Monitoring polysorbate 80 degradation in protein solutions using Total Holographic Characterization.

The degradation of polysorbate surfactants can limit the shelf life of biologic pharmaceutical products. Polysorbate is susceptible to degradation via either oxidation or hydrolysis pathways which releases free fatty acids (FFA) and other complex polymers. Degradants from Polysorbate 80 (PS80) can form particles and impact drug product quality. PS80 degradation products appear at low concentrations, and their refractive indexes are similar to that of the buffer, making them very challenging to detect. Furthermore, aggregates of FFA are similar in size and refractive index to protein aggregates adding complexity to characterizing these particles in protein solutions. Total Holographic Characterization (THC) is used in this work to characterize FFA particles of oleic acid and linoleic acid, the two most common degradation products of PS80. We demonstrate that the characteristic THC profile of the FFA oleic acid emulsion droplets can be used to monitor the degradation of PS80. THC can detect oleic acid at a concentration down to less than 100 ng/mL. Using the characteristic THC signal of oleic acid as a marker, the degradation of PS80 in protein solutions can be monitored quantitatively even in the presence of other contaminants of the same size, including silicone oil emulsion droplets and protein aggregates.

Label-free viability assay using in-line holographic video microscopy.

Total holographic characterization (THC) is presented here as an efficient, automated, label-free method of accurately identifying cell viability. THC is a single-particle characterization technology that determines the size and index of refraction of individual particles using the Lorenz-Mie theory of light scattering. Although assessment of cell viability is a challenge in many applications, including biologics manufacturing, traditional approaches often include unreliable labeling with dyes and/or time consuming methods of manually counting cells. In this work we measured the viability of Saccharomyces cerevisiae yeast in the presence of various concentrations of isopropanol as a function of time. All THC measurements were performed in the native environment of the sample with no dilution or addition of labels. Holographic measurements were made with an in-line holographic microscope using a 40[Formula: see text] objective lens with plane wave illumination. We compared our results with THC to manual counting of living and dead cells as distinguished with trypan blue dye. Our findings demonstrate that THC can effectively distinguish living and dead yeast cells by the index of refraction of individual cells.

An Interlaboratory Comparison on the Characterization of a Sub-micrometer Polydisperse Particle Dispersion.

The measurement of polydisperse protein aggregates and particles in biotherapeutics remains a challenge, especially for particles with diameters of ≈ 1 µm and below (sub-micrometer). This paper describes an interlaboratory comparison with the goal of assessing the measurement variability for the characterization of a sub-micrometer polydisperse particle dispersion composed of five sub-populations of poly(methyl methacrylate) (PMMA) and silica beads. The study included 20 participating laboratories from industry, academia, and government, and a variety of state-of-the-art particle-counting instruments. The received datasets were organized by instrument class to enable comparison of intralaboratory and interlaboratory performance. The main findings included high variability between datasets from different laboratories, with coefficients of variation from 13 % to 189 %. Intralaboratory variability was, on average, 37 % of the interlaboratory variability for an instrument class and particle sub-population. Drop-offs at either end of the size range and poor agreement on maximum counts of particle sub-populations were noted. The mean distributions from an instrument class, however, showed the size-coverage range for that class. The study shows that a polydisperse sample can be used to assess performance capabilities of an instrument set-up (including hardware, software, and user settings) and provides guidance for the development of polydisperse reference materials.

Holographic characterization and tracking of colloidal dimers in the effective-sphere approximation.

An in-line hologram of a colloidal sphere can be analyzed with the Lorenz-Mie theory of light scattering to measure the sphere's three-dimensional position with nanometer-scale precision while also measuring its diameter and refractive index with part-per-thousand precision. Applying the same technique to aspherical or inhomogeneous particles yields measurements of the position, diameter and refractive index of an effective sphere that represents an average over the particle's geometry and composition. This effective-sphere interpretation has been applied successfully to porous, dimpled and coated spheres, as well as to fractal clusters of nanoparticles, all of whose inhomogeneities appear on length scales smaller than the wavelength of light. Here, we combine numerical and experimental studies to investigate effective-sphere characterization of symmetric dimers of micrometer-scale spheres, a class of aspherical objects that appear commonly in real-world dispersions. Our studies demonstrate that the effective-sphere interpretation usefully distinguishes small colloidal clusters in holographic characterization studies of monodisperse colloidal spheres. The effective-sphere estimate for a dimer's axial position closely follows the ground truth for its center of mass. Trends in the effective-sphere diameter and refractive index, furthermore, can be used to measure a dimer's three-dimensional orientation. When applied to colloidal dimers transported in a Poiseuille flow, the estimated orientation distribution is consistent with expectations for Brownian particles undergoing Jeffery orbits.

