Optimization of Flow Imaging Microscopy Setting Using Spherical Beads with Optical Properties Similar to Those of Biopharmaceuticals.

Flow imaging microscopy (FIM) is widely used to characterize biopharmaceutical subvisible particles (SVPs). The segmentation threshold, which defines the boundary between the particle and the background based on pixel intensity, should be properly set for accurate SVP quantification. However, segmentation thresholds are often subjectively and empirically set, potentially leading to variations in measurements across instruments and operators. In the present study, we developed an objective method to optimize the FIM segmentation threshold using poly(methyl methacrylate) (PMMA) beads with a refractive index similar to that of biomolecules. Among several candidate particles that were evaluated, 2.5-µm PMMA beads were the most reliable in size and number, suggesting that the PMMA bead size analyzed by FIM could objectively be used to determine the segmentation threshold for SVP measurements. The PMMA bead concentrations measured by FIM were highly consistent with the indicative concentrations, whereas the PMMA bead size analyzed by FIM decreased with increasing segmentation threshold. The optimal segmentation threshold where the analyzed size was closest to the indicative size differed between an instrument with a black-and-white camera and that with a color camera. Inter-instrument differences in SVP concentrations in acid-stressed recombinant adeno-associated virus (AAV) and protein aggregates were successfully minimized by setting an optimized segmentation threshold specific to the instrument. These results reveal that PMMA beads can aid in determining a more appropriate segmentation threshold to evaluate biopharmaceutical SVPs using FIM.

Characterization of the Volume-based or Number-based Size Distribution for Silica Nanoparticles Using a Unique Combination of Online Dynamic Light Scattering Having a Uni-tau Multi-bit Correlator and High-resolution Centrifugal Field-Flow Fractionation Separator.

This paper presents a study of the size distributions of colloidal nanoparticles using an online dynamic light scattering (DLS) unit with a uni-tau multi-bit correlator (UMC) combined with a centrifugal field-flow fractionation (CF3) separator. Conventionally, the FFF-UV-MALS system utilizing field-flow fractionation (FFF) combined with a UV detector and multi-angle light scattering instrument (MALS) could be used to obtain the particle size distribution of colloidal nanoparticles. Lately, DLS as a technique to measure the size distributions of colloid materials has become prevalent. However, the DLS instrument will practically measure only the large particles in a multi-modal particle mixture. Therefore, the CF3-DLS w/UMC system that was developed consisted of a CF3 unit connected to an online DLS instrument with UMC. The system could measure the volume- or number-based size distribution with highly quantitative and accurate histograms for multi-modal samples. The size distributions were validated with size distributions obtained by images of an atomic force microscope (AFM). Two types of colloidal silica nanoparticles with different distribution widths were used in this study.

Determination of the Rayleigh Ratio with an Uncertainty Analysis by Static Light-Scattering Measurements of Certified Reference Materials for Molecular Weight.

The most important parameter for light-scattering measurements in the Rayleigh scattering region is the Rayleigh ratio, which is necessary to obtain the absolute scattered light intensity from the relative scattered light intensity. The absolute scattered light intensity is directly related to the molar masses of polymers, colloids, biomolecules, and the like. A new Rayleigh ratio was determined by measuring static light scattering from certified reference materials with highly accurate certified values of the molecular weight determined by several other techniques, such as MALDI-TOF mass spectrometry or size-exclusion chromatography. The new Rayleigh ratio can be used for evaluating the uncertainty of the molecular weight of polymers and macromolecules, as measured by light scattering.

Interlaboratory comparison of nanoparticle size measurements between NMIJ and NIST using two different types of dynamic light scattering instruments.

The question of how to relate particle sizes measured using a fixed-angle dynamic light scattering (DLS) instrument with those measured using a multi-angle DLS instrument is addressed. A series of nearly monodisperse polystyrene latex (PSL) particles with nominal diameters of 100 nm, 70 nm, 50 nm, and 30 nm were measured using two different types of DLS instruments: one owned by the National Metrology Institute of Japan (NMIJ) of the multi-angle type and the other owned by the National Institute of Standards and Technology (NIST) of the fixed-angle type. The mean particle size of the PSL particles was measured using the multi-angle-type instrument at various scattering angles and at various concentrations of particle suspension. These data were used to establish the functional dependence of the measured particle size on the scattering angle and particle concentration through the least-squares fitting method. The established function was then used to predict the mean particle sizes that would have been obtained if the same scattering angle and particle concentrations as those used at NIST had been selected at NMIJ. The mean particle sizes obtained at NIST and at NMIJ agreed quite well for all four PSL particle samples after compensating for the angle and concentration differences. The result of this study clearly demonstrates that consideration for the dependence of measured particle sizes on the scattering angle and particle concentration is crucial in intra-method comparisons of mean particle sizes obtained using DLS.

