Dark field microscopy

Dark-field microscopy (also called dark-ground microscopy) describes microscopy methods, in both light and electron microscopy, which exclude the unscattered beam from the image. As a result, the field around the specimen (i.e., where there is no specimen to scatter the beam) is generally dark.Dark-field illumination, sample contrast comes from light scattered by the sampleBright-field illumination, sample contrast comes from attenuation of light in the sampleCross-polarized light illumination, sample contrast comes from rotation of polarized light through the samplePhase-contrast illumination, sample contrast comes from interference of different path lengths of light through the sample Dark-field microscopy (also called dark-ground microscopy) describes microscopy methods, in both light and electron microscopy, which exclude the unscattered beam from the image. As a result, the field around the specimen (i.e., where there is no specimen to scatter the beam) is generally dark. In optical microscopy, dark-field describes an illumination technique used to enhance the contrast in unstained samples. 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. This produces the classic appearance of a dark, almost black, background with bright objects on it. The steps are illustrated in the figure where an inverted microscope is used. Dark-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. Considering the simplicity of the setup, the quality of images obtained from this technique is impressive. The main limitation of dark-field microscopy is the low light levels seen in the final image. This means that the sample must be very strongly illuminated, which can cause damage to the sample. Dark-field microscopy techniques are almost entirely free of artifacts, due to the nature of the process. However, 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.