In most of our microscope activities, we normally use microscopes that utilize white light for its illumination. These high power and low power microscopes are called brightfield microscopes. These types of microscopes are commonly used in most microscope activities, from simple kid microscopes, student microscopes to professional microscope applications. However, there are also microscopes that require a dark background in order to bring out the finer details of the specimen. This type of illumination technique is called dark field microscopy.
Darkfield microscopy is also called ‘darkground microscopy’. One of the oldest illumination technique in microscopy, darkfield microscopy is able to produce images of microscopic structures that are otherwise invisible or transparent in other forms of microscopy. This illumination technique can enhance the contrast in unstained microscope samples. Producing a dark to almost black background with bright specimens on it, darkfield microscopy works on a principle wherein the light will not be collected by the microscope objectives and therefore do not have a direct contribution in forming the image.
In darkfield microscopy, the image formation relies on the diffraction of light from the specimen which enters the microscope objective lens. Unlike in bright field microscopy wherein light rays directly enter the microscope objectives, the light rays in a microscope that employs darkfield microscopy do not directly pass through the objective lens. This can be seen on the images that are observed under the microscope. There are significant differences on the refractive indices between the object and its surroundings.
The specimens that are typically observed under a high power dark field microscope are those that contain fine filament structures, thin membranes, flagella and those that have areas that normally ‘disappears’ when illuminated under a bright light of a brightfield microscope.
The most important part of a darkfield microscope is the dark field condenser. The darkfield condenser blocks off the light that passes through its central area which forming a hollow cone of light. This cone of light is directed at an angle which misses the objective lens. The image formation relies on the angle of light rays that strike and be diffracted from the cells of the specimen.
The diffracted light rays produce a specimen image that appears brilliant and clear white against a black background. The images also have a very high contrast that enables minute structures to be very visible to the inexperienced eye. More often than not, the images seem to have white light surrounding the specimen, making it more apparent in size as well as more visible. These specimens do not usually need any form of staining or preparation and are usually examined in its natural living state under the microscope.
Having a microscope that works on darkfield microscopy entails a simple and easy set-up. Darkfield microscopy is an effective illumination technique that is ideal for biological specimens that are unstained and living. A smear from individual water borne microorganisms or tissue culture can be clearly observed under this microscope. Moreover, the quality of the images that darkfield microscopes produce is impressive.
Owning a darkfield microscope, however, requires the user to have a very dark working environment. This is to achieve images with better contrast and clearer quality. The specimen should also be strongly illuminated, which risks damage to the specimen but also results to a final image with poor low light levels if done otherwise.
The resulting image from a dark field microscope may seem to be a negative of a bright field microscope’s image but this is not the case. The features of a specimen under a brightfield microscope can only be visible when there is a shadow cast by the top lighting. If the raised features are too smooth for it to cast its own shadows, it will not be visible under the bright field microscope. Under a darkfield microscope, the light that is reflected off the feature’s sides appear visible.
Darkfield illumination and darkfield condensers are typically used in a high power live blood cell analysis microscope in order to produce a completely dark background, giving a maximum contrast between the blood specimen and the background. This is also employed in a low power gemological microscope wherein the gemstone specimen is illuminated from the sides to produce the desired image.