Quantitative Differentiation of Protein Aggregates From Other Subvisible Particles in Viscous Mixtures Through Holographic Characterization.

We demonstrate the use of holographic video microscopy to detect individual subvisible particles dispersed in biopharmaceutical formulations and to differentiate them based on material characteristics measured from their holograms. The result of holographic analysis is a precise and accurate measurement of the concentrations and size distributions of multiple classes of subvisible contaminants dispersed in the same product simultaneously. We demonstrate this analytical technique through measurements on model systems consisting of human IgG aggregates in the presence of common contaminants such as silicone oil emulsion droplets and fatty acids. Holographic video microscopy also clearly identifies metal particles and air bubbles. Being able to differentiate and characterize the individual components of such heterogeneous dispersions provides a basis for tracking other factors that influence the stability of protein formulations including handling and degradation of surfactant and other excipients.

The role of the medium in the effective-sphere interpretation of holographic particle characterization data.

The in-line hologram of a micrometer-scale colloidal sphere can be analyzed with the Lorenz-Mie theory of light scattering to obtain precise measurements of the sphere's diameter and refractive index. The same technique also can be used to characterize porous and irregularly shaped colloidal particles provided that the extracted parameters are interpreted with effective-medium theory to represent the properties of an equivalent effective sphere. Here, we demonstrate that the effective-sphere model consistently accounts for changes in the refractive index of the medium as it fills the pores of porous particles and therefore yields quantitative information about such particles' structure and composition. In addition to the sample-averaged porosity, holographic perfusion porosimetry gauges the polydispersity of the porosity. We demonstrate these capabilities through measurements on mesoporous spheres, fractal protein aggregates and irregular nanoparticle agglomerates, all of which are noteworthy for their industrial significance.

Holographic Characterization of Protein Aggregates in the Presence of Silicone Oil and Surfactants.

Characterizing protein aggregates in the presence of silicone oil is a long standing challenge for the pharmaceutical industry. Silicone oil is often used as a lubricant in devices that deliver and store therapeutic protein products and has been linked to protein aggregation, which can compromise a drug's effectiveness or cause autoimmune responses in patients. Most traditional technologies cannot quantitatively distinguish protein aggregates and silicone oil in their native formulations for sizes less than 5 µm. We use holographic video microscopy to study protein aggregation to demonstrate its capability to quantitatively distinguish protein aggregates and silicone oil in the presence of varying amounts of the surfactants SDS and polysorbate 80 in the size range of 0.5-10 µm without the need for dilution or special sample preparation. We show that SDS denatures proteins and stabilizes silicone oil. We also show that polysorbate 80 may limit protein aggregate formation if it is added to an IgG solution before introducing silicone oil.

Lifting degeneracy in holographic characterization of colloidal particles using multi-color imaging.

Micrometer sized particles can be accurately characterized using holographic video microscopy and Lorenz-Mie fitting. In this work, we explore some of the limitations in holographic microscopy and introduce methods for increasing the accuracy of this technique with the use of multiple wavelengths of laser illumination. Large high index particle holograms have near degenerate solutions that can confuse standard fitting algorithms. Using a model based on diffraction from a phase disk, we explain the source of these degeneracies. We introduce multiple color holography as an effective approach to distinguish between degenerate solutions and provide improved accuracy for the holographic analysis of sub-visible colloidal particles.

Holographic characterization of contaminants in water: Differentiation of suspended particles in heterogeneous dispersions.

