Special types of microscopy
Darkfield. A special condenser is used to highlight the contrasting structures of the unpainted material. Dark-field microscopy allows you to observe living objects. The observed object appears illuminated on a dark field. In this case, the rays from the illuminator fall on the object from the side, and only scattered rays enter the microscope lenses.
Phase contrast microscopy allows you to study living and unpainted objects. When light passes through painted objects, the amplitude of the light wave changes, and when light passes through unpainted objects, the phase of the light wave changes, which is used to obtain high-contrast images in phase-contrast and interference microscopy.
Polarization microscopy - imaging of unstained anisotropic structures (for example, collagen fibers and myofibrils).
Interference microscopy combines the principles of phase-contrast and polarization microscopy and is used to obtain contrast images of unpainted objects.
Fluorescence microscopy used for observing fluorescent (luminescent) objects. In a fluorescence microscope, light from a powerful source passes through two filters. One filter stops light in front of the sample and transmits light of the wavelength that excites fluorescence from the sample. Another filter allows light of the wavelength emitted by the fluorescent object to pass through. Thus, fluorescent objects absorb light of one wavelength and emit in another region of the spectrum.
Fluorescent dyes (fluorescein, rhodamine, etc.) selectively bind to specific macromolecules.
Electron microscopy
The theoretical resolution of transmission EM is 0.002 nm. The actual resolution of modern microscopes approaches 0.1 nm. For biological objects, the EM resolution in practice is 2 nm.
Translucent EM consists of a column through which electrons emitted by a cathode filament pass in a vacuum. A beam of electrons, focused by ring magnets, passes through the prepared sample. The nature of electron scattering depends on the density of the sample. Electrons passing through the sample are focused, observed on a fluorescent screen, and recorded using a photographic plate.
Scanning EM used to obtain a three-dimensional image of the surface of the object under study.
Chip method ( freezing-cleavage) is used to study the internal structure of cell membranes. The cells are frozen at liquid nitrogen temperature in the presence of a cryoprotectant and used for making chips. The cleavage planes pass through the hydrophobic middle of the lipid bilayer. The exposed inner surface of the membranes is shaded with platinum, and the resulting replicas are studied in a scanning electron microscope.
2. The main parts of a light microscope, their purpose and structure
The resolution of the microscope gives a separate image of two lines close to each other. The naked human eye has a resolution of about 1/10 mm or 100 microns. The best light microscope improves the capability of the human eye by about 500 times, i.e. its resolving power is about 0.2 µm or 200 nm.
Resolution and magnification are not the same thing. If you use a light microscope to take photographs of two lines located at a distance of less than 0.2 microns, then no matter how you enlarge the image, the lines will merge into one. You can get high magnification, but not improve its resolution.
There are useful and useless increases. By useful we mean such an increase in the observed object that it is possible to reveal new details of its structure. Useless is a magnification in which, by magnifying an object hundreds or more times, it is impossible to detect new structural details. For example, if an image obtained using a microscope (useful!) is enlarged many times more by projecting it onto a screen, then new, finer details of the structure will not be revealed, but only the size of existing structures will increase accordingly.
In educational laboratories, light microscopes are usually used, in which microscopic specimens are examined using natural or artificial light. The most common light biological microscopes are: BIOLAM, MIKMED, MBR (biological working microscope), MBI (biological research microscope) and MBS (stereoscopic biological microscope). They provide magnification ranging from 56 to 1350 times. A stereo microscope (MBS) provides a truly three-dimensional perception of a micro-object and magnifies from 3.5 to 88 times.
There are two systems in a microscope: optical and mechanical. The optical system includes lenses, eyepieces and a lighting device (a condenser with a diaphragm and a light filter, a mirror or an electric light).
Mechanical part of the microscope.
base (tripod) or solid leg (1);
box with micromechanism (2) and microscrew (3);
feed mechanism for rough aiming - macroscrew or ratchet (8);
stage (4);
screws (5, 6, 12, 13);
head (9); revolver (10); terminals; tube (11);
arc or tube holder (7);
Cremalier (macroscrew) – serves for approximate “rough” installation on the fo-
The mechanical system of the microscope consists of a stand, a box with a micrometer mechanism and a micrometer screw, a tube, a tube holder, a coarse aiming screw, a condenser bracket, a condenser moving screw, a revolver, and a sample stage.
