Finally, the cells illustrated in Figure 2(c) were imaged using Hoffman modulation contrast, where one side of the image is dark while its opposite side is bright, leading to the perception of a pseudo three-dimensional object. In this view, it is difficult to specify the edges of the cells and to interpret the cause of the dark and light areas in the image. Note the dark inner areas and the bright outer areas surrounding the edges of objects such as the cell membranes and nuclei (called halos, and are artifacts). These consist of the field aperture, condenser aperture, additional magnification for video detectors, electronic camera gamma, film gamma, printing paper gamma, image processing in real time, as well as specimen staining.Īn identical viewfield of the cheek cells using phase contrast optics is shown in Figure 2(b). There are several sites in the microscope that allow adjustment of contrast. Control of image contrast in a microscope optical system is dependent upon several factors, primarily the setting of aperture diaphragms, degree of aberration in the optical system, the optical contrast system employed, the type of specimen, and the optical detector. Contrast is not an inherent property of the specimen, but is dependent upon interaction of the specimen with light and the efficiency of the optical system coupled to its ability to reliably record this image information with the detector. At still lighter background colors ( I( b) > 0.20), image contrast is relatively insensitive to background intensity and large changes in I( b) produce only small increases or decreases in image contrast.Īlthough the optical systems found in modern microscopes may be capable of producing high resolution images at high magnifications, such a capability is worthless without sufficient contrast in the image. By lightening the background to a somewhat lighter gray color ( I( b) equals 0.10), small changes in image intensity provide a useful range of contrast. When the background is a very dark gray color ( I( b) equals 0.01), a small change in image intensity produces a large change in contrast. The graph shown in Figure 1 illustrates the effect of background intensity on contrast. From this equation, it is evident that specimen contrast refers to the relationship between the highest and lowest intensity in the image. Where I( b) is the intensity of the background and I( s) is the specimen intensity.
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