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DIGITAL HOLOGRAPHIC MICROSCOPY

Principles

DHM (Garcia-Sucerquia et al., 2006; Schnars and Juptner, 2002) is based on a technique originally outlined by Denis Gabor in 1949. A simple, in-line holographic microscope (Figure 1) consists of a laser, a camera, and an objective lens. Laser light scatters off structures in a microscopic sample, and the camera images the interference pattern between the scattered and unscattered light. The image is called a hologram.

To see how this technique can lead to a three-dimensional image, consider a coherent plane wave scattering off an idealized point particle. This particle represents anything that scatters light in the microscopic sample (e.g., a nanoparticle in solution or a subcellular structure). If we place a screen directly in front of the particle, we can see a fringe pattern that arises from interference between the scattered wave and the unscattered portion of the plane wave. This interference pattern has concentric circular fringes of varying intensity. The number and diameter of the fringes varies with the distance of the screen from the particle, and therefore, the interference pattern tells us about the location of the particle in all three dimensions.

FIGURE 1 Top left: Hologram formation from a point source showing a plane reference wave interfering with a spherical scattered wave to produce (center, calculated) a Gabor zone plate pattern. Right, calculated: A set of point scatterers yields the coherent superposition of zone plates. Bottom center: Diagram of in-line holographic instrument (bottom left); image of hologram from 1 μm diameter polystyrene particle in water.

FIGURE 1 Top left: Hologram formation from a point source showing a plane reference wave interfering with a spherical scattered wave to produce (center, calculated) a Gabor zone plate pattern. Right, calculated: A set of point scatterers yields the coherent superposition of zone plates. Bottom center: Diagram of in-line holographic instrument (bottom left); image of hologram from 1 μm diameter polystyrene particle in water.



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