Light-scattering gating and characterization of plasma microparticles

Abstract

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.

Chylomicrons against light scattering: The battle for characterization

“…It measures angle‐resolved light scattering in a form of the LSP for individual particles using a 405‐nm laser (30 mW, Radius, Coherent Inc., Santa Clara, California), which beams parallel to the cell flow, and the original light collection system. The 488‐nm laser (15 mW laser, FCD488‐020, JDS Uniphase Corporation, Milpitas, California) is used to produce forward (FSC) and side scattering signals (SSC) in a conventional manner . The operational angular ranges in which SFC measures light‐scattering information (angle‐resolved LSP, FSC and SSC) were determined for each experiment from analysis of 2 μm PS microspheres for the LSP and analysis of a mixture of different PS microspheres for the FSC and SSC, as described previously in , respectively.…”

“…The remaining part of detected plasma particles, including CMs and, possibly, cell‐derived microvesicles, seem to be mostly spherical . We applied the Mie theory to fit their LSPs, FSC and SSC amplitudes as described in . Thus, we identify and characterize individual CMs in platelet‐rich plasma by their spherical shape, size, and RI from the solution of ILS problem and construct distribution of CM population over these characteristics (see Figures and ).…”

“…The characterization is performed through the solution of the inverse light‐scattering (ILS) problem, given an appropriate particle shape model. This was successfully demonstrated for different blood cells and, recently, for microparticles in blood plasma . In the latter studies the LSP measurements were combined with conventional forward and side scattering (FSC and SSC), resulting in sub‐diffraction precision of microparticle sizing (about 20 nm) and a few thousandths precision in RI estimates.…”

“…In the latter studies the LSP measurements were combined with conventional forward and side scattering (FSC and SSC), resulting in sub‐diffraction precision of microparticle sizing (about 20 nm) and a few thousandths precision in RI estimates. Part of the measured microparticles was actually CMs , but this classification has not been discussed in details.…”

Single‐Particle Characterization by Elastic Light Scattering

“…[ 91 ] Later the SFC was improved by measuring polarized LSPs to detect particle non‐sphericity [ 92 ] and by simultaneous measurements of forward and side scattering for extremely precise characterization of homogeneous spheres. [ 93 ]…”