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Holographic Video Microscopy

Total Holographic Characterization® is based on holographic video microscopy, a non-invasive, non-destructive optical technique for individual particle characterization.

Holograms of particles are captured in videos and then analyzed pixel by pixel, using Lorenz-Mie theory of light scattering, resulting in the particle’s 3D position, its diameter, and its refractive index. The symmetry of the hologram reflects the symmetry of the particle. In this example, the particles are spheres. Non-spherical particles will have holograms that are not circularly symmetric. Deviations from circular symmetry provides additional information about the morphology of the particles.

This video shows holographic video microscopy data of a micrometer-diameter colloidal silica sphere diffusing in water as it sediments under gravity. The holographic image is fit to predictions of the Lorenz-Mie theory of light scattering, which yields the sphere's three-dimensional position with nanometer precision in each holographic snapshot. The animated plot shows the particle's trajectory reconstructed from the video. The experimental technique used to create this video is described in "Characterizing and tracking single colloidal particles with video holographic microscopy," S.-H. Lee, Y. Roichman, G.-R. Yi, S.-H. Kim, S.-M. Yang, A. van Blaaderen, P. van Oostrum and D. G. Grier, Optics Express 15, 18275-18282 (2007).

2 scientists using xSight
Our xSight, an in-line holographic video microscope, shown in use at Spheryx on the right and schematically in the Figure below, is based on an inverted microscope design outfitted with a high numerical aperture microscope objective. The conventional incandescent illumination is replaced with the collimated coherent beam from a gas/solid-state/diode laser.

Individual micrometer-scale colloidal spheres scatter a small proportion of the incident beam. The scattered light interferes with the unscattered portion and the light is collected by the microscope's objective lens. The microscope magnifies the interference pattern and projects it onto the face of a low-noise scientific video camera. The video stream is recorded as video with a digital video recorder for subsequent analysis with our own proprietary algorithms.
sketch of imaging process in xSight



In-line holographic video microscope.

A collimated laser beam illuminates the sample. Light scattered by the sample interferes with the unscattered portion of the beam in the focal plane of the objective lens. The interference pattern is magnified, recorded and then fit to predictions of Lorenz-Mie theory to obtain measurements of the particle's position, its size, and its refractive index.

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