Electron microscopy

Electron microscopy is a method of research structures that are out of sight of the light microscope and having a size of less than one micron (1 micron to 1-5+).
An electronic microscope (Fig) based on the use of a directional flow of electrons, which performs the role of a light beam in a light microscope, and the role of lenses play magnets (magnetic lenses).
Due to the fact that different areas of the investigated object differently delay the electrons on the screen electron microscope turns black and white image of the object under study, increased in tens and hundreds of thousands of times. In biology and medicine are mainly used transmission electron microscopes type.
Electron microscopy appeared in the 30-ies, when there were obtained the first images of some viruses (tobacco mosaic virus and bacteriophages). Currently, electron microscopy found the widest application in Cytology, Microbiology and Virology, causing the creation of new branches of science. Electron microscopy of biological objects use special methods of preparation. It is necessary to identify the individual components of the studied objects (cells, bacteria, virus, and so on), as well as for the conservation of their structure in conditions of high vacuum under the electron beam. Using electron microscopy studied the external shape of the object, the molecular organization of its surface, using the method of ultra-thin slices examined the internal structure of the object.
Electron microscopy in combination with biochemical, cytochemical methods, immunofluorescence, and x-ray structure analysis allows to judge about the composition and functions of the structural components of cells and viruses.
electronic microscope
Electron microscope 70-ies of the last century

Electron microscopy is the study of microscopic objects using an electronic microscope.
The electron microscope is electronic-optical instrument that has a resolution of several Angstrom and allowing to study the fine structure of microscopic structures, and even some molecules.
As a source of electrons to create electron beam, replacing light beam, is a three-electrode gun, consisting of a cathode, the control electrode and electrode (Fig. 1).

Fig. 1. Three-electrode gun: 1 - cathode; 2 - control electrode; 3 - electron beam; 4 - anode.

Electromagnetic lenses used in electron microscope instead optical, represent a multilayered solenoids, prisoners in mail from soft-magnetic material, having on the inside of the nonmagnetic gap (Fig. 2).

Fig. 2. Electromagnetic lens: 1 - pole piece; 2 - brass ring; 3 - winding; 4 - shell.

Electric and magnetic fields created in electronic microscope, are axially symmetric. Thanks to the action of these fields charged particles (electrons)that go from one point of the object within a small angle, again gather in the image plane. All of electron-optical system lies in the column electron microscope (Fig. 3).

Fig. 3. Electron-optical system: 1 - control electrode; 2 - aperture first condenser; 3 - aperture second capacitor; 4 - stigmator second capacitor; 5 - the object; 6 - the objective lens; 7 - stigmator objective lens; 8 - stigmator intermediate lenses; 9 - aperture projection lens; 10 - cathode; 11 - anode; 12 - the first condenser; 13 - second capacitor; 14 - corrector focus; 15 - table of objectutilities; 16 - aperture objective lens; 17 - selector aperture; 18 - intermediate lens; 19 - projection lens; a 20 - screen.

Created electron gun, the electron beam is directed into the field of action condenser lenses, which allow widely to change the density, diameter and the aperture of the beam falling on the object under examination. The camera object is set table, the construction of which moves object in mutually perpendicular directions. You consistently to explore the area equal to 4 mm2, and select the most interesting areas.
Behind the camera object is the lens, which allows to provide a sharp image of the object. It also gives the first enlarged image of an object, and using further, intermediate and projection, lenses overall increase can be increased to maximum. The image of the object appears on the screen, luminescense under the action of electrons. Behind the screen are photographic plates. The stability of the action of the electron gun and the clarity of the image along with other factors (constancy of high voltage and others) depends largely on the depth of vacuum in the electronic microscope column, so the performance is largely determined by a vacuum system (pumps, canals, pumping, cranes, valves, seals) (Fig. 4). The required negative pressure inside the column is achieved thanks to high efficiency vacuum pumps.
Preliminary underpressure in all vacuum system creates mechanical booster pump, then effective oil diffusion pump; both pumps connected in series and provide in column microscope high depression. The introduction of the system of electronic microscope oil booster pump enabled for a long time disable booster pump.