Simultaneous determination of the size and concentration of fine bubbles in water by laser-light scattering.

The preparation of nanoscale fine bubbles in water is an innovative technology, but no precise method for simultaneously measuring the size and concentration of such bubbles had previously been developed. We have developed a method for simultaneously determining the size and concentration of fine bubbles in water by a light-scattering technique. Dynamic light scattering gives the diffusion constant and particle size of fine bubbles, whereas static light scattering provides their concentration or molar mass. Static light scattering also provides the radius of gyration of the bubbles, thereby providing a means for validating measurements of the sizes of the fine bubbles.

Polymer analysis by supercritical fluid chromatography.

Synthetic polymers always have distributions of the degrees of polymerization. Supercritical fluid chromatography (SFC) is almost only one technique to be able to separate pure polymer that has exactly unique degree of polymerization. For typical examples of SFC separation of polymers, four kinds of polymers: polystyrene, poly(ethylene glycol), some nonionic surfactants, and polyprenol are described about the conditions of SFC. Separated pure polymers which have no molecular weight distribution, are called uniform polymer. Uniform polymers are really useful to calibrate or validate various detectors. Here, the applications of calibrating ultraviolet detector, evaporative light scattering detector, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry are summarized. These detectors have strong molecular weight dependence of their sensitivity coefficient, in contrast to the good linearity of sample concentration.

Accurate Size and Size-Distribution Determination of Polystyrene Latex Nanoparticles in Aqueous Medium Using Dynamic Light Scattering and Asymmetrical Flow Field Flow Fractionation with Multi-Angle Light Scattering.

Accurate determination of the intensity-average diameter of polystyrene latex (PS-latex) by dynamic light scattering (DLS) was carried out through extrapolation of both the concentration of PS-latex and the observed scattering angle. Intensity-average diameter and size distribution were reliably determined by asymmetric flow field flow fractionation (AFFFF) using multi-angle light scattering (MALS) with consideration of band broadening in AFFFF separation. The intensity-average diameter determined by DLS and AFFFF-MALS agreed well within the estimated uncertainties, although the size distribution of PS-latex determined by DLS was less reliable in comparison with that determined by AFFFF-MALS.

Development of a standard method for nanoparticle sizing by using the angular dependence of dynamic light scattering.

A standard method for nanoparticle sizing based on the angular dependence of dynamic light scattering was developed. The dependences of the diffusion coefficients for aqueous suspensions of polystyrene latex on the concentration and scattering angle were accurately measured by using a high-resolution dynamic light-scattering instrument. Precise measurements of the short-time correlation function at seven scattering angles and five concentrations were made for suspensions of polystyrene latex particles with diameters from 30 to 100 nm. The apparent diffusion coefficients obtained at various angles and concentrations showed properties characteristic of polystyrene latex particles with electrostatic interactions. A simulation was used to calculate a dynamic structure factor representing the long-range interactions between particles. Extrapolations to infinite dilution and to low angles gave accurate particle sizes by eliminating the effects of long-range interactions. The resulting particle sizes were consistent with those measured by using a differential mobility analyzer and those obtained by pulsed-field gradient nuclear magnetic resonance measurements.

Photochemistry of linear-shaped phenylacetylenyl- and (phenylacetylenyl)phenylacetylenyl-substituted aromatic enediynes.

The series of linear-shaped phenylacetylenyl- and (phenylacetylenyl)phenylacetylenyl-substituted aromatic enediynes 1-3 were synthesized as pure trans and cis isomers and their photochemistry explored. With expansion of the π-electron system, the absorption spectra red-shifted and the molar extinction coefficients dramatically increased up to 122000 M(-1) cm(-1) for trans-3. The absorption spectra of cis-2 and cis-3 consisted of two independent absorption bands. The fluorescence quantum yields of the molecules were high, even for the cis isomers (Φ(f) = 0.39-0.61). The fluorescence decay of each of the compounds was analyzed as a single exponential and the wavelength dependence of time constants was not observed, indicating a single emitting state in all cases. All isomers exhibited mutual cis-trans photoisomerization. The quantum yield of both trans-to-cis and cis-to-trans photoisomerization considerably decreased in 2 and 3, presumably due to an increased number of photochemical processes that yield nonreactive excited species and which result in nonradiative deactivation. Three energy minima exist in the excited triplet state, where the energy of planar conformation decreased with the extension of the phenyl acetylenyl chain, resulting in the promotion of nonradiative processes without conformational change.

