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. In optical microscopes a darkfield condenser lens must be used, which directs a cone of light away from the objective lens. 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. Objective lenses with a higher NA can be used but only if they have an adjustable diaphragm, which reduces the NA. Often these objective lenses have a NA that is variable from 0.7 to 1.25. 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. Light enters the microscope for illumination of the sample. A specially sized disc, the patch stop (see figure), blocks some light from the light source, leaving an outer ring of illumination. A wide phase annulus can also be reasonably substituted at low magnification. The condenser lens focuses the light towards the sample. The light enters the sample. Most is directly transmitted, while some is scattered from the sample. The scattered light enters the objective lens, while the directly transmitted light simply misses the lens and is not collected due to a direct-illumination block (see figure). Only the scattered light goes on to produce the image, while the directly transmitted light is omitted. 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.