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. Consequently, 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.

Light microscopy applications

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. Optical dark fields usually done with an condenser that features a central light-stop in front of the light source to prevent direct illumination of the focal plane, and at higher numerical apertures may require oil or water between the condenser and the specimen slide to provide an optimal refractive index.

The light's path

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.

Advantages and disadvantages

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.

One 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 halo or relief-style artifacts typical of differential interference contrast microscopy. This comes at the expense of sensitivity to phase information.

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. 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.

While the dark-field image may first appear to be a negative of the bright-field image, different effects are visible in each. 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. 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.

tissue paper