Determining the size distribution and composition of particles suspended in water can be challenging in heterogeneous multicomponent samples. Light scattering techniques can measure the distribution of particle sizes, but provide no basis for distinguishing different types of particles. Direct imaging techniques can categorize particles by shape, but offer few insights into their composition. Holographic characterization meets this need by directly measuring the size, refractive index, and three-dimensional position of individual particles in a suspension. The ability to measure an individual colloidal particle's refractive index is a unique capability of holographic characterization. Holographic characterization is fast enough, moreover, to build up population distribution data in real time, and to track time variations in the concentrations of different dispersed populations of particles. We demonstrate these capabilities using a model system consisting of polystyrene microbeads co-dispersed with bacteria in an oil-in-water emulsion. We also demonstrate how the holographic fingerprint of different contaminants can contribute to identifying their source.

Holographic characterization of colloidal fractal aggregates.

In-line holographic microscopy images of micrometer-scale fractal aggregates can be interpreted with an effective-sphere model to obtain each aggregate's size and the population-averaged fractal dimension. We demonstrate this technique experimentally using model fractal clusters of polystyrene nanoparticles and fractal protein aggregates composed of bovine serum albumin and bovine pancreas insulin.

Rapid, high-throughput tracking of bacterial motility in 3D via phase-contrast holographic video microscopy.

Tracking fast-swimming bacteria in three dimensions can be extremely challenging with current optical techniques and a microscopic approach that can rapidly acquire volumetric information is required. Here, we introduce phase-contrast holographic video microscopy as a solution for the simultaneous tracking of multiple fast moving cells in three dimensions. This technique uses interference patterns formed between the scattered and the incident field to infer the three-dimensional (3D) position and size of bacteria. Using this optical approach, motility dynamics of multiple bacteria in three dimensions, such as speed and turn angles, can be obtained within minutes. We demonstrated the feasibility of this method by effectively tracking multiple bacteria species, including Escherichia coli, Agrobacterium tumefaciens, and Pseudomonas aeruginosa. In addition, we combined our fast 3D imaging technique with a microfluidic device to present an example of a drug/chemical assay to study effects on bacterial motility.

Holographic deconvolution microscopy for high-resolution particle tracking.

Rayleigh-Sommerfeld back-propagation can be used to reconstruct the three-dimensional light field responsible for the recorded intensity in an in-line hologram. Deconvolving the volumetric reconstruction with an optimal kernel derived from the Rayleigh-Sommerfeld propagator itself emphasizes the objects responsible for the scattering pattern while suppressing both the propagating light and also such artifacts as the twin image. Bright features in the deconvolved volume may be identified with such objects as colloidal spheres and nanorods. Tracking their thermally-driven Brownian motion through multiple holographic video images provides estimates of the tracking resolution, which approaches 1 nm in all three dimensions.

Holographic particle-streak velocimetry.

We present a way to measure the positions and instantaneous velocities of micrometer-scale colloidal spheres using a single holographic snapshot obtained through in-line holographic video microscopy. This method builds on previous quantitative analyses of colloidal holograms by accounting for blurring that occurs as a sphere moves during the camera's exposure time. The angular variance of a blurred hologram's radial intensity profile yields both the magnitude and direction of a sphere's in-plane velocity. At sufficiently low speeds, the same hologram also can be used to characterize other properties, such as the sphere's radius and refractive index.

Strategies for three-dimensional particle tracking with holographic video microscopy.

The video stream captured by an in-line holographic microscope can be analyzed on a frame-by-frame basis to track individual colloidal particles' three-dimensional motions with nanometer resolution. In this work, we compare the performance of two complementary analysis techniques, one based on fitting to the exact Lorenz-Mie theory and the other based on phenomenological interpretation of the scattered light field reconstructed with Rayleigh-Sommerfeld back-propagation. Although Lorenz-Mie tracking provides more information and is inherently more precise, Rayleigh-Sommerfeld reconstruction is faster and more general. The two techniques agree quantitatively on colloidal spheres' in-plane positions. Their systematic differences in axial tracking can be explained in terms of the illuminated objects' light scattering properties.

Rotational and translational diffusion of copper oxide nanorods measured with holographic video microscopy.