Stand- This is the base of the microscope.
Box with micrometer mechanism m, built on the principle of interacting gears, is fixedly attached to the stand. The micrometer screw serves to slightly move the tube holder, and, consequently, the lens over distances measured in micrometers. A full turn of the micrometer screw moves the tube holder by 100 microns, and a turn of one division lowers or raises the tube holder by 2 microns. To avoid damage to the micrometer mechanism, it is allowed to turn the micrometer screw in one direction by no more than half a turn.
Tube or tube - cylinder, into which eyepieces are inserted from above. The tube is movably connected to the head of the tube holder; it is fixed with a locking screw in a certain position. By loosening the locking screw, the tube can be removed.
Revolver designed for quickly changing lenses that are screwed into its sockets. The centered position of the lens is ensured by a latch located inside the revolver.
Screw rough aiming is used to significantly move the tube holder, and, consequently, the lens in order to focus the object at low magnification.
The object table is intended to place the drug on it. In the middle of the table there is a round hole into which the front lens of the condenser fits. There are two springy terminals on the table - clamps that secure the drug.
Condenser bracket movably connected to the micrometer mechanism box. It can be raised or lowered by a screw that rotates a gear that fits into the grooves of a comb-cut rack.
Microscope(from Greek mikros- small and skopeo- I look) - an optical device for obtaining an enlarged image of small objects and their details invisible to the naked eye.
The first known microscope was created in 1590 in the Netherlands by hereditary opticians Zechariah And Hans Jansen , who mounted two convex lenses inside one tube. Later Descartes in his book “Dioptrics” (1637), he described a more complex microscope, composed of two lenses - a flat-concave (eyepiece) and a biconvex (objective). Further improvement of optics made it possible Anthony van Leeuwenhoek in 1674, made lenses with a magnification sufficient to carry out simple scientific observations and, for the first time in 1683, described microorganisms.
A modern microscope (Figure 1) consists of three main parts: optical, lighting and mechanical.
Main details optical part The microscope consists of two systems of magnifying lenses: an eyepiece facing the researcher’s eye and a lens facing the specimen. Eyepieces They have two lenses, the upper one is called the main one, and the lower one is called the collective lens. The eyepiece frames indicate what they produce. increase(×5, ×7, ×10, ×15). The number of eyepieces on a microscope may vary, and therefore monocular And binocular microscopes (designed to observe an object with one or two eyes), as well as trinoculars , allowing you to connect documentation systems (photo and video cameras) to the microscope.
Lenses are a system of lenses enclosed in a metal frame, of which the front (front) lens produces magnification, and the corrective lenses behind it eliminate defects in the optical image. The numbers on the lens frame also indicate what they produce. increase (×8, ×10, ×40, ×100). Most models intended for microbiological research are equipped with several lenses with different degrees of magnification and a rotating mechanism designed for quick change - turret , often called " turret ».
Lighting part is designed to create a light flux that allows you to illuminate an object in such a way that the optical part of the microscope performs its functions with extreme precision. The lighting part of a direct transmitted light microscope is located behind the object under the lens and includes Light source (lamp and electrical power supply) and optical-mechanical system (condenser, field and aperture adjustable diaphragm). Condenser consists of a system of lenses that are designed to collect rays coming from a light source at one point - focus , which must be in the plane of the object under consideration. In its turn d diaphragm located under the condenser and is designed to regulate (increase or decrease) the flow of rays passing from the light source.
Mechanical part The microscope contains parts that combine the optical and lighting parts described above, and also allow the placement and movement of the specimen under study. Accordingly, the mechanical part consists of grounds microscope and holder , to the top of which are attached tube - a hollow tube designed to accommodate the lens, as well as the above-mentioned turret. Below is stage , on which slides with the samples being studied are mounted. The stage can be moved horizontally using an appropriate device, as well as up and down, which allows for adjusting image sharpness using gross (macrometric) And precision (micrometric) screws.
Increase, which the microscope produces is determined by the product of the objective magnification and the eyepiece magnification. In addition to light-field microscopy, the following are widely used in special research methods: dark-field, phase-contrast, luminescent (fluorescent) and electron microscopy.
