Light microscopy

To understand how the microscope is indispensable in microbiological studies need to remember the limitations of the eye as an optical instrument. The apparent magnitude of an object is directly proportional to the angle at which the subject is seen. So, if the distance from the eye to the object is halved, the apparent size of the object will double. However, the human eye can not focus on objects that are at a distance of less than about 25 cm. This is the distance at which the maximum resolution. To all you could see the object, its angular size should be at least 1 ‘. At a distance of 25 cm, this corresponds to a particle size of about 0.1 mm.

Most of the cells (and therefore most of unicellular organisms) are too small to be seen with the naked eye. Therefore, in order to detect these organisms and to consider their form and structure, requires a microscope. Purpose of the lens system of this device, disposed between the object and the eye, is to increase the apparent angle subtended by the object in the microscope. In addition to the increase, it is also important contrast and resolution. To object could be distinguished under the microscope, it requires a certain degree of contrast between the object and the surrounding background, and to obtain a clear image magnified microscope must have sufficient resolution, which would allow to separately take a very close point of the image.

Light Microscope

Antonie van Leeuwenhoek discovered the world of microbes, using a simple microscope with a short-lenticular lens. To design is now used sophisticated microscopes and improvements took almost two centuries of research in applied optics.

In the modern complex microscope has three separate lens system. A condenser disposed between the light source and the object, collects the rays of light in the microscope field.

A simple two optical lenses inherent defect. They are unable to focus simultaneously the entire field microscope (spherical aberration) and create a colored border around the image (chromatic aberration). These defects can be almost completely eliminated by placing near the main lens of the additional corrective lenses. Therefore, the lens and the eyepiece of the microscope modern complex is composed of multiple lenses to reduce these aberrations are minimized.

To obtain a clear image is very important to adjust the con densornuyu lens. At high magnification, you must install the condenser to provide critical coverage of the field of the microscope: the rays of light from the source should be focused in the object plane, and light field should take the lens almost completely.

Limit resolution

Maximum useful increase achievable by light microscopy, the physical properties of light. Since light has a wave nature, a very small object will be seen through a microscope as a disk surrounded by a number of light and dark rings. Two adjacent points of the object can «solve» t. E., They will be perceived separately, only if the surrounding rings do not overlap. The limit of resolution is the smallest distance between two points at which they can still be seen separately. It is this distance and is determined by the maximum useful increase light microscope.

The value of the limiting resolution (d) gives the equation d = 0,5A / nsinoc where X — the wavelength of the light source, a — half-angle lens, t. E. The angle between the rays coming from the object to the edges of the lens, and n — index refraction of the medium between the subject (the drug) and the lens. Denominator (nsina), commonly called numerical aperture (NA), reflects the properties of the lens. Up to a certain limit resolution increases with numerical aperture. If between the drug and the lens is air, then the value of NA can reach about 0.65 and is limited really possible diameter of the objective lens. The value of N can be increased if the space between the specimen and the lens to fill with oil, which has an index of refraction considerably greater than air. And applying immersion oil immersion lens, NA value can be increased to 1.4, although it is usually in this case reaches about 1.25. If you use a visible light with the shortest wavelength (about 426 to them), the best possible conditions at a maximum resolution of a light microscope is close to 200 nm. In other words, using a light microscope can not obtain separate images of the two points, the distance between which is less than 200 nm.

The contrast in the light microscope, and its increase

Small living biological objects, such as microbial cells, viewed under a microscope is typically a thin layer of water environment, it concluded between the object and cover glasses. At the same time the visibility of the object due to the fact that he misses the light is less than its environment, and as a result between the object and the environment created some contrast. The object transmits less light, firstly because of the light is absorbed by the cell, and secondly, because part of it is derived from the optical path of the microscope as a result of differences in refractive index and the cell environment. Except for some highly colored structures (what, for example, chloroplasts), the biological objects are usually very weakly absorbing in the visible region of the spectrum. Therefore, the contrast of living cells is almost exclusively due to the refraction of light. However, the degree of contrast can be greatly enhanced by applying a stain. Treatment of such dyes that selectively bind, the entire cell or certain of its components, resulting in a much stronger absorption of light. Most staining techniques cause cell death, and therefore before staining cells normally fixed, t. E. Spend some of their chemical treatment to reduce the possibility of structural changes in the cells after their death. This typically used solutions containing osmic acid and aldehydes, particularly glutaraldehyde frequently.

