Scattered Light Imaging: Resolving the substructure of nerve fiber crossings in whole brain sections with micrometer resolution.

For developing a detailed network model of the brain based on image reconstructions, it is necessary to spatially resolve crossing nerve fibers. The accuracy hereby depends on many factors, including the spatial resolution of the imaging technique. 3D Polarized Light Imaging (3D-PLI) allows the three-dimensional reconstruction of nerve fiber tracts in whole brain sections with micrometer in-plane resolution, but leaves uncertainties in pixels containing crossing fibers. Here we introduce Scattered Light Imaging (SLI) to resolve the substructure of nerve fiber crossings. The measurement is performed on the same unstained histological brain sections as in 3D-PLI. By illuminating the brain sections from different angles and measuring the transmitted (scattered) light under normal incidence, light intensity profiles are obtained that are characteristic for the underlying brain tissue structure. We have developed a fully automated evaluation of the intensity profiles, allowing the user to extract various characteristics, like the individual directions of in-plane crossing nerve fibers, for each image pixel at once. We validate the reconstructed nerve fiber directions against results from previous simulation studies, scatterometry measurements, and fiber directions obtained from 3D-PLI. We demonstrate in different brain samples (human optic tracts, vervet monkey brain, rat brain) that the 2D fiber directions can be reliably reconstructed for up to three crossing nerve fiber bundles in each image pixel with an in-plane resolution of up to 6.5 µm. We show that SLI also yields reliable fiber directions in brain regions with low 3D-PLI signals coming from regions with a low density of myelinated nerve fibers or out-of-plane fibers. This makes Scattered Light Imaging a promising new imaging technique, providing crucial information about the organization of crossing nerve fibers in the brain.

Understanding Nanomedicine Size and Biological Response Dependency: What Is the Relevance of Previous Relationships Established on Only Batch-Mode DLS-Measured Sizes?

PURPOSE: Most relationships between size and nanomedicine performance and safety were established before the early 2010s' when batch-mode dynamic light scattering (batch-mode DLS) was the only easy size measurement method for colloids available. They are basis for the rational design of nanomedicines, but misunderstood contrasting results are reported. This work aimed to investigate whether these relationships can be used with confidence knowing that batch-mode DLS can be tricky when measuring sizes of polydisperse systems. METHODS: A polydisperse dispersion of polymer nanoparticles ranging from 100 to 465 nm was synthesized. The particles were separated in 4 fractions by successive centrifugations. The capacity of each fraction and parent dispersion to activate the complement system was evaluated by Crossed immuno-electrophoresis. RESULTS: Each fraction was a population of particles with a distinct size. It showed a different capacity to activate the complement system. Particles of the fractions showing the strongest capacity to activate the complement systems had a different size evaluated by batch-mode DLS then that of the parent particles. CONCLUSION: Particles activating the complement system in the parent dispersion were not those that were detected by batch-mode DLS while measuring its size. This work pointed out that previously established relationships between nanomedicine size and their biological response should be taken with caution if sizes were only measured by batch-mode DLS.

The NISTmAb Reference Material 8671 value assignment, homogeneity, and stability.

The NISTmAb Reference Material (RM) 8671 is intended to be an industry standard monoclonal antibody for pre-competitive harmonization of best practices and designing next generation characterization technologies for identity, quality, and stability testing. It must therefore embody the quality and characteristics of a typical biopharmaceutical product and be available long-term in a stable format with consistent product quality attributes. A stratified sampling and analysis plan using a series of qualified analytical and biophysical methods is described that assures RM 8671 meets these criteria. Results for the first three lots of RM 8671 highlight the consistency of material attributes with respect to size, charge, and identity. RM 8671 was verified to be homogeneous both within and between vialing lots, demonstrating the robustness of the lifecycle management plan. It was analyzed in concert with the in-house primary sample 8670 (PS 8670) to provide a historical link to this seminal material. RM 8671 was verified to be fit for its intended purpose as a technology innovation tool, external system suitability control, and cross-industry harmonization platform. Graphical abstract The NISTmAb Reference Material (RM) 8671 is intended to be an industry standard monoclonal antibody for pre-competitive harmonization of best practices and designing next generation characterization technologies for identity, quality, and stability testing.

Development of orthogonal NISTmAb size heterogeneity control methods.

