Types of microscopes: description, main characteristics, purpose. How is an electron microscope different from a light microscope? The main parts of the microscope: mechanical, optical and lighting Parts of the microscope and their significance

Botany Lab #1

Topic: “The structure of the microscope. Preparation of temporary preparations. The structure of a plant cell. Plasmolysis and deplasmolysis.

Purpose: 1. To study the structure of the 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. To 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 the same and different features in them.

Equipment: microscope, microcopying kit, sodium chloride or sucrose solution, iodine solution in potassium iodide, filter paper strips, glycerin, methylene blue, slices of watermelon, tomato, onion with anthocyanin. microscope preparation cell

1. Get acquainted with the device of the biological microscope MBR - 1 or Biolam. Write down the purpose of the main parts.
2. Get acquainted with the device of stereoscopic microscopes MBS - 1.
3. Write down the rules for working with a microscope.
4. Learn the technique of making temporary preparations.
5. Prepare 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 exposure to 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 device of this device is quite simple, but inept use of the microscope leads to its damage. That is why it is necessary to learn the structure of the microscope, 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.

Objectives serve to magnify the image of an object and consist of a system of lenses. The degree of magnification of the lens is in direct proportion to the number of lenses. A high magnification lens has 8 to 10 lenses. The first lens facing the preparation is called the frontal. The MBR-1 microscope is equipped with three objectives. The magnification of the lens is indicated on it with numbers: 8x, 40x, 90x. Distinguish between the working state of the lens, 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. Higher magnification lenses must be handled very carefully and carefully so as not to damage the front lens in any way. With the help of 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 is used to enlarge 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 magnification of the objective by the magnification of the eyepiece.

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.

The mirror serves to collect and direct the rays of light falling from the mirror onto the 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 macro screws, a tube holder, a revolver and an object table. The micrometer screw is used to slightly move the tube holder, as well as the lens, over distances measured in micrometers (µm). A full turn of the microscrew moves the tube holder by 100 µm, and a turn by one division by 2 µm. To avoid damage to the micrometer mechanism, it is allowed to turn the micrometer screw to the side no more than half a turn.

The macro screw 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 to quickly change lenses that are screwed into its sockets. The centered position of the lens is provided by a latch located inside the revolver.

The object table is designed to place the preparation on it, which is fixed on it with the help of two locks.

Rules for working with a microscope

1. Wipe the optical part of the microscope with a soft cloth.
2. put 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 the items necessary for work are placed to the right of the microscope.
3. fully open the diaphragm. The condenser is placed in a semi-lowered position.
4. With the help of a mirror, set up a sunny "bunny", looking into the hole of the object table. To do this, the lens of the condenser located under the opening of the stage must be brightly illuminated.
5. transfer the microscope at low magnification (8x) to the working position - set the lens at a distance of 1 cm from the object stage and, looking into the eyepiece, check the illumination of the field of view. It must be brightly lit.
6. Place the object under study on the stage and slowly raise the microscope tube until a clear image appears. View the entire drug.
7. To study any part of the object at high magnification, first put this part in the center of the field of view of a small lens. After that, turn the revolver so that the 40x lens takes its working position (do not raise the lens!). With the help of a microscope, a clear visibility of the image of the object is achieved.
8. after finishing work, transfer the revolver from a large increase to a small one. The object is removed from the working table, the microscope is put into a non-working state.

Method for preparing a micropreparation

1. A drop of liquid (water, alcohol, glycerin) is applied to a glass slide.
2. With a dissecting needle, take a part of the object and place it in a drop of liquid. Sometimes a cut of the organ under study is made with a razor. Then, choosing the thinnest section, put it on a glass slide in a drop of liquid.
3. cover the object with a cover slip so that air does not get under it. To do this, the coverslip is taken by the edges with two fingers, the lower edge is drawn to the edge of the liquid drop and smoothly lowered, holding it with a dissecting needle.
4. the drug is placed on the object table and examined.