Primary(own) fluorescence occurs without special treatment of drugs and is inherent in a number of biologically active substances, such as aromatic amino acids, porphyrins, chlorophyll, vitamins A, B2, B1, some antibiotics (tetracycline) and chemotherapeutic substances (acriquin, rivanol). Secondary (induced) fluorescence occurs as a result of processing microscopic objects with fluorescent dyes - fluorochromes. Some of these dyes are diffusely distributed in cells, others selectively bind to certain cell structures or even to certain chemicals.
To carry out this type of microscopy, special luminescent (fluorescent) microscopes , differing from a conventional light microscope by the presence of a powerful light source (ultra-high pressure mercury-quartz lamp or halogen incandescent quartz lamp), emitting predominantly in the long-wave ultraviolet or short-wave (blue-violet) region of the visible spectrum.
This source is used to excite fluorescence before the light it emits passes through a special exciting (blue-violet) light filter and is reflected interference beam splitter record , almost completely cutting off longer wavelength radiation and transmitting only that part of the spectrum that excites fluorescence. At the same time, in modern models of fluorescent microscopes, exciting radiation hits the specimen through the lens (!) After excitation of fluorescence, the resulting light again enters the lens, after which it passes through the one located in front of the eyepiece locking (yellow) light filter , cutting off short-wave exciting radiation and transmitting luminescence light from the drug to the observer's eye.
Due to the use of such a system of light filters, the glow intensity of the observed object is usually low, and therefore fluorescence microscopy should be carried out in special darkened rooms .
An important requirement when performing this type of microscopy is also the use non-fluorescent immersion And enclosing media . In particular, to quench the intrinsic fluorescence of cedar or other immersion oil, small amounts of nitrobenzene are added to it (from 2 to 10 drops per 1 g). In turn, a buffer solution of glycerol, as well as non-fluorescent polymers (polystyrene, polyvinyl alcohol) can be used as containing media for drugs. Otherwise, when performing luminescence microscopy, ordinary glass slides and coverslips are used, which transmit radiation in the used part of the spectrum and do not have their own luminescence.
Accordingly, important advantages of fluorescence microscopy are:
1) color image;
2) high degree of contrast of self-luminous objects on a black background;
3) the possibility of studying cellular structures that selectively absorb various fluorochromes, which are specific cytochemical indicators;
4) the ability to determine functional and morphological changes in cells in the dynamics of their development;
5) the possibility of specific staining of microorganisms (using immunofluorescence).
Electron microscopy
The theoretical foundations for using electrons to observe microscopic objects were laid W. Hamilton , who established an analogy between the passage of light rays in optically inhomogeneous media and the trajectories of particles in force fields, as well as de Broglie , who put forward the hypothesis that the electron has both corpuscular and wave properties.
Moreover, due to the extremely short wavelength of electrons, which decreases in direct proportion to the applied accelerating voltage, the theoretically calculated resolution limit , which characterizes the ability of the device to separately display small, maximally located details of an object, for an electron microscope is 2-3 Å ( Angstrom , where 1Å=10 -10 m), which is several thousand times higher than that of an optical microscope. The first image of an object formed by electron beams was obtained in 1931. German scientists M. Knollem And E. Ruska .
In the designs of modern electron microscopes, the source of electrons is metal (usually tungsten), from which, after heating to 2500 ºС, the result is thermionic emission electrons are emitted. With the help of electric and magnetic fields, the formed electron flow You can speed up and slow down, as well as deflect in any direction and focus. Thus, the role of lenses in an electron microscope is played by a set of appropriately designed magnetic, electrostatic and combined devices called “ electronic lenses" .
A necessary condition for the movement of electrons in the form of a beam over a long distance is also the creation of vacuum , since in this case the average free path of electrons between collisions with gas molecules will significantly exceed the distance over which they must move. For these purposes, it is sufficient to maintain a negative pressure of approximately 10 -4 Pa in the working chamber.
According to the nature of studying objects, electron microscopes are divided into translucent, reflective, emissive, raster, shadow And mirrored , among which the first two are the most commonly used.
