Dark field Microscopy.pdf

DARK FIELD MICROSCOPY Introduction Bright field microscope : Field is brightly illuminated and object is dark/opaque/ colored. Limitations of bright field microscope – Transparent and semitransparent objects are not readily visibl...

DARK FIELD MICROSCOPY Introduction Bright field microscope : Field is brightly illuminated and object is dark/opaque/ colored. Limitations of bright field microscope – Transparent and semitransparent objects are not readily visible as visibility depends on contrast between the object and its background. Dark field microscope: Background is dark and object is self-luminous. Ordinary compound microscope equipped for dark field illumination by substituting a dark field condenser for the ordinarily used Abbe condenser. Principle of dark field microscope If the aperture of the condenser is opened completely and dark-field stop is inserted below the condenser, the light rays reaching object form a hollow cone. If a stop of suitable size is selected ,all direct rays from the condenser can be made to pass outside the object. Any object within this beam of light will reflect some light into objective and becomes visible. The only light that reaches the lens is that scattered by the specimen, and thus the specimen appears light on a dark background.The method of illuminating an object , where object appears self- luminous against a dark background is known as Dark – Field Illumination. Ray diagram of a Dark field microscope Figure: Ray diagram of a dark field microscopy. Only the light rays that strikes on the object are directed through the objective.  Construction of a Dark field microscope It is a modification of bright field microscope. The difference lies only in the condenser. Three types of condensers are used for dark field illumination:- 1. The Abbe condenser 2. The paraboloid condenser 3. The cardiod condenser 1. The Abbe condenser Most commonly employed than other two. Suitable for object which do not need higher magnification, to make them visible. It may be employed by any of the following method: 1.Inserting a dark field stop below the condenser. 2. Unscrewing the top part of the condenser and substituting for it a dark field element. 2. The paraboloid condenser Designed to be used with oil immersion objective and an intense source of light. It is necessary to place cedar wood oil or glycerin between the condenser and the slide. Specimen must be mounted in liquid. The N.A. of the objective must not be greater than that of condenser. 3.The Cardioid condenser Most refined type of dark field illumination. Specially designed to be used for the examination of colloidal solution i.e. particles measuring less than 0.25 microns in diameter. Advantages of a Dark field microscope Organisms can be seen in their natural live state as staining is not used to get the contrast(Staining process kills organisms). Delicate organisms like spirochetes such as Treponema pallidum can be observed with better contrast. Motility of bacteria and other organisms can be observed. Resolution by dark-field microscopy is better than light microscopy and phase contrast microscopy. Limitations of dark- field microscopy : Perfect alignment of all parts of the system is essential. For accurate , high power work, immersion oil is necessary between slide , objective and condenser lens.