DARK FIELD METHOD (Greek mikros small + skopeo to look, investigate) — a method of microscopic examination of the objects which are not absorbing light and differing on optical properties from the environment.
uses of T. the m both with the diagnostic purpose, and in research work is generally intravital studying of spirochetes, treponemas, leptospir, the flagellar device of bacteria and even separate flagellums, detection of large viruses. So, T. m apply to diagnosis of a typhinia on «a phenomenon of loading» (Brusin's reaction — Rikkenberg), a hay fever on morphology leptospir and an agglutination test and a lysis, syphilis on morphology of treponemas etc. In studying of physiology of a cell of T. the m can be used for overseeing by influence various physical. - chemical factors on structure of cytoplasm. In the drawing outward of a leptospira at T is shown. m.
Principle T. the m is that an object is lit with slanting beams or a side beam of light. At the same time only the beams disseminated by the objects which are under review get to a lens of a microscope. Therefore the observer sees them brightly shining against a dark background. T. the m allows to find much smaller microobjects, than microscopy in the light field. At T. the m cannot define an exact form of fine details of an object as the darkfield image represents the shining aura around an object.
For the first time T. the m were carried out in 1902 by Zhigmondi and F. W. Ziedentopf by means of a slot-hole ultramicroscope; an object at the same time was lit with a side beam of light, perpendicular an optical axis of a microscope. In such device the most fine particles, size to-rykh lies outside resolving power of a light microscope, became visible thanks to Tyndall's effect (similar to the motes lit with a narrow beam of light). This method of lighting did not find application for studying biol. objects, but it is used in the ultramicroscope (see) intended for a research of colloid systems.
Further various ways of obtaining effect of a dark field were developed. In biological T. the m is applied preferential a method of the central blackout in the condenser. It can be reached by an easy way — blackout of the central part of the ordinary svetlopolny condenser by the placement of a circle of black paper on its lower lens or on glass under the condenser in the holder for filters or by means of the central diaphragm from dense materials — a cardboard, metal. Diameter of the central darkened site shall be such that its image completely blocked an output pupil of the used lens. For each lens it is selected by practical consideration.
Higher quality of the image can be reached at use special lens and mirror a paraboloid - and cardioids - condensers, especially the cardioid condenser in combination with high-aperture (to 0,85) immersion objectives. For receiving darkfield lighting in incident light use epikondensor with a ring-shaped mirror.
The domestic industry releases special condensers for T. m: low-aperture (And — 0,7) the condenser of a light and dark field with a long working distance (OI-Yu) for work with lenses of small increase and high-aperture (And — 1,2) the condenser of a dark field (OI-13) for work with any lenses (with an aperture not higher than 0,85).
At T. the aperture of a lens shall be lower than m, than an aperture of the condenser in order that extreme beams could not get from the condenser directly to a lens. Apply diaphragms of various types to reduction of an aperture of immersion objectives: or in the form of funnels inserts, or iris, allowing to change smoothly an aperture from 1 — 1,25 to 0,7 — 0,85. The lenses with a variable aperture supplied with an iris diaphragm can be used both for svetlopolny microscopy, and for darkfield. For increase in an aperture of the condenser of N of improvement of quality of the image it is necessary to immergirovat the condenser, placing a drop of immersion liquid between a frontal lens of the condenser and a lower surface of a slide plate.
Slide and cover plates for T. m shall be absolutely pure, without scratches as all these defects come to light at T. m also interfere with observation of objects. Thickness of slide plates shall be no more than 1,1 mm, cover — 0,17 mm. During the use of thicker slide plates the beams which are going out of the condenser cannot be focused on drug since their crossing will appear in the thickness of glass, and in a microscope not the dark field with brightly shining objects, and a dark circle with the shining periphery — an optical cut of a cone of the dispersing beams will be visible. The same picture can be observed during the use of too thin slide plates (crossing of beams is above the plane of drug); in this case situation can be improved by lowering of the condenser.
For obtaining the good darkfield image it is necessary to use lighters with the light sources having big superficial brightness and working in the mode of the maximum heat and also to carry out a tsentrirovka of light, about a cut it is possible to judge by a luminescence of air traps in drug (at the decentered lighting they shine unevenly — in the form of a sickle, and at centered — have evenly shining rim).
Darkfield lighting in a microscope is carried out as follows: after installation of light in the light field according to Köhler (see. Microscopic methods of a research) replace the condenser with darkfield. Apply a drop of immersion liquid on a frontal lens of the condenser (water, an immersion .masl) and lift the condenser before contact with a lower surface of the slide plate with drug which is on a stage of microscope. Observing in an eyepiece, focus on drug a lens with small increase. Vertical movement of the condenser try to obtain the minimum size of a light spot. By means of tsentrirovochny adaptation of the condenser remove a light spot in a midfield of sight, establish a lens of the necessary increase and in addition center the condenser.
Drug is prepared by a method the crushed drop (see); thickness of a nappe in drug between subject and cover glasses shall be minimum.
Darkfield microphotographing and filming of moving objects (leptospir, flagellums) presents considerable difficulties because of the insufficient brightness of the image. Their overcoming is possible by means of electron-optical and television amplifiers of brightness of the image (see. Luminescent microscopy ).
In a submicroscopy the effect of a dark field was also gained. However this method did not find application for studying biol. objects.
See also Microscope .
Bibliography: Appelt. Introduction to methods of microscopic examination, the lane with it., page 116, M., 1959; R about with - by Monday G. I. and L e in and neon L. B. Mikroskopicheskaya of the technician, page 37, M., 1957; The Guide to microbiological diagnosis of infectious diseases, under the editorship of K. I. Matveev, M., 1973; With to in about r-G. E. ts, etc. Microscopes, page 83, L., 1969; N about t a n i H. Light microscope study of mixed helices in reconstituted Salmonella flagella, J. molec. Biol., v. 106, p. 151, 1976.
M. Ya. Korn.