Method for investigation of histological specimen surface relief

FIELD: biology, medicine, in particular histological investigation of shells with natural surface by using light-optical microscope.

SUBSTANCE: claimed method includes surface relief investigation by using plane field microscope in reflected light at one-side falling shadow-forming lighting. Light-reflecting ability of specimen surface is provided by silver impregnation. Said impregnation is carried out by specimen hold in 10 % silver nitrogen solution for 10 min followed by reducing thereof with 1 % ascorbic acid solution for 1 min.

EFFECT: method for investigation of natural shell surface relief of improved quality due to visual representation of surface three-dimension image on tissue level.

3 cl, 4 dwg

 

The method relates to biology and medicine, namely to methods histological studies of membranes with a natural surface, such as the peritoneum, pleura, synovial membrane using optical microscopes.

There is a method of studying the surface of biological objects optical binary stereomicroscopes with lenses from 0.6 to 4 fold increase. They give a small increase in micromicrocuries dodania level research.

Traditional microscopy of histological preparations at the tissue level studies carried out by the flat field microscopes (modern classification of traditional bio-medical microscopes O. Egorov WITH a microscope. SPb., 2000, p.42) when illuminated by the light. In this study the contours and internal structure selectively coated parts in the thickness of the irradiated product. However, microscopic clearance does not give a picture of the surface of the drug. There is a clear methodological contradiction in the ideology microscopy the surface of various structures in the lumen. It is impossible to study on the lumen surface shape of the drug, which is a kind of interface.

To study the surface topography of the object you want to visualize the structural form of the surface in the bulk izobrazheniya obtain three-dimensional images of the surface topography of histological preparations at the tissue level studies using optical microscopes must create two conditions:

1. The illumination of the surface of histological preparation on the part of the observer - lens.

2. Making the surface of histological preparation light reflecting ability.

The objective of the invention is to improve the quality of the study of the surface topography of natural membranes at the expense of rendering three-dimensional images of their surface at the tissue level.

This object is achieved by a method for studying the surface topography of histological preparations microscopes flat field, which is in contrast to the prototype surface preparation give the light reflecting ability by impregnation of silver, and microscopy performed in reflected light when unilateral falling tenebrosum lighting. The silver impregnation is performed by keeping the drug in a 10% solution of silver nitrate for 10 minutes and then restoring it in the metallic state with 1% ascorbic acid solution for 1 minute.

It is advisable to set the angle of incidence of the light beam on the surface of the drug 75-85° relative to the axis of the lens, and in the research process to change the azimuth of the light beam by rotation of the object stage of the microscope on ±180° on the South zero azimuth.

For microscopy of histological preparations in the reflected svetoslava surface preparation must have light reflecting ability. Good light reflecting ability to have the metals, and the best among them the silver, the coefficient reflecting up to 95% (Olenin S., G. Fadeev. Inorganic chemistry. M., 1979).

Well-known methods of deposition of metals on the surface of biological tissues. These methods are unduly expensive and time-consuming for large areas on the light level microscopy.

The famous "reaction of the silver mirror" is added to the ammonia solution of silver nitrate a few drops of formaldehyde or glucose solution leads to the restoration of silver ions in solution to metallic silver (Glinka D., General chemistry. L., 1977. - S). In morphology of the conventional methods of impregnation - introduction of silver in tissues in the form of solutions of ammonium salts with subsequent restoration by the structures of the solutions of formalin or glucose (Romas B. Microscopic techniques. M., 1954)

However, all known methods of impregnation of histological preparations of silver, designed for light optical microscopy transmission using high light-absorbing ability of the recovered silver and intermediate products of its recovery unsuitable for microscopy in reflected light of the surface topography of the drug.

First, the recovery of silver is that is are on separate structures, as background, the remainder of his of the matrix is washed out, that is an intermediate substance that is involved in the formation of surface preparation is not rendered.

Secondly, the recovery of silver is not brought to the metallic state, stopping at the intermediate stages of the reaction it is restored to yellow-brown color of the product - colors are not reflective of the oxides of silver.

Third, these methods include impregnation megatape laundering silver with surface preparation (wiring drugs through several portions of distilled water after the sensitization phase nitrate of silver, and after exposure in ammoniacal solution of silver). This prevents deposition of silver on the surface of the drug and reduces the impregnation surface lying its structures. Otherwise the product becomes opaque and unsuitable for microscopy on clearance due to the deposition of silver on the surface of the drug on both sides and preimpregnate its surface structures during the diffusion of fluids in the depth of the preparation. Especially this refers to the impregnation of the relatively large thickness of film preparations of biological membranes, when megatape laundering silver is more durable and the manifestation of it is in the regenerating solutions (form is in very low concentration, with detergent effect.

Thus, known methods of impregnation of silver provide selective detection of certain elements tissues (vessels, nerves, collagen fibers), which are generally not involved in the formation of luminale surface membranes. This recovery of silver is not possible to give them light reflecting ability, not impregnants surface structure that, in the aggregate, do not provide a reliable picture of the surface topography of drugs in the reflected light.

The proposed method provides a plating surface preparation on the basis of total (non-selective) impregnation of all structures involved in the formation of this surface. For this impregnation of silver in the preparation is in the form of a 10% solution of nitrate salts thereof, are not characterised by the selective deposition on separate structures. Usually applied ammoniacal solutions of silver nitrate deposited selectively on argentophile structures, in addition, they are unstable and easily dissociate into stable oxides that form in the subsequent non-reflective dark brown artifacts on the surface of the drug. Recovery of silver is 1% aqueous solution of ascorbic acid (excluding megatoy leaching). Commonly used formaldehyde and glucose is not the vos is tanglefoot silver from its nitrate salt to the metallic state. It is known that the strong reducing properties of ascorbic acid (Apirs. The histochemistry. M., 1962, s). Produced by the proposed method drugs matte-view mirror with gray-blue color, indicating high-quality and complete recovery of the impregnated silver, and have good light reflecting ability.