Characterization of fullerene colloidal suspension in a cell culture medium for in vitro toxicity assessment.

To elucidate important parameters for in vitro toxicity assessment of C(60) and C(70) fullerene colloidal particles, experiments were carried out in culture medium using pulsed field gradient nuclear magnetic resonance (PFG-NMR), asymmetrical flow field-flow fractionation (AFFFF), and dynamic light scattering (DLS) methods. First, the amounts of total and bulk bovine serum albumin (BSA) molecules in C(60) and C(70) fullerene colloidal suspensions were determined using the PFG-NMR and AFFFF methods. Because the amount of bulk BSA molecules in the cell culture medium is a significant factor in inducing cell growth and because BSA can strongly adsorb onto the fullerene particles, this value is an important parameter for toxicological assessment. It was found that most of the BSA molecules are freely diffusing for both fullerene colloidal suspensions, at least in the range of fullerene concentration from 0.0025-0.15 mg mL(-1). Second, structural analysis of the fullerene colloidal nanoparticles was successfully performed using AFFFF-multi angle light scattering (MALS) and DLS methods. Based on the observed light scattering profiles obtained from a narrow size distribution of colloidal particles collected after AFFFF separation, it was estimated that the fullerene colloidal nanoparticles of both C(60) and C(70) did not adopt a hard spherical structure in the culture medium. The results from combined analysis using the AFFFF-MALS and DLS methods also supported this conclusion and indicated that the fullerene colloidal particles adopted a more flexible structure in culture medium. Since carbon nanomaterials with different geometric structures exhibit quite different cytotoxicity and bioactivity, these results are important for in vitro toxicity assessment.

Dispersion characteristics of various metal oxide secondary nanoparticles in culture medium for in vitro toxicology assessment.

The aim of this study is to characterize the dispersion characteristics of various metal oxide nanoparticles and secondary nanoparticle formation in culture medium. Many studies have already investigated the in vitro toxicities of various metal oxide nanoparticles; however, there have been few discussions about the particle transport mode to cells during a period of toxicity assessment. The particle transport mode would strongly affect the amount of uptake by cells; therefore, estimation of the transport mode for various metal oxide particles is important. Fourteen different metal oxide nanoparticle dispersions in a culture medium were examined. The sizes of the secondary nanoparticles were observed to be larger than 100 nm by dynamic light scattering (DLS). According to Stokes law and the Stokes-Einstein assumption, pure metal oxide particles with such sizes should gravitationally settle faster than diffusion processes; however, the secondary metal oxide particles examined in this study exhibited unexpectedly slower gravitational settling rates. The slow gravitational settling kinetics of particles was estimated to be caused by the inclusion of protein into the secondary nanoparticles, which resulted in lower densities than the pure metal oxide particles. The ratios of metal oxide to protein in secondary particles could be affected by the protein adsorption ability of the corresponding metal oxide primary particles. To the best of our knowledge, it was clarified for the first time that stably dispersed secondary metal oxide nanoparticles with slow gravitational settling kinetics are induced by secondary nanoparticles consisting of small amounts of metal oxide particles and large amounts of protein, which results in lower particle densities than the pure metal oxide particles. The estimation of particle dynamics in culture medium using this method would be significant to recognize the inherent toxicity of nanoparticles.

Evaluation of a condensation nucleation light scattering detector for the analysis of synthetic polymer by supercritical fluid chromatography.

A condensation nucleation light scattering detector (CNLSD) was adapted for use as a detector for supercritical fluid chromatography. The performance of the CNLSD was evaluated and compared to evaporative light-scattering detector (ELSD) using a well-defined equimass mixture of uniform poly(ethylene glycol) oligomers and a certified reference material of poly(ethylene glycol) 1000. The CNLSD was able to detect a 10 times less concentrated solution of uniform oligomers compared to the ELSD. The quantitativeness of CNLSD was high enough to obtain the molecular mass distribution of poly(ethylene glycol) 1000 without any calibrations; on the other hand, the original data measured by ELSD was about 4% smaller than the certified value of poly(ethylene glycol) 1000. The CNLSD was suitable for supercritical fluid chromatography as a mass concentration detector.