Optical design transmission (transmission) electron microscope is entirely equivalent to the corresponding optical microscope design in which the light beam is replaced by an electron beam and glass lens systems are replaced by electron lens systems. Accordingly, a transmission electron microscope consists of the following main components: lighting system, object camera, focusing system And final image registration block , consisting of a camera and a fluorescent screen.
All these nodes are connected to each other, forming a so-called “microscope column”, inside which a vacuum is maintained. Another important requirement for the object under study is its thickness of less than 0.1 microns. The final image of the object is formed after appropriate focusing of the electron beam passing through it onto photographic film or fluorescent screen , coated with a special substance - phosphor (similar to the screen in TV picture tubes) and turning the electronic image into a visible one.
In this case, the formation of an image in a transmission electron microscope is associated mainly with different degrees of electron scattering by different areas of the sample under study and, to a lesser extent, with differences in electron absorption by these areas. Contrast is also enhanced by using “ electronic dyes "(osmium tetroxide, uranyl, etc.), selectively binding to certain areas of the object. Modern transmission electron microscopes, designed in a similar way, provide maximum useful magnification up to 400,000 times, which corresponds to resolution at 5.0 Å. The fine structure of bacterial cells revealed using transmission electron microscopy is called ultrastructure .
IN reflective (scanning) electron microscope the image is created using electrons reflected (scattered) by the surface layer of an object when it is irradiated at a small angle (approximately a few degrees) to the surface. Accordingly, the formation of an image is due to the difference in electron scattering at different points of an object depending on its surface microrelief, and the result of such microscopy itself appears in the form of the structure of the surface of the observed object. Contrast can be enhanced by sputtering metal particles onto the surface of the object. The achieved resolution of microscopes of this type is about 100 Å.
Laboratory lesson in botany No. 1
Topic: “Structure of a microscope. Preparation of temporary preparations. The structure of a plant cell. Plasmolysis and deplasmolysis."
Purpose: 1. To study the structure of a microscope (brands - MBR, MBI, Biolam), the purpose of its parts. Learn the rules of working with a microscope.
- 2. Learn the technique of preparing temporary preparations.
- 3. Study the structural main components of a plant cell: membrane, cytoplasm, nucleus, plastids.
- 4. Get acquainted with the phenomenon of plasmolysis and deplasmolysis.
- 5. Learn to compare cells of different tissues with each other, find identical and different features in them.
Equipment: microscope, microcopying kit, sodium chloride or sucrose solution, iodine solution in potassium iodide, strips of filter paper, glycerin, methylene blue, slices of watermelon, tomato, onion with anthocyanin. microscope preparation cell
- 1. Familiarize yourself with the design of the biological microscope MBR-1 or Biolam. Write down the purpose of the main parts.
- 2. Familiarize yourself with the design of stereoscopic microscopes MBS - 1.
- 3. Write down the rules for working with a microscope.
- 4. Learn the technique of making temporary preparations.
- 5. Make a preparation of the epidermis of juicy onion scales and examine at low magnification a section of the epidermis consisting of a single layer of cells with clearly visible nuclei.
- 6. Study the structure of the cell at high magnification, first in a drop of water, then in a solution of iodine in potassium iodide.
- 7. Induce plasmolysis in onion scale cells by treating with a sodium chloride solution. Then transfer to a state of deplasmolysis. Sketch.
General remarks
A biological microscope is a device with which you can examine various cells and tissues of a plant organism. The design of this device is quite simple, but inept use of the microscope leads to its damage. That is why it is necessary to understand the structure of a microscope and the basic rules for working with it. In a microscope of any brand, the following parts are distinguished: optical, lighting and mechanical. The optical part includes: lenses and eyepieces.
Lenses serve to magnify the image of an object and consist of a system of lenses. The degree of lens magnification is directly dependent on the number of lenses. A high magnification lens has 8 - 10 lenses. The first lens facing the preparation is called the frontal lens. The microscope MBR - 1 is equipped with three lenses. The lens magnification is indicated on it by numbers: 8x, 40x, 90x. The operating state of the lens is distinguished, i.e., the distance from the cover glass to the front lens. The working distance with an 8x lens is 13.8 mm, with a 40x lens - 0.6 mm, with a 90x lens - 0.12 mm. It is necessary to handle higher magnification lenses very carefully and carefully so as not to damage the front lens in any way. Using a lens in a tube, an enlarged, real, but inverse image of the object is obtained and the details of its structure are revealed. The eyepiece serves to magnify the image coming from the lens and consists of 2 - 3 lenses mounted in a metal cylinder. The magnification of the eyepiece is indicated on it by the numbers 7x, 10x, 15x.
