Holographic video microscopy is a non-invasive, non-destructive optical technique for individual particle characterization.
Holographic videos of individual spheres are analyzed pixel by pixel, using Lorenz-Mie theory of light scattering, resulting in the particle’s 3D position, its radius, and its refractive index.
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 stream. 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).
Our 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 colloidal spheres scatter a small proportion of the incident beam, and the scattered light interferes with the unscattered portion in the focal plane of the microscope's objective lens. The microscope magnifies the interference pattern and projects it onto the face of a low-noise scientific video camera, with a total system magnification. The video stream is recorded as uncompressed digital video with a digital video recorder for subsequent analysis with our own proprietary approaches.
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.