The course of the laboratory lesson

Cut a small piece (about 1 cm 2) from the fleshy scales of the bulb with a scalpel. Remove the transparent film (epidermis) from the inner side (concave) with tweezers. Put in the prepared drop and apply a coverslip.

With a low magnification, find the most illuminated place (the least damaged, without wrinkles and bubbles). Change to high magnification. Consider and draw one cell. Mark the membrane with pores, the parietal layer of the cytoplasm, the nucleus with nucleoli, the vacuole with cell sap. Then, a solution of sodium chloride (plasmolytic) is dripped from one side of the coverslip. On the opposite side, without moving the preparation, they begin to suck out water with pieces of filter paper, while looking through a microscope and monitoring what is happening in the cells. A gradual detachment 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 takes full plasmolysis of the cell. Then the plasmolytic is replaced with water. To do this, carefully place a drop of water on the border of the coverslip with the subject slowly wash the drug from the plasmolytic. It is observed that gradually the cell sap fills the entire volume of the vacuole, the cytoplasm is applied to the cell membrane, i.e. deplasmolysis occurs.

It is necessary to draw a cell in plasmolyated and deplasmolyated states, to designate all parts of the cell: nucleus, membrane, cytoplasm.

According to the tables, draw a diagram of the submicroscopic structure of a plant cell, designate all the components.

onion peel

Cytoplasm nucleus envelope

Onion peel. cell organelles.

The cytoplasm is an obligatory component of the cell, in which complex and diverse processes of synthesis, respiration, and growth take place.

The nucleus is one of the most important organelles of the cell.

A shell is a surface layer that wraps around something.

Plasmolysis by adding sodium chloride solution

Plasmolysis is the lagging of the cytoplasm from the cell membrane, which occurs as a result of the loss of water by the vacuole.

Deplasmolysis

Deplasmolysis is a phenomenon in which the protoplast returns to its reverse state.

Plasmolysis with the addition of sucrose

Deplasmolysis with the addition of sucrose

Conclusion: Today we got acquainted with the device of a biological microscope, we also learned the method of preparing temporary preparations. We studied the main structural components of a plant cell: membrane, cytoplasm, nucleus using onion skin as an example. And got acquainted with the phenomenon of plasmolysis and deplasmolysis.

Questions for self-control

1. What parts of a cell can be seen 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. What are the differences between a plant cell and an animal cell?
6. How to prove the permeability of the cell membrane?
7. Significance of plasmolysis and deplasmolysis for a plant cell?
8. How is the connection between the nucleus and the cytoplasm?
9. Place of study of the topic "Cell" in the course of general biology of high school.

Literature

1. A.E. Vasiliev and others. Botany (anatomy and morphology of plants), "Enlightenment", M, 1978, p.5-9, p.20-35
2. Kiseleva N.S. Anatomy and morphology of plants. M. "Higher School", 1980, p.3-21
3. Kiseleva N.S., Shelukhin N.V. Atlas of plant anatomy. . "High School", 1976
4. Khrzhanovsky V.G. and other Atlas of anatomy and morphology of plants. "Higher School", M., 1979, p.19-21
5. Voronin N.S. Guide to laboratory studies in anatomy and morphology of plants. M., 1981, p.27-30
6. Tutayuk V.Kh. Anatomy and morphology of plants. M. "Higher School", 1980, p.3-21
7. D.T. Konysbayeva WORKSHOP ON ANATOMY AND MORPHOLOGY OF PLANTS

Topic: Microscope Work No. 1. The device of a light microscope

Equipment: microscope, permanent preparation, pencil case.

Design of work: Write down the device of the microscope, the purpose of its parts, the rules of work.

A microscope is an optical-mechanical device that allows you to magnify the object in question (object, preparation).

In a microscope, optical and mechanical systems are distinguished.

OPTICAL SYSTEM:

The objective lens is the most important part of the microscope and is screwed to the bottom of the tube. The lens in the microscope is in close proximity to the object in question, for which it got its name. It consists of a system of optical lenses inserted into a brass frame and requires very careful handling and careful maintenance (in no way should you press the lens on the specimen lying on the stage, as this can cause damage or even fall out of the lens).

