their ability to penetrate the blood-brain barrier (BBB) in mice brain. The new obtained nanoparticles were characterized by field emission scanning electron microscope (FE-SEM), dynamic light scattering (DLS) and Fourier transform infrared (FTIR-ATR) analysis. The BBB penetration and distribution of tailored SNPs in mice brain were examined using confocal laser scanning microscopy (CLSM), flow cytometer (FACS) and transmission electron microscopy (TEM). The promising results obtained by in vivo experiments, point out that silica nanoparticle derivatives are an efficient permeable delivery vehicle that are able to cross the BBB and reach the brain tissues via specific and non-specific mechanisms. These findings will enrich the knowledge to rationally engineer multifunctional nanoparticles, and bring new insights into BBB permeability.

The in vitro study of Her-2 targeted gold nanoshell liquid fluorocarbon poly lactic-co-glycolic acid ultrasound microcapsule for ultrasound imaging and breast tumor photothermal therapy

Antibody-mediated targeting therapy has been successful in treating patients with breast cancer by improving the specificity and clinical efficacy. In this study, we constructed the human epidermal growth factor receptor-2 (Her2) antibody-conjugated ultrasound contrast agent with lactic-co-glycolic acid (PLGA) as film forming and perfluorocty bromide (PFOB) as internal material, which was coated by gold nanoshell (Her2-PFOB@PLGA@Au), to realize the integration of diagnosis and treatment. The contrast agent was spherical, with the diameter was 256.8 ± 53.4 nm, and had a good dispersion; Ultrasound imaging experiments in vitro showed that the gold nanoshell polylactic acid microcapsule was suitable for ultrasound contrast imaging with the exquisite and uniform dot intensive high echo. The agent had a great photothermal effect under the near-infrared (NIR) with no obvious biological toxicity for both Her2-positive and negative tumor cells; Moreover, both the results of laser scanning confocal microscope (LSCM) and flow cytometer (FCM) demonstrated the great specificity of Her2-PFOB@PLGA@Au conjugating with Her2 positive breast cancer cells (SKBR3). In conclusion, the successful synthesis of the Her2-PFOB@PLGA@Au microcapsule, offered a new therapeutic strategy of combining diagnosis with therapy for fighting against the breast cancer.

Advanced consumable-free morphological analysis of intact red blood cells by a compact scanning flow cytometer.

Whereas modern automated blood cell analyzers measure the volume of individual red blood cells (RBCs), leading to four RBC indices (mean corpuscular volume, MCV; mean corpuscular hemoglobin, MCH; mean corpuscular hemoglobin concentration, MCHC; red cell distribution width, and RDW), the RBC shape has not been assessed by clinical screening tools. We applied the scanning flow cytometer (SFC) for complete characterization of intact RBC morphology in terms of diameter, maximal and minimal thicknesses, volume, surface area, sphericity index, spontaneous curvature, hemoglobin concentration, and content. The above-mentioned individual RBC characteristics were measured without fluorescent markers and other chemicals by a SFC equipped only with 660 nm laser for RBC illumination and single detector for measurement of angle-resolved light scattering. The distributions over all RBC characteristics were constructed and processed statistically to form the novel 31 RBC indices for 22 donor samples. Our results confirm the possibility of precise, label-free, enhanced morphological analysis of individual intact RBCs with compact single-detector flow cytometer. Detailed characterization of RBCs with high statistics and precision can be used to increase the value of screening examinations and to reveal pathologies accompanied by abnormality of RBC shape. © 2017 International Society for Advancement of Cytometry.

Spectral solution of the inverse Mie problem

We developed a fast method to determine size and refractive index of homogeneous spheres from the power Fourier spectrum of their light-scattering patterns (LSPs), measured with the scanning flow cytometer. Specifically, we used two spectral parameters: the location of the non-zero peak and zero-frequency amplitude, and numerically inverted the map from the space of particle characteristics (size and refractive index) to the space of spectral parameters. The latter parameters can be reliably resolved only for particle size parameter greater than 11, and the inversion is unique only in the limited range of refractive index with upper limit between 1.1 and 1.25 (relative to the medium) depending on the size parameter and particular definition of uniqueness. The developed method was tested on two experimental samples, milk fat globules and spherized red blood cells, and resulted in accuracy not worse than the reference method based on the least-square fit of the LSP with the Mie theory. Moreover, for particles with significant deviation from the spherical shape the spectral method was much closer to the Mie-fit result than the estimated uncertainty of the latter. The spectral method also showed adequate results for synthetic LSPs of spheroids with aspect ratios up to 1.4. Overall, we present a general framework, which can be used to construct an inverse algorithm for any other experimental signals.

Synthesis, characterization, and in vitro evaluation of targeted gold nanoshelled poly(d,l-lactide-co-glycolide) nanoparticles carrying anti p53 antibody as a theranostic agent for ultrasound contrast imaging and photothermal therapy

Breast cancer is the leading cause of cancer-related deaths in women and earlier detection can substantially reduce deaths from breast cancer. Polymers with targeted ligands are widely used in the field of molecular ultrasound imaging and targeted tumor therapy. In our study, the nanotheranostic agent was fabricated through filling perfluoropropane (C3F8) into poly(d,l-lactic-co-glycolic acid) nanoparticles (PLGA NPs), followed by the formation of gold nanoshell on the surface, then conjugated with anti p53 antibody which has high specificity with the p53 protein overexpressing in breast cancer. The average diameter of the gold nanoshelled PLGA NPs carrying anti p53 antibody (p53-PLGA@Au NPs) was 247 ± 108.2 nm. The p53-PLGA@Au NPs had well-defined spherical morphology and hollow interiors observed by electron microscope, and had a good photothermal effect under the irradiation of an 808 nm laser. The results of laser scanning confocal microscope (LSCM) and flow cytometer (FCM) indicated the specific targeting of p53-PLGA@Au NPs conjugating with breast cancer MCF-7 cells overexpressing p53 protein in vitro. Also the ultrasound imaging experiments in vitro showed that p53-PLGA@Au NPs were suitable for ultrasound contrast imaging. In conclusion, the p53-PLGA@Au NPs are demonstrated to be novel targeted UCAs and may have potential applications in the early diagnosis and targeted near-infrared (NIR) photothermal therapy of breast cancer in the future.

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.