To determine the total magnification, multiply the objective magnification by the eyepiece magnification.
The lighting device consists of a mirror, a condenser with an iris diaphragm and is designed to illuminate an object with a beam of light.
A mirror serves to collect and direct rays of light falling from the mirror onto an object. The iris diaphragm is located between the mirror and the condenser and consists of thin metal plates. The diaphragm serves to regulate the diameter of the light flux directed by the mirror through the condenser to the object.
The mechanical system of the microscope consists of a stand for micro- and macroscrews, a tube holder, a revolver and a stage. The micrometer screw serves to slightly move the tube holder and the lens over distances measured in micrometers (µm). A full turn of the microscrew moves the tube holder by 100 microns, and a turn by one division by 2 microns. To avoid damage to the micrometer mechanism, it is allowed to turn the micrometer screw to the side no more than half a turn.
A macroscrew is used to significantly move the tube holder. It is usually used when focusing an object at low magnification. Eyepieces are inserted into the tube-cylinder from above. The revolver is designed for quickly changing lenses that are screwed into its sockets. The centered position of the lens is ensured by a latch located inside the revolver.
The object table is designed to place a drug on it, which is fixed on it using two locks.
Rules for working with a microscope
- 1. Wipe the optical part of the microscope with a soft cloth.
- 2. Place the microscope at the edge of the table so that the eyepiece is opposite the experimenter’s left eye and do not move the microscope during operation. The notebook and all items necessary for work are placed to the right of the microscope.
- 3. open the aperture completely. The condenser is placed in a half-lowered position.
- 4. Using a mirror, adjust the sun “beam”, looking into the hole of the object table. To do this, the condenser lens located under the opening of the stage must be brightly illuminated.
- 5. Move the microscope at low magnification (8x) to the working position - install the lens at a distance of 1 cm from the stage and, looking through the eyepiece, check the illumination of the field of view. It should be brightly lit.
- 6. The object being studied is placed on the stage and the microscope tube is slowly raised until a clear image appears. Review the entire drug.
- 7. To study any part of an object at high magnification, first place this area in the center of the field of view of a small lens. After this, turn the revolver so that the 40x lens takes the working position (do not lift the lens!). Using a microscope, a clear image of an object is achieved.
- 8. After finishing the work, transfer the revolver from high magnification to low magnification. The object is removed from the work table and the microscope is placed in an inoperative state.
Method of preparation of a microslide
- 1. Apply a drop of liquid (water, alcohol, glycerin) to a glass slide.
- 2. Use a dissecting needle to take a part of the object and place it in a drop of liquid. Sometimes a section of the organ being studied is made using a razor. Then, having selected the thinnest section, place it on a glass slide in a drop of liquid.
- 3. cover the object with a cover glass so that no air gets under it. To do this, take the cover glass by the edges with two fingers, draw the bottom edge to the edge of the drop of liquid and smoothly lower it, holding it with a dissecting needle.
- 4. The specimen is placed on the stage and examined.
Progress of the laboratory lesson
Using a scalpel, cut a small piece (about 1 cm2) from the fleshy scales of the onion. Remove the transparent film (epidermis) from the inner (concave) side with tweezers. Place in the prepared drop and apply a cover glass.
With low magnification, find the most illuminated place (least damaged, without folds or bubbles). Switch to high magnification. Examine and sketch one cell. Mark the membrane with pores, the wall layer of cytoplasm, the nucleus with nucleoli, the vacuole with cell sap. Then a solution of sodium chloride (plasmolytic) is dripped onto one side of the cover glass. On the opposite side, without moving the preparation, they begin to suck out the water with pieces of filter paper, while you need to look through a microscope and monitor what is happening in the cells. A gradual departure of the protoplast from the cell membrane is detected, due to the release of water from the cell sap. There comes a moment when the protoplast inside the cell is completely separated from the membrane and undergoes complete plasmolysis of the cell. Then replace the plasmolytic with water. To do this, carefully place a drop of water on the border of the cover glass with the slide and slowly wash the drug from the plasmolytic. It is observed that the cell sap gradually fills the entire volume of the vacuole, the cytoplasm is applied to the cell membrane, i.e. deplasmolysis occurs.