Size effect on UV-Vis absorption properties of colloidal C(60) particles in water.

An obvious size effect of colloidal C(60) particles on UV-Vis absorption spectra was found after size fractionation of colloidal C(60) particles in water using the Field Flow Fractionation method.

Reliable size determination of nanoparticles using dynamic light scattering method for in vitro toxicology assessment.

Dynamic light scattering (DLS) is widely used for the evaluation of the particle size in the toxicity assessment of nanoparticles. However, the many types of DLS instruments and analytical procedures sometimes give different apparent sizes of particles and make it complicated to understand the size dependence on particles for the toxicity assay. In this study, we established an evaluation method of secondary nanoparticle sizes using a DLS analysis. First, we established a practical method for determining size with an appropriate evaluation of uncertainties. This proposed method could be a universal protocol for toxicity assessment that would allow researchers to achieve some degree of concordance on the size of nanoparticles for an assessment. Second, we investigated the processes associated with particles in suspension by examining the changes in the size and the light scattering intensity of secondary nanoparticles during an in vitro toxicity assessment, since the transport mode of particles to cells is significant in understanding in vitro nano-toxicity. In this study, these two points were investigated on TiO(2) nanoparticles suspension as an example. The secondary particles of TiO(2) with a light scattering intensity-averaged diameter (d(l)) of 150-250 nm were characterized with appropriate uncertainties. The sizes were found to be comparable with values determined using other analytical procedures and other instruments. It is suggested that d(l) could be an effective size parameter for toxicity assessments. Furthermore, TiO(2) secondary nanoparticle suspensions are well dispersed with slow gravity settling, no agglomeration, with the diffusion process as the primary transport mode of particles to cells.

Quantitative comparison of a corona-charged aerosol detector and an evaporative light-scattering detector for the analysis of a synthetic polymer by supercritical fluid chromatography.

A corona-charged aerosol detector (CAD) was developed to improve the sensitivity, reproducibility and quantitativeness of detection as compared to evaporative light-scattering detector (ELSD) for liquid chromatography. Our laboratory used the corona CAD as a detector for supercritical fluid chromatography (SFC) and evaluated its performance compared to the ELSD by using a certified reference material of poly(ethylene glycol) (PEG) and a well-defined equimass mixture of uniform PEG oligomers. The corona CAD was able to detect a 10 times more dilute solution of uniform oligomers compared to the ELSD. Although the original data of molecular mass by ELSD was 4.6% smaller than the certified value of PEG 1000, molecular mass distribution obtained by corona CAD was virtually almost the same as the certified value without any calibrations.

Certification and uncertainty evaluation of the certified reference materials of poly(ethylene glycol) for molecular mass fractions by using supercritical fluid chromatography.

Poly(ethylene glycol) (PEG) is a useful water-soluble polymer that has attracted considerable interest in medical and biological science applications as well as in polymer physics. Through the use of a well-calibrated evaporative light-scattering detector coupled with high performance supercritical fluid chromatography, we are able to determine exactly not only the average mass but also all of the molecular mass fractions of PEG samples needed for certified reference materials issued by the National Metrology Institute of Japan. In addition, experimental uncertainty was determined in accordance with the Guide to the expression of uncertainty in measurement (GUM). This reference material can be used to calibrate measuring instruments, to control measurement precision, and to confirm the validity of measurement methods when determining molecular mass distributions and average molecular masses. Especially, it is suitable for calibration against both masses and intensities for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Calibration of an evaporative light-scattering detector as a mass detector for supercritical fluid chromatography by using uniform Poly(ethylene glycol) oligomers.

The quantitativeness of an evaporative light-scattering detector (ELSD) for supercritical fluid chromatography (SFC) was evaluated by using an equimass mixture of uniform poly(ethylene glycol) (PEG) oligomers. Uniform oligomers, in which all molecules have an identical molecular mass, are useful for the accurate calibration of detectors. We calibrated the SFC-ELSD system for various concentrations and molecular masses by using an equimass mixture of PEG oligomers. ELSD not only showed a good linear response to the injected concentration over a wide concentration range, from 10(-4) to 10(-1)g/mL, but also showed a strong dependence on the molecular mass of the solute. By using chromatograms of the equimass mixture of uniform oligomers to calibrate SFC-ELSD, it was possible to determine exact values of not only the average mass but also the molecular-mass distribution for a PEG 1540 sample. The average molecular mass was shifted to a higher value by several percentage points after calibration of the ELSD.