![]() ![]() Analysis of intensities in such images may then be used to estimate the amount of that bending. In single-crystal specimens, single-reflection dark-field images of a specimen tilted just off the Bragg condition allow one to "light up" only those lattice defects, like dislocations or precipitates, that bend a single set of lattice planes in their neighborhood. Dark-field images, under these conditions, allow one to map the diffracted intensity coming from a single collection of diffracting planes as a function of projected position on the specimen and as a function of specimen tilt. Weak-beam DF of strain around nuclear track coresĭark-field studies in transmission electron microscopy play a powerful role in the study of crystals and crystal defects, as well as in the imaging of individual atoms.īriefly, imaging involves tilting the incident illumination until a diffracted, rather than the incident, beam passes through a small objective aperture in the objective lens back focal plane. Comparison of transillumination techniques used to generate contrast in a sample of tissue paper (1.559 μm/pixel when viewed at full resolution).Raised features that are too smooth to cast shadows will not appear in bright-field images, but the light that reflects off the sides of the feature will be visible in the dark-field images. In bright-field microscopy, features are visible where either a shadow is cast on the surface by the incident light or a part of the surface is less reflective, possibly by the presence of pits or scratches. While the dark-field image may first appear to be a negative of the bright-field image, different effects are visible in each. In general the dark-field image lacks the low spatial frequencies associated with the bright-field image, making the image a high-passed version of the underlying structure. The interpretation of dark-field images must be done with great care, as common dark features of bright-field microscopy images may be invisible, and vice versa. This comes at the expense of sensitivity to phase information. This means that the sample must be very strongly illuminated, which can cause damage to the sample.ĭark-field microscopy techniques are almost entirely free of halo or relief-style artifacts typical of DIC and phase-contrast imaging. One limitation of dark-field microscopy is the low light levels seen in the final image. Considering the simplicity of the setup, the quality of images obtained from this technique is impressive. The steps are illustrated in the figure where an inverted microscope is used.ĭark-field microscopy produces an image with a dark backgroundĭark-field microscopy is a very simple yet effective technique and well suited for uses involving live and unstained biological samples, such as a smear from a tissue culture or individual, water-borne, single-celled organisms. This produces the classic appearance of a dark, almost black, background with bright objects on it. It works by illuminating the sample with light that will not be collected by the objective lens and thus will not form part of the image. In optical microscopy, dark-field describes an illumination technique used to enhance the contrast in unstained samples. Often these objective lenses have a NA that is variable from 0.7 to 1.25. Objective lenses with a higher NA can be used but only if they have an adjustable diaphragm, which reduces the NA. To maximize the scattered light-gathering power of the objective lens, oil immersion is used and the numerical aperture (NA) of the objective lens must be less than 1.0. In optical microscopes a darkfield condenser lens must be used, which directs a cone of light away from the objective lens. As a result, the field around the specimen (i.e., where there is no specimen to scatter the beam) is generally dark. Operating principle of dark-field and phase-contrast microscopiesĭark-field microscopy (also called dark-ground microscopy) describes microscopy methods, in both light and electron microscopy, which exclude the unscattered beam from the image. ![]()
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