An optical microscopy illumination technique used to enhance the contrast in unstained samples is called darkfield microscopy. It works on the principle of illuminating the sample with light that will not be collected by the objective lens, so not form part of the image. This produces the classic appearance of a dark, almost black, background with bright objects on it. A bright specimen on a dark background is presented by darkfield illumination. Research microscopes are equipped with special darkfield condensers. Gladly, it is possible to improvise darkfield illumination on almost any compound microscope. Darkfield microscopy techniques are normally used by scientists to, due to the simplicity of the setup, are almost entirely free of artefacts. However the interpretation of dark field images must be done with care as common dark features of brightfield microscopy images may be invisible, and vice versa. 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 qualities of images obtained from this technique are impressive. The low light levels seen in the final image is the main limitation of dark field microscopy. This means the sample must be very strongly illuminated, and can cause damage to the sample. The path of light goes as light enters the microscope for illumination of the sample. The condenser lens focuses the light towards the sample. A specially sized disc, the patch stop blocks some light from the sample, leaving an outer ring of illumination. The light enters the sample.

Nearly all 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. Only the scattered light goes on to produce the image, while the directly transmitted light is omitted. Darkfield illumination presents a bright specimen on a dark background. Research microscopes are equipped with special darkfield condensers. Gladly, it is possible to improvise darkfield illumination on almost any compound microscope. When trying to set up darkfield illumination, be sure that both the field diaphragm and the aperture diaphragm are sufficiently open so that light can reach the periphery of the condenser. The image of the lamp filament should be on the plane of the specimen, critical illumination, instead of on the aperture diaphragm as in Kohler illumination. If the microscope has an Abbe condenser, a stop in the filter holder may work for objectives as powerful as 40X. For microscopes fitted with a fixed condenser, it is also possible to improvise darkfield illumination for higher power objectives. Place a stand magnifier with a lens of wide diameter, or any high power magnifying lens, near the condenser. A filter caddy can be used, or the magnifier can be set on the lamp housing. Experiment with different stops centered on top of the lens of the magnifier. Darkfield illumination with a 100X objective cannot be improvised by any simple method. If you must have darkfield at this magnification, you will have to buy a darkfield condenser and possibly a 100X objective with a built in diaphragm. Before investing in such a condenser, be sure that your substage hardware can accept it. Darkfield illumination is more than a simple value reversal of brightfield illumination. Only diffracted, reflected or refracted light from objects in the specimen can reach the eye. Since most of the light that reaches the eye is light scattered from the specimen, the light source should be very bright. Removal of the light’s frosted filter as discussed earlier may be helpful. Darkfield illumination, along with polarization and several other types of illumination, is an optical contrasting technique. Borders between objects with different refractive indices are strongly represented by reflected and diffracted light in the absence of axially transmitted light. The darkfield effect is sometimes more three-dimensional, and many structures that are invisible under brightfield illumination are distinctly visible. Some objects that are too small for the microscope to resolve as an image will show up as bright spots of diffracted light. Leeuwenhoek was secretive about his manner of observation, but a passage in one of his early letters suggests that he had hit on a method of darkfield illumination for his simple lenses. He likens a view of blood corpuscles to sand grains scattered on black silk — a perfect description of a low power darkfield view of a blood smear. The use of darkfield illumination would explain how he got a apparent view of certain objects before other microscopists were able to see them at all.