Purpose of the lens:

1) To build an image in the microscope tube that is geometrically similar to the object being studied.

2) Enlarge the image by a certain number of times.

3) Reveal details that are not visible to the naked eye. Lenses in Quantity 2-3 pieces are screwed into a special device called a revolver (4).

Eyepiece - inserted into the upper part of the tube. It considers the image of the object (and not the object), directed upward by the lens. It consists of a system of lenses inserted into a metal cylinder. The eyepiece builds an image, enlarges it, but does not reveal the details of the structure.

Condenser - collects and concentrates in the plane of the preparation all the light reflected from the mirror. The condenser consists of a cylinder (frame) inside which there are 2 lenses. Raising and lowering the condenser, you can adjust the illumination of the drug.

Diaphragm - located at the bottom of the condenser. Just like the condenser, it serves to regulate the intensity of light.

Mirror - serves to capture light from a light source. It is movably attached under the table, rotating around a horizontal axis. The mirror on one side is flat, on the other side it is concave.

MECHANICAL SYSTEM:

base (tripod) or massive leg (1); box with micromechanism (2) and microscrew (3);

feed mechanism for rough aiming - a macro screw or rack (8); object table (4);

screws (5, 6, 12, 13);

head (9); revolver (10); terminals; tube (11);

arc or tube holder (7); Cremalera (macroscrew)- serves for an approximate "rough" setting on the photo

Microscrew - serves for finer and more accurate aiming.

Subject table- attached to the front of the column, on which the test object is placed. There are 2 terminals on the table; with their help, the drug is fixed. The movement of the drug is carried out with the help of screws that are located on the side of the table.

Tube - serves to connect the lens and the eyepiece, and is connected to the tripod in such a way that it can be raised and lowered. The movement of the tube is carried out with the help of two screws: macrometric and micrometric.

Tripod - connects all the above parts of the microscope.

Determining the overall magnification of a microscope

Lens	10x	15x

Determining the focal length

F8=0.9cm~1cm

F40=1.2mm~1mm

Auxiliary equipment (remember the names):

1. glass slides and coverslips;

2. glass or cone for water, pipette;

3. razor (blade), dissecting needles;

4. filter paper strips, napkin.

Rules for working with a microscope:

Work with a microscope should be done without hasty and abrupt movements. Keep the microscope clean and tidy. Keep the microscope away from dust and dirt.

1. The transfer of the microscope is carried out with two hands: with one hand - by the tube holder, the other - from below by the base.

2. The microscope is installed directly in front of the worker, opposite his left eye, and does not move.

3. On the right side are the necessary tools, materials and sketchbook.

4. Before starting work, the eyepiece, lens, mirror are wiped from dust with a soft (preferably cambric) cloth.

5. Putting the microscope in a permanent place, lower the tube of the microscope with the help of a microscrew, while looking from the side of the microscope, so that the low magnification objective is at a distance of ~ 1 cm from the glass slide.

6. Each object is studied first at a low magnification, and then transferred to a large one.

7. Natural light is used for lighting, but not direct, solar or electric, matte is better.

8. Lighting installation:

a) remove the frosted glass under the condenser; b) install the condenser with the front lens at the level of the microscope stage (under-

take it out with a screw; c) fully open the diaphragm;

d) install a low magnification lens; e) direct the light by moving the mirror so that, after passing through the lens, the beam of light

it completely illuminated the plane of the entrance pupil of the lens.

9. After setting the illumination, we place the preparation on the object table so that the object under consideration is under the front lens of the low magnification objective. Then we lower the tube again with the help of a rack so that there is a distance between the front lens of the small objective and the cover slip of the preparation. 3-4 mm (when lowering the tube, you need to look not into the eyepiece, but from the side of the lens).

10. Looking into the eyepiece with the left eye (without closing the right one), we smoothly turn the cremaler screw with our right hand, we find the image, at the same time we give the object an advantageous position with the left hand.