It is necessary to sketch the cell in plasmolated and deplasmolated states, to designate all parts of the cell: nucleus, membrane, cytoplasm.
Using the tables, draw a diagram of the submicroscopic structure of a plant cell and identify all components.
Onion skin
Cytoplasm core shell
Onion skin. Cell organelles.
Cytoplasm is an essential component of the cell in which complex and diverse processes of synthesis, respiration, and growth occur.
The nucleus is one of the most important organelles of the cell.
A shell is a skin-tight surface layer covering something.
Plasmolysis by adding sodium chlorine solution
Plasmolysis is the detachment of the cytoplasm from the cell membrane, which occurs as a result of water loss from the vacuole.
Deplasmolysis
Deplasmolysis is a phenomenon in which the protoplast returns to its reverse state.
Plasmolysis upon addition of sucrose
Deplasmolysis upon addition of sucrose
Conclusion: Today we got acquainted with the structure of a biological microscope, and also learned the technique of preparing temporary preparations. We studied the main structural components of a plant cell: membrane, cytoplasm, nucleus using the example of onion skin. And we got acquainted with the phenomenon of plasmolysis and deplasmolysis.
Questions for self-control
- 1. What parts of a cell can be viewed with an optical microscope?
- 2. Submicroscopic structure of a plant cell.
- 3. What organelles make up the submicroscopic structure of the nucleus?
- 4. What is the structure of the cytoplasmic membrane?
- 5. Differences between a plant cell and an animal cell?
- 6. How to prove the permeability of the cell membrane?
- 7. The importance of plasmolysis and deplasmolysis for a plant cell?
- 8. How is the connection between the nucleus and the cytoplasm accomplished?
- 9. Place of study of the topic “Cell” in the general biology course of high school.
Literature
- 1. A.E. Vasiliev et al. Botany (anatomy and morphology of plants), “Enlightenment”, M, 1978, pp. 5-9, pp. 20-35
- 2. Kiseleva N.S. Anatomy and morphology of plants. M. "Higher School", 1980, pp. 3-21
- 3. Kiseleva N.S., Shelukhin N.V. Atlas of plant anatomy. . "Higher School", 1976
- 4. Khrzhanovsky V.G. and others. Atlas on the anatomy and morphology of plants. "Higher School", M., 1979, pp. 19-21
- 5. Voronin N.S. Guide to laboratory exercises in plant anatomy and morphology. M., 1981, p.27-30
- 6. Tutayuk V.Kh. Anatomy and morphology of plants. M. "Higher School", 1980, pp. 3-21
- 7. D.T. Konysbaeva PRACTICUM ON ANATOMY AND MORPHOLOGY OF PLANTS
Functional parts of a microscope
The microscope includes three main functional parts:
1. Lighting part
Designed to create a light flux that allows you to illuminate an object in such a way that subsequent parts of the microscope perform their functions with extreme precision. The illumination part of a transmitted light microscope is located behind the object under the lens in direct microscopes and in front of the object above lens V inverted. The lighting part includes a light source (lamp and electrical power supply) and an optical-mechanical system (collector, condenser, field and aperture adjustable/iris diaphragms).
2. Reproducing part
Designed to reproduce an object in the image plane with the image quality and magnification required for research (i.e., to construct an image that would reproduce the object with the appropriate optics as accurately as possible and in all details microscope resolution, magnification, contrast and color rendering). The reproducing part provides the first stage of magnification and is located after the object to the microscope image plane.
The reproducing part includes lens and an intermediate optical system.
Modern microscopes of the latest generation are based on optical systems lenses, corrected to infinity. This additionally requires the use of so-called tube systems, which provide parallel beams of light emerging from lens, “collected” in the image plane microscope.
3. Visualization part
Designed to obtain a real image of an object on the retina of the eye, photographic film or plate, on the screen of a television or computer monitor with additional magnification (second stage of magnification).
