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Holographic particle characterization treats holographic microscopy of colloidal particles as an inverse problem whose solution yields the diameter, refractive index and three-dimensional position of each particle in the field of view, all with exquisite precision. This rich source of information on the composition and dynamics of colloidal dispersions has created new opportunities for fundamental research in soft-matter physics, statistical physics and physical chemistry, and has been adopted for product development, quality assurance and process control in industrial applications. Aberrations introduced by real-world imaging conditions, however, can degrade performance by causing systematic and correlated errors in the estimated parameters. We identify a previously overlooked source of spherical aberration as a significant source of these errors. Modeling aberration-induced distortions with an operator-based formalism identifies a spatially varying phase factor that approximately compensates for spherical aberration in recorded holograms. Measurements on model colloidal dispersions demonstrate that phase-only aberration compensation greatly improves the accuracy of holographic particle characterization without significantly affecting measurement speed for high-throughput applications. 

The intensity distribution of a holographically-projected optical trap can be tailored to the physical properties of the particles it is intended to trap. Dynamic optimization is especially desirable for manipulating dark-seeking particles that are repelled by conventional optical tweezers, and even more so when dark-seeking particles coexist in the same system as light-seeking particles. We address the need for dexterous manipulation of dark-seeking particles by introducing a class of “dark” traps created from the superposition of two out-of-phase Gaussian modes with different waist diameters. Interference in the difference-of-Gaussians (DoG) trap creates a dark central core that is completely surrounded by light and therefore can trap dark-seeking particles rigidly in three dimensions. DoG traps can be combined with conventional optical tweezers and other types of traps for use in heterogeneous samples. The ideal hologram for a DoG trap being purely real-valued, we introduce a general method based on the Zernike phase-contrast principle to project real-valued holograms with the phase-only diffractive optical elements used in standard holographic optical trapping systems. We demonstrate the capabilities of DoG traps (and Zernike holograms) through experimental studies on high-index, low-index and absorbing colloidal particles dispersed in fluid media. 

Holographic molecular binding assays use holographic video microscopy to directly detect molecules binding to the surfaces of micrometer-scale colloidal beads by monitoring associated changes in the beads’ light-scattering properties. Holograms of individual spheres are analyzed by fitting to a generative model based on the Lorenz-Mie theory of light scattering. Each fit yields an estimate of a probe bead’s diameter and refractive index with sufficient precision to watch a population of beads grow as molecules bind. Rather than modeling the molecular-scale coating, however, these fits use effective medium theory, treating the coated sphere as if it were homogeneous. This effective-sphere analysis is rapid and numerically robust and so is useful for practical implementations of label-free immunoassays. Here, we assess how measured effective-sphere properties reflect the actual properties of molecular-scale coatings by modeling coated spheres with the discrete-dipole approximation and analyzing their holograms with the effective-sphere model. 

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.