We use holographic video microscopy to track the three-imensional translational and rotational diffusion of copper oxide nanorods suspended in water. Rayleigh-Sommerfeld back-propagation of a single holographic snapshot yields a volumetric reconstruction of the nanorod's optical scattering pattern, from which we estimate both its dimensions and also its instantaneous position and orientation. Analyzing a video sequence yields measurements of the freely diffusing nanorod's dynamics, from which we estimate the technique's resolution.

Flow visualization and flow cytometry with holographic video microscopy.

The video stream captured by an in-line holographic microscope can be analyzed on a frame-by-frame basis to track individual colloidal particles' three-dimensional motions with nanometer resolution, and simultaneously to measure their sizes and refractive indexes. Through a combination of hardware acceleration and software optimization, this analysis can be carried out in near real time with off-the-shelf instrumentation. An efficient particle identification algorithm automates initial position estimation with sufficient accuracy to enable unattended holographic tracking and characterization. This technique's resolution for particle size is fine enough to detect molecular-scale coatings on the surfaces of colloidal spheres, without requiring staining or fluorescent labeling. We demonstrate this approach to label-free holographic flow cytometry by detecting the binding of avidin to biotinylated polystyrene spheres.

Switchable self-protected attractions in DNA-functionalized colloids.

Surface functionalization with DNA is a powerful tool for guiding the self-assembly of nanometre- and micrometre-sized particles. Complementary 'sticky ends' form specific inter-particle links and reproducibly bind at low temperature and unbind at high temperature. Surprisingly, the ability of single-stranded DNA to form folded secondary structures has not been explored for controlling (nano) colloidal assembly processes, despite its frequent use in DNA nanotechnology. Here, we show how loop and hairpin formation in the DNA coatings of micrometre-sized particles gives us in situ control over the inter-particle binding strength and association kinetics. We can finely tune and even switch off the attractions between particles, rendering them inert unless they are heated or held together--like a nano-contact glue. The novel kinetic control offered by the switchable self-protected attractions is explained with a simple quantitative model that emphasizes the competition between intra- and inter-particle hybridization, and the practical utility is demonstrated by the assembly of designer clusters in concentrated suspensions. With self-protection, both the suspension and assembly product are stable, whereas conventional attractive colloids would quickly aggregate. This remarkable functionality makes our self-protected colloids a novel material that greatly extends the utility of DNA-functionalized systems, enabling more versatile, multi-stage assembly approaches.

Multicolored carbon nanotubes: decorating patterned carbon nanotube microstructures with quantum dots.

In this work, techniques to create patterned array of multiwalled nanotube (MWNT) microstructures decorated with quantum dots (QDs) were presented. Using aligned array of intertwined MWNTs as the supporting template, a droplet of solution comprising QDs was deposited onto the MWNTs. When the solution evaporated away, QDs were left behind on the MWNT template. Coupled with the technique of laser pruning, a wide variety of QDs-decorated MWNT microstructures were created. In addition, the aligned array of MWNTs was found to be an effective nanosieve that could effectively sort out QDs with a size difference of approximately 0.5 nm. In this case, a droplet of solution comprising QDs of different sizes was placed on aligned array of MWNTs. As the solution spread across as well as trickled down the MWNTs, the smaller QDs were found to venture further and deeper into the MWNTs. Again coupled with laser pruning, fluorescence microscopy revealed multicolored MWNT microstructures due to preferential decoration of these QDs with difference sizes. As a result, multicolored/multicomponents hybrid functional materials were achieved.

Size selective assembly of colloidal particles on a template by directed self-assembly technique.

We report a simple and effective approach to organize micron- and submicron-sized particles in a size selective manner. This approach utilizes the template assisted directed self-assembly technique. A topographically patterned photoresist surface is fabricated and used to create an ordered array of colloidal particles from their aqueous suspensions. Assembly of particles on this template is then achieved by using a conventional spin coating technique. Feasibility of this technique to form a large area of patterned particle assemblies has been investigated. To arrange the particles on the template, the physical confinement offered by the surface topography must overcome a joint effect of centrifugal force and the hydrophobic nature of the photoresist surface. This concept has been extended to the size selective sorting of colloidal particles. The capability of this technique for sorting and organizing colloidal particles of a particular diameter from a mixture of microspheres is demonstrated.