The visualizing part is located between the image plane of the lens and the eyes of the observer ( camera, camera). The imaging part includes a monocular, binocular or trinocular visual attachment with an observation system ( eyepieces, which work like a magnifying glass).
In addition, this part includes additional magnification systems (magnification wholesaler/change systems); projection attachments, including discussion attachments for two or more observers; drawing apparatus; image analysis and documentation systems with corresponding adapter (matching) elements.
Structural and technological parts
Modern microscope consists of the following structural and technological parts:
optical;
mechanical;
electric.
Mechanical part of the microscope
The main structural and mechanical block of the microscope is tripod. The tripod includes the following main blocks: base And tube holder.
Base is a block on which the entire microscope. In simple microscopes, lighting mirrors or overhead illuminators are installed on the base. In more complex models, the lighting system is built into the base without or with a power supply.
Types of microscope bases
base with lighting mirror;
so-called “critical” or simplified lighting;
Keller lighting.
change unit lenses, having the following design options - turret device, threaded device for screwing lens, “sled” for threadless fastening lenses using special guides;
focusing mechanism for coarse and fine adjustment of the microscope for sharpness - mechanism for focusing movement of lenses or stages;
attachment point for replaceable object tables;
mounting unit for focusing and centering movement of the condenser;
attachment point for replaceable attachments (visual, photographic, television, various transmitting devices).
Microscopes may use stands to mount components (for example, a focusing mechanism in stereo microscopes or an illuminator mount in some models of inverted microscopes).
The purely mechanical component of the microscope is stage, intended for fastening or fixing an observation object in a certain position. Tables can be fixed, coordinated and rotating (centered and non-centered).
The study of microbial cells invisible to the naked eye is only possible with the help of microscopes. These devices make it possible to obtain images of the objects under study, magnified hundreds of times (light microscopes), tens and hundreds of thousands of times (electron microscopes).
A biological microscope is called a light microscope because it provides the ability to study an object in transmitted light in a light and dark field of view.
The main elements of modern light microscopes are mechanical and optical parts (Fig. 1).
The mechanical part includes a tripod, tube, revolving attachment, micromechanism box, object stage, macrometric and micrometric screws.
Tripod consists of two parts: the base and the tube holder (column). Base The rectangular microscope has four support platforms at the bottom, which ensures a stable position of the microscope on the surface of the work table. Tube holder connects to the base and can be moved in a vertical plane using macro- and micrometer screws. When the screws are rotated clockwise, the tube holder is lowered; when rotated counterclockwise, it rises from the drug. In the upper part of the tube holder it is reinforced head with a socket for a monocular (or binocular) attachment and a guide for a revolving attachment. The head is attached screw.
Tube – This is a microscope tube that allows you to maintain a certain distance between the main optical parts - the eyepiece and the lens. An eyepiece is inserted into the tube at the top. Modern models of microscopes have an inclined tube.
Turret nozzle is a concave disk with several slots into which 3 are screwed – 4 lenses. By rotating the revolving attachment, you can quickly install any lens into the working position under the hole in the tube.
Rice. 1. Microscope structure:
1 – base; 2 – tube holder; 3 – tube; 4 – eyepiece; 5 – revolving attachment; 6 – lens; 7 – object table; 8 – terminals pressing the drug; 9 – condenser; 10 – condenser bracket; 11 – handle for moving the condenser; 12 – folding lens; 13 – mirror; 14 – macroscrew; 15 – microscrew; 16 – box with micrometric focusing mechanism; 17 – head for attaching the tube and revolving nozzle; 18 – screw for fastening the head
Micromechanism box carries on one side a guide for the condenser bracket, and on the other, a guide for the tube holder. Inside the box is the microscope focusing mechanism, which is a system of gear wheels.
Subject table serves to place a drug or other research object on it. The table can be square or round, movable or fixed. The movable table moves in a horizontal plane using two side screws, which allows you to view the drug in different fields of view. On a fixed table, to examine an object in different fields of view, the specimen is moved by hand. In the center of the stage there is a hole for illumination from below by light rays directed from the illuminator. The table has two spring terminals, intended for fixing the drug.