Microbial cells often not necessary to paint in order to make them visible. The easiest way to monitor microorganisms in a living state, in a wet preparation, and for many purposes this is sufficient. The main value of staining is that it makes it possible to obtain specific information about the internal structure of the cell or its chemical properties. For example, deoxyribonucleic acid specific staining reveals the structure and localization of the nucleus. Many special staining techniques used to obtain data on the intracellular deposition of reserve materials, such as glycogen, polyphosphate, and poly-p-hydroxybutyric acid. Gram stain and strong acidic dyes allow to obtain information on the composition of the layers of the cell wall of bacteria. Sometimes the detection of surface layers with very low refractive index, such as mucus or capsules, often surrounding the microbial cells, using so-called negative dyes which do not penetrate into the cell. Such layers can be detected by adding to the medium in which cells are suspended, mascara. It contains coal particles can not penetrate the capsule, and it is detected as a light zone surrounding the cell.

Darkfield microscopy

When covering a small portion of the light scattered by the object, and the object becomes visible as a point of light on a dark background. This phenomenon is used in the method of dark field illumination, enabling to detect those objects which are too small to be seen with the ordinary light, or for some reason do not produce sufficient contrast. This coverage is achieved by using a special condenser, which focuses on the preparation of a hollow cone of light, radiating beams which do not fall into the lens. It passes through the lens and creates an image of only the light that is scattered by the object.

Illumination of the sample in the method of dark field microscopy can be carried out in the incident light. The image in this case, a light scattered by the inhomogeneities of the sample.

Phase-contrast microscope

The relatively low contrast when observing living cells in a conventional light microscope can be strengthened considerably if you use the device with a modified optical system — the so-called phase-contrast microscope. The system of rings in the condenser lens and separates those rays are diffracted (deflected) in the facility, from those who are not diffracted. After the diffracted beams pass through the glass ring (phase plate), introducing an additional phase shift, they recombine with the rays, which are not diffracted. With such modifications optics can dramatically enhance the contrast of cells or intercellular structures, very little difference in the refractive index of the surrounding environment.

UV and fluorescence microscopy

Since the resolution of a light microscope is directly dependent on the wavelength of the light used, you can slightly increase the resolution (about half), if the object is to cover the ultraviolet rays. For short-wave UV opaque glass, so the lens in such a microscope must be made of quartz; the image has to take pictures as the eye does not see UV rays. Because of the high cost of equipment and the complexity of ultraviolet microscopy finds only limited application. However, its modification, so-called fluorescent microscopy is often used for a number of important biological questions.

Some chemical compounds absorb ultraviolet rays by emitting the absorbed energy in the form of light with longer wavelengths already belonging to the visible region of the spectrum. This phenomenon is called fluorescence. When irradiated with ultraviolet fluorescent object, it will shine brightly on a dark background. This is the principle of fluorescence microscopy. Since fluorescent object emits visible light, which passes through the microscope, in this case made of quartz to be manufactured only one condenser. In biology, fluorescence microscopy is used mainly in the immunofluorescence method. If the specific antigen to immunize an animal (e.g., a particular bacterium), then it will contain the serum antibody specifically reacts with the antigen. If the antibody molecules chemically attach a fluorescent dye, they acquire the ability to fluoresce intensely; and when such antibodies bind to a specific antigen, the whole complex is fluorescent. Therefore, by fluorescence microscopy can specifically detect a particular type of bacteria cells in a mixed population of microbes, if the treated population fluorescent antisera to the bacterium. This method was also used in studies of cell wall growth in bacteria).

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