The NISTmAb is a monoclonal antibody Reference Material from the National Institute of Standards and Technology; it is a class-representative IgG1κ intended to serve as a pre-competitive platform for harmonization and technology development in the biopharmaceutical industry. The publication series of which this paper is a part describes NIST's overall control strategy to ensure NISTmAb quality and availability over its lifecycle. In this paper, the development of a control strategy for monitoring NISTmAb size heterogeneity is described. Optimization and qualification of size heterogeneity measurement spanning a broad size range are described, including capillary electrophoresis-sodium dodecyl sulfate (CE-SDS), size exclusion chromatography (SEC), dynamic light scattering (DLS), and flow imaging analysis. This paper is intended to provide relevant details of NIST's size heterogeneity control strategy to facilitate implementation of the NISTmAb as a test molecule in the end user's laboratory. Graphical abstract Representative size exclusion chromatogram of the NIST monoclonal antibody (NISTmAb). The NISTmAb is a publicly available research tool intended to facilitate advancement of biopharmaceutical analytics. HMW = high molecular weight (trimer and dimer), LMW = low molecular weight (2 fragment peaks). Peak labeled buffer is void volume of the column from L-histidine background buffer.

Evaluation of Miniature Dynamic Light Scattering Technology for the Assessment of Hemodynamic Status During Graded Hemorrhage and Retransfusion in Pigs.

BACKGROUND: Hemorrhagic shock with occult hypoperfusion is a key challenge to prehospital staff during triage and transfer of patients, especially during mass casualty incidents. Recent advances in Dynamic Light Scattering (DLS), and miniaturization of this technology, has resulted in noninvasive sensors capable of continuously monitoring tissue perfusion. This study evaluated the ability of miniature DLS (mDLS) sensors to assess hemodynamic status in a porcine model of hemorrhage. METHODS: Following ethics committee approval, anesthetized and ventilated pigs underwent graded hemorrhage and then retransfusion. Standard vital signs were monitored in conjunction with a thermodilution cardiac output (CO), central venous pressure (CVP), and arterial blood gases. The mDLS sensor was attached to each animal's leg and all monitoring measurements were taken 5 minutes after completion of each period of hemorrhage and retransfusion to allow equilibration. RESULTS: All measured parameters changed during bleeding and retransfusion. During bleeding; p value were 0.011 for heart rate, 0.07 for CVP, <0.001 for both mean arterial pressure, and mDLS. During retransfusion; p values were 0.023 for heart rate, 0.008 for CVP, and <0.001 for both mean arterial pressure and mDLS. Pearson correlation between changes in mDLS and CO demonstrated r value of 0.917 during hemorrhage and 0.965 during retransfusion. Changes in hemoglobin were not statistically significant during bleeding (p = 0.331) but were during retransfusion (p = 0.0001). Changes of bicarbonate, base excess, and lactate were found to be statistically significant during both phases of the experiment (p = 0.001). CONCLUSIONS: In an animal model of hemorrhagic shock, the mDLS sensor strongly correlates with traditional measures of CO. This initial assessment supports further investigation of this technology in human studies.

Measuring proteins with greater speed and resolution while reducing sample size.

A multi-angle light scattering (MALS) system, combined with chromatographic separation, directly measures the absolute molar mass, size and concentration of the eluate species. The measurement of these crucial properties in solution is essential in basic macromolecular characterization and all research and production stages of bio-therapeutic products. We developed a new MALS methodology that has overcome the long-standing, stubborn barrier to microliter-scale peak volumes and achieved the highest resolution and signal-to-noise performance of any MALS measurement. The novel design simultaneously facilitates online dynamic light scattering (DLS) measurements. As National Institute of Standards and Technology (NIST) new protein standard reference material (SRM 8671) is becoming the benchmark molecule against which many biomolecular analytical techniques are assessed and evaluated, we present its measurement results as a demonstration of the unique capability of our system to swiftly resolve and measure sharp (20~25 µL full-width-half-maximum) chromatography peaks. Precise measurements of protein mass and size can be accomplished 10 times faster than before with improved resolution. In the meantime the sample amount required for such measurements is reduced commensurately. These abilities will have far-reaching impacts at every stage of the development and production of biologics and bio-therapeutic formulations.

Development and validation of a HILIC-ELSD method for simultaneous analysis of non-substituted and acetylated xylo-oligosaccharides.

