What is darkfield microscopy? How small can a cell be seen?
Dark field microscope is a type of optical microscope, also called ultramicroscope. There is a light shield in the center of the condenser of a dark field microscope so that the illumination light does not directly enter the human lens. Only the light reflected and diffracted by the specimen is allowed to enter the objective lens. Therefore, the background of the field of view is black and the edges of the objects are bright. of. This kind of microscope can see particles as small as 4~200nm, and the resolution can be 50 times higher than that of ordinary microscopes.
principle
Dark field microscope is modified based on the structure of ordinary optical microscope by utilizing the principle of Tyndall optical effect. Darkfield condensers allow the central beam of the light source to be blocked. It is not possible to enter the objective lens through the specimen from bottom up. As a result, the light changes its path and irradiates obliquely on the observed specimen. The specimen is reflected or scattered when encountering the light. The scattered light is thrown into the objective lens, so the entire field of view is dark.
The basic principle of dark field microscopy is the Tyndall effect. When a beam of light passes through a dark room, a bright dust "pathway" can be observed in the air from the direction perpendicular to the incident light. This phenomenon is the Tyndall effect. After a dark-field microscope is replaced with a dark-field condenser on an ordinary optical microscope, due to the occlusion of the internal parabolic structure of the condenser, the light irradiating on the surface of the object to be inspected cannot directly enter the objective lens and eyepiece, and only scattered light can pass through, thus reducing the field of view. is dark. Since the dark field microscope does not emit transparent light into the direct observation system, when there is no object, the field of view is dark and it is impossible to observe any object; when there is an object, the light diffracted back by the object and the scattered light are brightly visible in the dark background. . When observing an object in a dark field, most of the illumination light is refracted. Due to the location, structure and thickness of the object (specimen), the scattering and refraction of light vary greatly.
Structure and modification
The basic structure of a dark field microscope is an ordinary microscope optical set plus a light barrier. An ordinary microscope can be converted into a dark-field microscope as long as the condenser is removable and the diameter of the bracket is suitable for installing a dark-field condenser. When there is no dark field condenser, you can use thick black paper to make a central light shield and place it on the filter frame under the condenser of an ordinary microscope to obtain the dark field effect. The light shield is used to block the light in the middle of the light source so that the light can only enter the specimen from the surroundings. The size is about the same as the aperture size. Different magnifications use different apertures, so different light blocks need to be made.
Practical application
Darkfield microscopy is often used to observe unstained transparent samples. Because these samples have a refractive index similar to that of the surrounding environment, they are difficult to see clearly under ordinary bright field conditions. Therefore, dark field conditions are used to improve the contrast between the sample itself and the background. This kind of microscope can see particles as small as 4~200nm. It can only see the existence, movement and surface characteristics of the object, but cannot distinguish the fine structure of the object. Clinically, darkfield microscopy is commonly used to examine pallid spirochetes. This is a pathogen test, which is of great significance for the diagnosis of early syphilis.
