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CORNELL BIOPL 4440 - Study Notes

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CHAPTER 10. Differential Interference Contrast (DIC) MicroscopyCHAPTER 10. Differential Interference Contrast (DIC) MicroscopyIn the last chapter I discussed the Mach-Zehnder interferometer-based Leitz interference microscope in which two beams, one produced by a point in the specimen and one produced by a coherent reference beam, interfere at the image plane to produce a single interference image. I also discussed image duplication interference microscopes, where two coherent and laterally separated images of a given point in the object are formed in the image plane. The two coherent and laterally separated image points are produced by an interferometer, which is a combination of a beam splitter and a beam recombiner. Here I will discuss differential interference contrast microscopy (DIC) which also employs an interferometer. However, in a differential interference contrast microscope, the two images of a given point in a specimen are laterally displaced a distance that is smaller than the resolving power of the objective lens, and thus the two images of the point overlap and interfere in the image plane. By producing two coherent and laterally displaced overlapping images, a differential interference contrast microscope is able to convert gr adients in the optical path lengths of nearby points in the object into variations in light intensity. Steep gradients in optical path lengths appear very bright or very dark, while areas that have a uniform optical path length appear gray. A differential interference contrast microscope can also be used to change gradients in optical path lengths in transparent objects into spectacular differences in color. Differential interference contrast microscopes have been designed and developed by F. Smith (Patents from 1947-1950, first published in 1955), M. Francon (1952,1953,1957), G. Nomarski (1955), and H. Beyer (1967,1971) and G. Schöppe (1987). Most differential interference contrast microscopes utilize birefringent prisms to split and recombine the beams. By contrast, the Zeiss Jena interference microscope uses a Mach-Zehnder type interferometer. In this ingenious design, the separation of the beams can be controlled so that two completely separate images are formed. Because of this, the microscope can be used as a quantitative image duplication interference microscope. Additionally, if the beams are separated a distance less than the minimum resolvable distance of the lens, the images overlap and the microscope can be used qualitatively as a differential interference contrast microscope.Before I discuss the design of a differential interference contrast microscope, I would like to show that gradients in the optical path length are equivalent to the derivative of the optical path length.While a differential interference contrast microscope can be considered to be a microscope that gives the derivative of the optical path length of a specimen, an image duplication interference microscope can be considered to be a microscope that gives the integral of the optical path length.1. Design of a Transmitted Light Differential Interference Contrast Microscope.The polarizing microscope, the image duplication interference microscope based on polarized light,and the differential interference microscope based on polarized light have a certain degree of 276similarity (Lang, 1968; Allen et al., 1969). In a polarizing microscope, a single beam of linearly polarized light that is produced by a polarizer passes through a birefringent specimen that is oriented so that its slow axis is at a 45° angle relative to the azimuth of the polarizer. Thus the single beam of incident light is split into two beams that vibrate orthogonally to each other and are not or barely displaced laterally relative to each other. The two beams that leave the specimen are recombined in the second prism and turned into elliptically polarized light which passes through theanalyzer in the crossed position. The optical path difference between the two beams, which is the cause of the elliptically polarized light that leaves the specimen, is due to the difference in optical path length caused by the anisotropic bonds in a single molecule.In an image duplication interference microscope based on polarized light, the linearly polarized light from the polarizer is further acted upon by a calcite crystal or a Wollaston prism which laterally separates the ordinary and extraordinary rays anywhere from about 10 µm to about 500 µm, depending on the objective lens. One beam passes throught the specimen and the other passes through the surround. The two beams are recombined in the beam recombiner and are turned into elliptically polarized light, which passes through the analyzer in the crossed position. The optical path difference between the two beams, which is the cause of the elliptically polarized light that leaves the specimen, is due to the difference in optical path length between a point in the specimen and a point in the background.In a differential interference contrast microscope, the linearly polarized light from the polarizer is acted upon by a prism that laterally separates the ordinary ray and the extraordinary ray. However,277in a differential interference contrast microscope, the beams are separated by only 0.2 µm to about 1.3 µm, depending on the objective lens. The two beams are recombined in the beam recombiner and are turned into elliptically polarized light, which passes through the analyzer in the crossed position. The optical path difference between the two beams, which is the cause of the elliptically polarized light that leaves the specimen, is due to the difference in optical path length between two points along a given azimuth in the specimen that are separated about 0.2 µm to 1.3 µm, dependingon the objective lens.Wollaston prisms or modified Wollaston prisms, also known as Nomarski prisms, are used to makethe interferometers that separate and recombine the beams in differential interference contrast microscopes. In order to produce an interference image, the first prism must laterally separate the incident light into two beams, and the second prism recombines the two beams laterally and separates the two beams axially. Wollaston prisms are constructed from two wedges of either calcite or quartz, which are cemented together so that their optic axes are perpendicular to each other. Wollaston prisms are often too thick to be placed in front of the condenser


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CORNELL BIOPL 4440 - Study Notes

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