A new HILIC-ELSD method was developed for compositional analysis of both xylo-oligosaccharides (XOS) with degree of polymerization (DP) from 2 to 8 and acetylated XOS with DP from 3 to 8. The method was carried out on a zwitterionic HILIC column using ELSD as a detector. The influences of mobile phase composition, column temperature and flow rate on the retention time and resolution of XOS were investigated. An excellent separation result was achieved with a linear gradient elution of 75%-50% acetonitrile in 30min, at a flow rate of 1mL/min and the column temperature at 35°C. In addition, LC-ESI-MS was employed to determine the structural information of X7, X8 and acetylated XOS. The proposed method was simple, reliable, and no derivatization procedure was needed. It is suitable for compositional analysis and quality control of XOS.

Investigation of the binding of AuNPs-6-mercaptopurine and the sensitive detection of 6-mercaptopurine using resonance Rayleigh light scattering.

A highly sensitive method for the detection of 6-mercaptopurine (MP) by resonance Rayleigh light scattering (RLS) method was developed. Gold nanoparticles (AuNPs) were synthesized by a modified seed method and characterized using transmission electron microscopy (TEM). AuNPs were bound to MP via covalent bonding to form the MP-AuNPs complex, which increased the RLS intensity of MP at 347 nm (increased by 65.7%). Under optimum conditions, the magnitude of the enhanced RLS intensity for MP-AuNPs was proportional to MP concentration in the range 0.0681-1.702 µg mL-1 . The linear regression equation was represented as follows: ΔIRLS = 9.31 + 82.42c (r = 0.9948). The limit of detection (LOD, 3σ) was 3.32 ng mL-1 . The system was applied successfully to detect MP in pharmaceuticals. MP recoveries were 99.9-101.7% with a relative standard deviation (RSD) (n = 5) of 0.59-0.77% for three synthetic samples, and 97.5-110.0% with an RSD of 0.98-2.10% (n = 5) for tablet samples.

Statistical Optimization of Evaporative Light Scattering Detection for Molten Sucrose Octaacetate and Comparison With Ultraviolet Diode Array Detection Validation Parameters Using Tandem HPLC Ultraviolet Diode Array Detection/Evaporative Light Scattering Detection-Specific Stability-Indicating Method.

A sucrose octaacetate (SOA) gradient HPLC evaporative light scattering detection (ELSD) and low-wavelength UV-diode array detection (UV-DAD)-specific stability-indicating method development and validation comparison is reported. A central composite response surface design and multicriteria optimization was used to maximize molten SOA area-under-the-curve response and signal-to-noise ratio. The ELSD data were also analyzed using multivariate principal component analysis, analysis of variance, and standard least squares effects modeling. The method suitability and validation parameters of both methods were compared. To the authors' knowledge, this is the first report that validates an ELSD method using a molten analyte. SOA exhibited a low molar absorptivity of 439 absorption units/cm/M in water at 210 nm requiring low-wavelength UV-DAD detection. The low-wavelength UV-DAD method provided substantially better intraday and interday precision, intraday and interday goodness-of-fit, detection limit, and quantitation limit than ELSD. ELSD exhibited a 60-fold greater area-under-the-curve response, better resolution, and 58% more theoretical plates. On balance, the UV-DAD method was chosen for SOA chemical kinetic studies. This study illustrates that ELSD may not always be the best alternative to gradient HPLC low-wavelength UV detection.

Optical focusing deep inside dynamic scattering media with near-infrared time-reversed ultrasonically encoded (TRUE) light.

Focusing light deep inside living tissue has not been achieved despite its promise to play a central role in biomedical imaging, optical manipulation and therapy. To address this challenge, internal-guide-star-based wavefront engineering techniques--for example, time-reversed ultrasonically encoded (TRUE) optical focusing--were developed. The speeds of these techniques, however, were limited to no greater than 1 Hz, preventing them from in vivo applications. Here we improve the speed of optical focusing deep inside scattering media by two orders of magnitude, and focus diffuse light inside a dynamic scattering medium having a speckle correlation time as short as 5.6 ms, typical of living tissue. By imaging a target, we demonstrate the first focusing of diffuse light inside a dynamic scattering medium containing living tissue. Since the achieved focusing speed approaches the tissue decorrelation rate, this work is an important step towards in vivo deep tissue noninvasive optical imaging, optogenetics and photodynamic therapy.