The surface topography of such a drug is studied using microscopes flat field in the reflected light when unilateral incident light. In this light, the surface preparation on the part of the observer (lens) surface topography is visualized in a three-dimensional image. The flat field microscopes capable of forming a three-dimensional image within the depth of the sharp image of the lenses of the order of 100-200 microns (Egorova O. WITH a microscope. SPb., 2000, p.40). The light source is placed on the side and above the level of the table of the microscope. Optimal tenebrosus is the angle of incidence of the light beam on the drug within 75-85° relative to the axis of the lens. The magnitude of the angle of incidence of the beam depends on several factors: the length of the focal length and diameter of the lens being used, the amount of detail and the nature of the ruggedness of the surface topography of the drug, from the necessity of lighting the bottom of a deep and narrow pits, Mosh the spine of the source, light reflecting ability of surface preparation and other Such one-sided incident light creates a gradient of illumination of the individual elements of the surface topography of the drug depending on the angle of incidence on them light rays, i.e. the slopes of the elevations and recesses facing the light source is illuminated more than the other slopes. At large angles of incidence of light opposite to the light source, the slopes of the individual elevations and recesses with angle, large angle of incidence, be not illuminated by direct rays of light. This increase chiaroscuro lighting effect (tenebrioninae) as contrast illumination elements of the surface topography allows you to identify the contours of the structures with gentle slopes, individual fibers and cells. The change of azimuth lighting turn table of the microscope allows to clarify the structural features of the surface detail with complex asymmetrical shapes. For example, the lighting of furrows and ridges perpendicular to or along their direction reveals various details of their structure. The change of azimuth coverage allows you to avoid the consequences of azimuthal effect" (G. Skvortsov et al. Microscopes. L. 1969, p.72). The azimuthal angle is measured from the South zero azimuth as positive in the direction of clockwise and as a negative is positive against clockwise movement.

The method is as follows.

Before microscopy, the surface of the drug is subjected to plating to give it a light reflecting ability.

For this fixed in formalin film preparations shells with natural surface (peritoneum, pleura, mucous, synovial membrane, and others), straightened the pieces of the diaphragm, the intestinal wall, the capsules of the joints, the area of the articular cartilage, etc. are washed in tap water during the day and opolaskivaetsya in distilled water. The thickness of the preparation for microscopy in reflected light does not matter.

Then carry out the exposure of the slices in a 10% solution of silver nitrate for 10 minutes, restore silver with 1% ascorbic acid solution for 1 minute.

Then as usual: stop manifestations in ammonia water, rinse in distilled water, the conclusion in balsam.

Prepared in this way drugs are examined through a microscope flat field lenses up to 50-fold increase (overall increase of up to 350-500 times), allowing to visualize the volumetric picture of the terrain surface at the tissue level studies. Microscopy of the drug in the reflected light when unilateral falling tenebrosum lighting. The light source is placed on the side and enter the level of the table of the microscope. Optimal tenebrosus is the angle of incidence of the light beam on the drug within 75-85°. To identify the structural features of individual structures of the relief varies the angle of incidence and the azimuth of the light beam.

The photographs show examples obtained using the proposed method of images, which clearly visualized image and the surface topography of the drug.

Figure 1 - the pleural surface of a person with a relief of the folds and contours of the cells mesothelia. The lens 50, the eyepiece 6,3; the lighting on the right is the angle of incidence of the light beam 80°, the azimuthal angle is 90°.

Figure 2 - the surface of the basal membrane of the peritoneum person with the underlying fat lobules forming a rounded elevation with steep slopes 1. Lens 12, an eyepiece 12,5; the lighting on the right is the angle of incidence of the light beam 80°, the azimuthal angle is 90°.

Figure 3 - surface basement membrane diaphragmatic peritoneum person with parallel and relatively regular (wavelength 15-20 μm) narrow folds Lens 25, eyepiece 12,5; the lighting on the left is the incidence angle of the light beam 75°, azimuthal angle (+70°.

Figure 4 - surface of the synovial membrane nagdadalantao synovial bags person (formed along with synoviocyte and intercellular substance). Through holes visualized the presence of Ni is sustained fashion cavity. The length of the shadow from the edge of a large hole at the bottom of the cavity determenirovana depth of the cavity and the angle of incidence of the light flux. Lens 6,3; eyepiece 12,5; the lighting on the left is the incidence angle of the light beam 75°, azimuthal angle (+120°.

The obtained images are of high quality visualization of volumetric surface topography down to the detail of cellular structures.

Thus, the proposed method can improve the quality of research of the surface topography of histological preparations, watching them in a three-dimensional image at the tissue level, allowing for more reliable information about the condition of a biological object.

1. Method for studying the surface topography of histological preparations microscopes flat field, characterized in that the surface preparation give the light reflecting ability by impregnation of silver, and microscopy preparation is carried out in reflected light when unilateral falling tenebrosum lighting, and silver impregnation is performed by maintaining the drug in 10%solution of nitric acid silver for 10 min and subsequent restoration of its 1%ascorbic acid solution for 1 minute

2. The method according to claim 1, characterized in that the set angle of incidence of the light beam on the surface of the Reparata 75-85° relative to the axis of the lens.

3. The method according to claim 2, characterized in that the research process to change the azimuth of the light beam by rotation of the object stage of the microscope on ±180° on the South zero azimuth.



 

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