Fig. 4. Vacuum diagram of the electron microscope: 1 - trap cooled by liquid nitrogen (hladoprodukt); 2 - high vacuum valve; 3 - diffusion pump; 4 - bypass valve; 5 - small buffer tank; 6 - booster pump; 7 - mechanical booster pump preliminary underpressure; 8 - way valve valve; 9 - a large buffer tank; 10 - column electron microscope; 11 - valve inlet air in a column of a microscope.

Electric diagram of microscope consists of a source of high voltage, heated cathode, power electromagnetic lenses, as well as systems providing network variables voltage motor booster pump, furnace diffusion pump and lighting control. Power supply the device is very high demands: for example, high-resolution electron microscope degree of instability of a high-voltage must not exceed 5 x 10-6 per 30 seconds.
Intense electron beam is formed as a result of thermoemission. Source filament cathode, which is a V-shaped tungsten filament, is a high-frequency generator. The generated voltage with frequency 100-200 kHz provides obtaining monochromatic electron beam. Power lens electron microscope is provided by the constant vysokotarifitsirovannyh shock.

Fig. 5. Electron microscope UEMW-B for the study of living organisms.

Available instruments (Fig. 5) guaranteed-resolution + 4,5; on a separate unique images obtained the permission of 1.27 + approaching the size of the atom. A useful increase is equal to 200 000.
The electron microscope is a precision device that requires special methods of preparation. Biological objects Malcontenta, so you have to artificially increase the contrast. There are several ways to improve contrast agents. When shaded preparation under an angle of platinum, tungsten, carbon, and so on, it becomes possible to determine on electron microscopic images of size in all three spatial axes of the coordinate system. If a positive contrast, the drug is connected with water-soluble salts of heavy metals (Oranjestad, monoxide, lead, potassium permanganate and others). When negative contrast, the drug is surrounded by a thin layer of amorphous material of high density, impenetrable for electrons (molybdate ammonium, oranjezicht, phosphorus-tungsten acid and others).
Electron microscopy viruses (versascopy) led to significant progress in the study of ultra-thin, molecular structure of viruses (see). Along with the physical, biochemical and genetic methods research application of electron microscopy has also contributed to the emergence and development of molecular biology. The subject of study for this new section of biology is submicroscopic organization and function of human cells, animals, plants, bacteria and Mycoplasma, and also the organization Rickettsia, and viruses (Fig. 6). Viruses, large molecules of protein and nucleic acids (RNA, DNA), separate fragments of cells (for example, the molecular structure of a membrane of bacterial cells) can be investigated by means of an electronic microscope after special treatment: shadowing metal, positive or negative contrast uranilnitrata or phosphorus-tungsten acid and other compounds (Fig. 7).

Fig. 6. Cell tissue culture hearts monkeys, cynomolgus, infected with a virus variola (X 12 000): 1 - engine; 2 - mitochondria; 3 - the cytoplasm; 4 - the virus.
Fig. 7. Influenza a (negative contrast (H 000): 1 - shell; 2 - ribonucleoprotein.

By the method of negative contrast on the surface of many viruses were found naturally located group of protein molecules - capsomere (Fig. 8).

Fig. 8. As part of its surface capsid herpes virus. See individual capsomere (X500 000): 1 - side view; 2 - the view from the top.
Fig. 9. Ultra-thin slice of Salmonella typhimurium (h 000): 1 - engine; 2 - cover; 3 - the cytoplasm.

The internal structure of bacteria and viruses, and other larger biological objects can be studied only after dissecting them with ultratome and preparation of very thin slices thickness of 100-300+. (Fig. 9). Due to improved methods of fixation, fill and polymerization of biological objects, the use of diamond and glass knives with ultraluminous and use vysokoskorostnaya connections to colouring of serial sections managed to get ultra-thin sections of not only large but also the smallest viruses humans, animals, plants and bacteria.