Special receptions of microscopy of biological objects Special methods of light microscopy (illumination and supervision). Method of a dark field.

M ethods of microscopy are selected depending on character and properties of studied objects as they, influence picture contrast.

The method of a dark field in passing light (Dark-field microscopy) is used for receiving of images of transparent not absorbing objects, which cannot be visible, if to apply a method of a light field. Frequently it is biological objects. Light from the lighter and mirrors goes on a preparation with condenser a special design - condenser of a dark field. After an output from condenser the basic part of the light rays, which have not changed the direction at passage through a transparent preparation, forms a bunch in the form of a hollow cone and does not pass in an objective (which is inside of this cone). The image in a microscope is formed by means of only small part of the beams dispersed by microparticles of a preparation being on subject glass inside of a cone and the past through an objective. Dark-field microscopy is based on Tyndall effect, known example is detection of motes in air at illumination by their narrow beam of a s unlight.

On a dark background light images of elements of structure of the preparation, differing from an environment a parameter of refraction are visible. Using this method, it is impossible to define by the form images, particles are transparent or opaque, they have a greater or smaller parameter of refraction in comparison with an environment. At carrying out dark-field research subject glasses should be not thicker than 1,1-1,2 mm, cover glass- 0,17 mm, without scratchs and dirtying. At manufacturing of a preparation it is necessary to avoid presence bubbles and large particles (these defects will be visible brightly luminescent and will not allow to observe a preparation).

Method of ultramicroscopy

In a basis of a method of ultramicroscopy the same principle lays - preparations in ultramicroscopes are shined perpendicularly to a direction of supervision. At this method it is possible to find out (but "not observe") very small particles which sizes lay far outside resolvability of the strongest microscopes. Presence at a preparation of particles with the size 2×10^-9 m probably to register by means of immersion ultramicroscopes. It is impossible to define the form and the exact sizes of such particles by means of this method. Their images are represented the observer in the form of diffractive spots which sizes depend not on the sizes and the form of particles, and from the aperture of an objective and increase in a microscope. Such particles disseminate not enough light, therefore for their illumination extremely strong light sources, for example a coal electric arch are required. Ultramicroscopes are applied basically in colloidal chemistry.

Method of a light field and its version

T he method of a light field in passing light is applied at studying transparent preparations with included in them absorbing (absorbing light) particles and details. It can be, for example, the thin painted cuts of animal and vegetative tissues. At absence of a preparation the bunch of light from condenser, passing through an objective, gives in regular intervals shined field near to a focal plane of an ocular. At presence in a preparation of an absorbing element there is a partial absorption and partial dispersion of light falling on it, it causes occurrence of the image. Application of this method is possible at supervision of not absorbing objects, but only in the event that they disperse shining bunch so strongly, that its significant part does not get in an objective. Method of slanting illumination - a version of the previous method.

Difference between them consists that on object direct light under a greater corner to a direction of supervision. Sometimes it helps to reveal «relief» object due to formation of shadows.

The method of a light field in reflected light is applied at research of opaque objects reflecting light, for example metals or ores. Illumination of a preparation (from the gaffer and a translucent mirror) is made from above, through an objective which simultaneously plays also a role condenser. In the image created by an objective, the structure of a preparation is visible because there is distinction in reflecting ability of its elements; on a light field also the heterogeneity, dispersive light falling on them are allocated.