Principle

Highly refractive structures bend light to a much greater angle than do structures of low refractive index. The same properties that cause the light to bend also delay the passage of light by a quarter of a wavelength or so. In a light microscope in bright field mode, light from highly refractive structures bends farther away from the center of the lens than light from less refractive structures and arrives about a quarter of a wavelength out of phase. Light from most objects passes through the center of the lens as well as to the periphery. Now if the light from an object to the edges of the objective lens is retarded a half wavelength and the light to the center is not retarded at all, then the light rays are out of phase by a half wavelength. They cancel each other when the objective lens brings the image into focus. A reduction in brightness of the object is observed. The degree of reduction in brightness depends on the refractive index of the object.

Applications for phase contrast microscopy

Phase contrast is preferable to bright field microscopy when high magnifications (400x, 1000x) are needed and the specimen is colorless or the details so fine that color does not show up well. Cilia and flagella, for example, are nearly invisible in bright field but show up in sharp contrast in phase contrast. Amoebae look like vague outlines in bright field, but show a great deal of detail in phase. Most living microscopic organisms are much more obvious in phase contrast.

Figure. (a) organelles are nearly invisible in bright field although they have different refractive indexes; (b) light is bent and retarded more by objects with a high refractive index; (c) in phase contrast a phase plate is placed in the light path. Barely refracted light passes through the center of the plate and is not retarded. Highly refracted light passes through the plate farther from center and is held back another one quarter wavelength.; (d) The microscope field shows a darker background (in this case the cell cytoplasm has a higher refractive index than the contractile vacuole), with the organelles in sharp contrast.

Using phase contrast

Phase contrast condensers and objective lenses add considerable cost to a microscope, and so phase contrast is often not used in teaching labs except perhaps in classes in the health professions and in some university undergraduate programs. This is unfortunate since the images obtainable in phase contrast mode can be very dramatic. To use phase contrast the light path must be aligned. An element in the condenser is aligned with an element in a specialized phase contrast lens. This usually involves sliding a component into the light path or rotating a condenser turret. The elements are either lined up in a fixed position or are adjusted by the observer until the phase effect is optimized. Generally, more light is needed for phase contrast than for corresponding bright field viewing, since the technique is based on a diminishment of brightness of most objects.

Phase contrast microscopy, first described in 1934 by Dutch physicist Frits Zernike, is a contrastenhancing optical technique that can be utilized to produce high-contrast images of transparent specimens, such as living cells (usually in culture), microorganisms, thin tissue slices, lithographic patterns, fibers, latex dispersions, glass fragments, and subcellular particles (including nuclei and other organelles). In effect, the phase contrast technique employs an optical mechanism to translate minute variations in phase into corresponding changes in amplitude, which can be visualized as differences in image contrast. One of the major advantages of phase contrast microscopy is that living cells can be examined in their natural state without previously being killed, fixed, and stained. As a result, the dynamics of ongoing biological processes can be observed and recorded in high contrast with sharp clarity of minute specimen detail. Presented in Figure 1 is a cut-away diagram of a modern upright phase contrast microscope, including a schematic illustration of the phase contrast optical train. Partially coherent illumination produced by the tungsten-halogen lamp is directed through a collector lens and focused on a specialized annulus (labeled condenser annulus) positioned in the substage condenser front focal plane. Wavefronts passing through the annulus illuminate the specimen and either pass through undeviated or are diffracted and retarded in phase by structures and phase gradients present in the specimen. Undeviated and diffracted light collected by the objective is segregated at the rear focal plane by aphase plate and focused at the intermediate image plane to form the final phase contrast image observed in the eyepieces. Prior to the invention of phase contrast techniques, transmitted brightfield illumination was one of the most commonly utilized observation modes in optical microscopy, especially for fixed, stained specimens or other types of samples having high natural absorption of visible light. Collectively, specimens readily imaged with brightfield illumination are termedamplitude objects (or specimens) because the amplitude or intensity of the illuminating wavefronts is reduced when light passes through the specimen. The addition of phase contrast optical accessories to a standard brightfield microscope can be employed as a technique to render a contrast-enhancing effect in transparent specimens that is reminiscent of optical staining (see Figure 2). Light waves that are diffracted and shifted in phase by the specimen (termed a phase object) can be transformed by phase contrast into amplitude differences that are observable in the eyepieces. Large, extended specimens are also easily

visualized with phase contrast optics due to diffraction and scattering phenomena that occur at the edges of these objects. The performance of modern phase contrast microscopes is so refined that it enables specimens containing very small internal structures, or even just a few protein molecules, to be detected when the technology is coupled to electronic enhancement and post-acquisition image processing. Presented in Figure 2 is a comparison of living cells in culture imaged in both brightfield and phase contrast illumination. The cells are human glial brain tissue grown in monolayer culture bathed with a nutrient medium containing amino acids, vitamins, mineral salts, and fetal calf serum. In brightfield illumination (Figure 2(a)), the cells appear semi-transparent with only highly refractive regions, such as the membrane, nucleus, and unattached cells (rounded or spherical), being visible. When observed using phase contrast optical accessories, the same field of view reveals significantly more structural detail (Figure 2(b)). Cellular attachments become discernable, as does much of the internal structure. In addition, the contrast range is dramatically improved.