The Phase-Contrast Microscopes

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This diagram of a phase-contrast microscope illustrates phase differences between light passing through the object and background. These differences are produced by passing the rays through different parts of a phase plate. The light rays are superimposed in the image plane, producing contrast due to their interference.

Source: OpenStax Microbiology

OpenStax Microbiology

Phase-contrast microscopes use refraction and interference caused by structures in a specimen to create highcontrast, high-resolution images without staining. It is the oldest and simplest type of microscope that creates an image by altering the wavelengths of light rays passing through the specimen. To create altered wavelength paths, an annular stop is used in the condenser. The annular stop produces a hollow cone of light that is focused on the specimen before reaching the objective lens. The objective contains a phase plate containing a phase ring. As a result, light traveling directly from the illuminator passes through the phase ring while light refracted or reflected by the specimen passes through the plate. This causes waves traveling through the ring to be about one-half of a wavelength out of phase with those passing through the plate. Because waves have peaks and troughs, they can add together (if in phase together) or cancel each other out (if out of phase). When the wavelengths are out of phase, wave troughs will cancel out wave peaks, which is called destructive interference. Structures that refract light then appear dark against a bright background of only unrefracted light. More generally, structures that differ in features such as refractive index will differ in levels of darkness.

Because it increases contrast without requiring stains, phase-contrast microscopy is often used to observe live specimens. Certain structures, such as organelles in eukaryotic cells and endospores in prokaryotic cells, are especially well visualized with phase-contrast microscopy.

This figure compares a brightfield image (left) with a phase-contrast image (right) of the same unstained simple squamous epithelial cells. The cells are in the center and bottom right of each photograph (the irregular item above the cells is acellular debris). Notice that the unstained cells in the brightfield image are almost invisible against the background, whereas the cells in the phase-contrast image appear to glow against the background, revealing far more detail.

Source: OpenStax Microbiology

Source:

Parker, N., Schneegurt, M., Thi Tu, A.-H., Forster, B. M., & Lister, P. (n.d.). Microbiology. Houston, Texas: OpenStax. Access for free at: https://openstax.org/details/books/microbiology

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