, photographed with an oil dark field condenser with a
numerical aperture of 1.40 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 microscopies 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. Image:Paper_Micrograph_Dark.png|Dark-field illumination, sample contrast comes from light
scattered by the sample Image:Paper_Micrograph_Bright.png|
Bright-field illumination, sample contrast comes from
attenuation of light in the sample Image:Paper_Micrograph_Cross-Polarised.png|
Cross-polarized light illumination, sample contrast comes from rotation of
polarized light through the sample Image:Paper_Micrograph_Phase.png|
Phase-contrast illumination, sample contrast comes from
interference of different path lengths of light through the sample
Use in computing Dark-field microscopy has been applied in
computer mouse pointing devices to allow the mouse to work on transparent glass by imaging microscopic flaws and dust on the glass's surface. ==Transmission electron microscope applications==