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Light Microscopy: Comparison of Optics

Author(s): David R. Caprette, PhD

Phase Contrast Microscopy

There is too little difference in transparency or color among most intracellular structures to enable them to be seen in a bright field light microscope. However, structures such as cell nuclei, cytoplasm, contractile vacuoles, cilia, and flagella differ markedly in refractive index. A phase contrast microscope causes images of such structures to differ in brightness, producing high contrast with similar resolution to that of a bright field instrument.
    
Light passing through an object with high refractive index is scattered so that the light path is longer than for objects that do not refract light as much. Scattered light also loses some velocity when traveling through the object. The maximum difference in path length between biological structures of low versus high refractive index typically is 1/4 of a wavelength. A phase contrast lens contains an element, called a "phase plate," that is matched to an annular diaphragm in the condenser. A hollow cone of light passes through the diaphragm, the condenser lens, and then through the specimen. Light that is not significantly scattered by the specimen passes through the central part of the phase plate, which does not alter the properties of the light. Highly scattered light passes through a ring in the phase plate with high refractive index, causing the light to lose another 1/4 wavelength.
    
The image comes to a focus at the back of the objective lens, where the altered light is recombined with the unobstructed light. Light that has been retarded a full 1/2 wavelength is completely out of phase with the unobstructed light, causing interference that shows up as a dark spot. Unscattered light recombines in phase and thus shows up as a bright spot. By centering the condenser so that the annular diaphragm is concentric with the phase plate, one can optimize the dynamic range (i.e., obtain maximum contrast in brightness among parts of a specimen with varying indexes of refraction). Organelles that are nearly or completely invisible in bright field often are strikingly apparent in phase contrast.