Fook Chiong Cheong

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.

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 nanorods 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 nanorods dynamics, from which we estimate the techniques resolution.

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 cameras exposure time. The angular variance of a blurred holograms radial intensity profile yields both the magnitude and direction of a spheres in-plane velocity. At sufficiently low speeds, the same hologram also can be used to characterize other properties, such as the spheres radius and refractive index.

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 aggregates 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.

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 particles 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 particles in turbid media

Holographic particle characterization uses in-line holographic microscopy and the Lorenz-Mie theory of light scattering to measure the diameter and the refractive index of individual colloidal particles in their native dispersions. This wealth of information has proved invaluable in fields as diverse as soft-matter physics, biopharmaceuticals, wastewater management, and food science but so far has been available only for dispersions in transparent media. Here, we demonstrate that holographic characterization can yield precise and accurate results even when the particles of interest are dispersed in turbid media. By elucidating how multiple light scattering contributes to image formation in holographic microscopy, we establish the range conditions under which holographic characterization can reliably probe turbid samples. We validate the technique with measurements on model colloidal spheres dispersed in commercial nanoparticle slurries.

Three-dimensional Nanorod Tracking with Holographic Video Microscopy

We use holographic video microscopy to track the three-dimensional translational and rotational diffusion of copper oxide nanorods suspended in water. Analyzing a video sequence yields measurements of the freely diffusing nanorod’s dynamics.

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 drugs 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.

Label-free Molecular Binding Assays using Holographic Video Microscopy

Inline holographic video microscopy’s resolution for single particle characterization is fine enough to detect avidin coating on biotinylated colloidal surfaces, without requiring staining or fluorescent labeling.

Single frame holographic particle image velocimetry

We present a novel method for single frame particle image velocimetry of micron scale spheres based on holographic video microscopy. Our approach takes advantage of the blurring that recorded holograms suffer when a sphere moves during the exposure period of the camera. By measuring the angular variance in intensity of the blurred hologram, we extract a modelindependent metric for the particle velocity. We find this to be accurate for speeds that permit characterization of other properties of the sphere, such as radius and refractive index through Lorenz-Mie mocroscopy. Singl-frame holographic velocimetry yields information on the dynamics of a particle, without sacrificing any other measurements.