Some microscope systems are equipped with a drug driver, which is necessary when examining the surface of a drug or when counting cells. The drug driver allows the drug to move in two mutually perpendicular directions. The drug dispenser has a system of rulers - verniers, with the help of which you can assign coordinates to any point of the object under study.
Macrometric screw(macroscrew) serves for preliminary approximate installation of the image of the object in question. When the macroscrew is rotated clockwise, the microscope tube lowers; when rotated counterclockwise, it rises.
Micrometer screw(microscrew) is used to accurately position the image of an object. The micrometer screw is one of the most easily damaged parts of the microscope, so it must be handled with care - do not rotate it to roughly set the image to avoid spontaneous lowering of the tube. When the microscrew is fully rotated, the tube moves 0.1 mm.
The optical part of the microscope consists of main optical parts (lens and eyepiece) and an auxiliary lighting system (mirror and condenser).
Lenses(from lat. objektum- object) is the most important, valuable and fragile part of the microscope. They are a system of lenses enclosed in a metal frame, on which the degree of magnification and numerical aperture are indicated. The outer lens, with its flat side facing the preparation, is called the frontal lens. It is she who provides the increase. The remaining lenses are called correction lenses and serve to eliminate deficiencies in the optical image that arise when examining the object under study.
Lenses are dry and immersion, or submersible. Dry A lens that has air between the front lens and the object being viewed is called a lens. Dry lenses usually have a long focal length and magnification of 8x or 40x. Immersion(submersible) is a lens that has a special liquid medium between the front lens and the specimen. Due to the difference between the refractive indices of glass (1.52) and air (1.0), some of the light rays are refracted and do not enter the observer's eye. As a result, the image is unclear and smaller structures remain invisible. Scattering of the light flux can be avoided by filling the space between the preparation and the front lens of the lens with a substance whose refractive index is close to the refractive index of glass. These substances include glycerin (1.47), cedar (1.51), castor (1.49), flaxseed (1.49), clove oil (1.53), anise oil (1.55) and other substances. Immersion lenses are marked on the frame: I (immersion) – immersion, NI (homogeneous immersion) – homogeneous immersion, OI (oilimmersion) or MI– oil immersion. Currently, synthetic products that match the optical properties of cedar oil are more often used as immersion liquids.
Lenses are distinguished by their magnification. The magnification value of the lenses is indicated on their frame (8x, 40x, 60x, 90x). In addition, each lens is characterized by a certain working distance. For an immersion lens this distance is 0.12 mm, for dry lenses with magnification 8x and 40x - 13.8 and 0.6 mm, respectively.
Eyepiece(from lat. ocularis- ophthalmic) consists of two lenses - ophthalmic (upper) and field (lower), enclosed in a metal frame. The eyepiece serves to magnify the image produced by the lens. The magnification of the eyepiece is indicated on its frame. There are eyepieces with working magnification from 4x to 15x.
When working with a microscope for a long time, you should use a binocular attachment. The nozzle bodies can move apart within the range of 55–75 mm, depending on the distance between the observer’s eyes. Binocular attachments often have their own magnification (about 1.5x) and correction lenses.
Condenser(from lat. condenso– compact, thicken) consists of two or three short-focus lenses. It collects the rays coming from the mirror and directs them to the object. Using a handle located under the stage, the condenser can be moved in a vertical plane, which leads to an increase in the illumination of the field of view when the condenser is raised and a decrease in it when the condenser is lowered. To adjust the light intensity, the condenser has an iris (petal) diaphragm, consisting of steel crescent-shaped plates. When the diaphragm is fully open, it is recommended to consider colored preparations; when the diaphragm opening is reduced, uncolored ones are recommended. Below the condenser is located flip-up lens in a frame, used when working with low magnification lenses, for example, 8x or 9x.
Mirror has two reflective surfaces - flat and concave. It is hinged at the base of the tripod and can be easily rotated. In artificial lighting, it is recommended to use the concave side of the mirror, in natural lighting – the flat side.
Illuminator acts as an artificial light source. It consists of a low-voltage incandescent lamp mounted on a tripod and a step-down transformer. On the transformer body there is a rheostat handle that regulates the intensity of the lamp and a toggle switch for turning on the illuminator.
In many modern microscopes, the illuminator is built into the base.