11. Turning to a high magnification, we transfer the revolver and put a 40 lens in place of a small magnification X . At high magnification, by rotating the microscrew, a clear image is achieved (the microscrew is rotated no more than half a turn). Remember that turning the micro and macro screws clockwise lowers the lens barrel, while turning it back raises it.

12. After work, we again install a low magnification lens.

13. Only at low magnification should the specimen be removed from the microscope stage. After work, the microscope should be wiped with a napkin and placed under the cover.

Work number 2. Working with a microscope at low and high magnification.

Design of work: Write down the technique for preparing preparations.

Preparations and their preparation.

Medicines can be temporary or permanent. When making a temporary preparation, the object is placed in a drop of a transparent liquid - water or glycerin. Ta-

which drugs are not subject to long-term storage. In the case when the object of study is placed in a drop of hot glycerin-gelatin or Canada balsam, which hardens when cooled. It turns out a permanent drug that can be stored for years.

In practical classes in plant anatomy, students use both permanent and temporary preparations made by them on their own. To make a temporary preparation, you must:

o using a pipette, apply a drop of water or glycerin to the center of the glass slide; o with a dissecting needle, place the object in a drop of the prepared liquid;

o carefully cover the object with a thin (fragile) coverslip. The top of the coverslip must remain dry, i.e. water should not go beyond it. Excess water is removed with a strip of filter paper. If there is little liquid under the glass, you can add it by bringing the pipette to the edge of the coverslip without lifting it.

o the preparation often contains air bubbles that enter it together with the object or, when the cover slip is abruptly lowered, and interfere with the study of the object with their contours. They can be removed by adding water from one side of the coverslip while simultaneously removing it from the opposite side, or by lightly tapping the coverslip with a dissecting needle, holding the preparation almost vertically.

SCHOOL USE

The acquired knowledge and practical skills are used in the school biology course at the lesson "Introduction to magnifying instruments" and in the process of teaching the entire course of botany and other biological disciplines.

HOMEWORK: Learn the device of a microscope, the rules for working with it and the technique for preparing preparations.

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The design of the microscope directly depends on its purpose. As you probably already guessed, microscopes are different, and an optical microscope will differ significantly from an electron or X-ray microscope. This article will discuss in detail the structure optical light microscope, which is currently the most popular choice of amateurs and professionals, and with which you can solve many research problems.

Optical microscopes also have their own classification and may differ in their structure. However, there is a basic set of parts that go into any optical microscope. Let's look at each of these details.

In a microscope, optical and mechanical parts can be distinguished. The optics of a microscope includes objectives, eyepieces, and a lighting system. A tripod, a tube, an object table, fastenings of the condenser and light filters, mechanisms for adjusting the object table and the tube holder make up the mechanical part of the microscope.

Let's start with perhaps optical part .

Eyepiece. That part of the optical system that is directly connected with the eyes of the observer. In the simplest case, the lens consists of a single lens. Sometimes, for greater convenience, or, as they say, "ergonomics", the lens can be equipped, for example, with an "eyecup" made of rubber or soft plastic. Stereoscopic (binocular) microscopes have two eyepieces.
Lens. Perhaps the most important part of the microscope, providing the main magnification. The main parameter is aperture, what it is is described in detail in the "Basic parameters of microscopes" section. Objectives are divided into "dry" and "immersion", achromatic and apochromatic, and even in cheap simple microscopes they are a rather complex lens system. Some microscopes have unified lens mounting elements, which allows you to complete the device in accordance with the tasks and budget of the consumer.
Illuminator. Very often, an ordinary mirror is used, which makes it possible to direct daylight onto the test sample. Currently, special halogen lamps are often used, which have a spectrum close to natural white light and do not cause gross color distortions.
Diaphragm. Basically, microscopes use so-called "iris" diaphragms, so named because they contain petals similar to those of an iris flower. By shifting or expanding the petals, you can smoothly adjust the strength of the light flux that enters the sample that is not being studied.
Collector. With the help of a collector located near the light source, a light flux is created that fills the condenser aperture.
Condenser. This element, which is a converging lens, forms a light cone directed at the object. The intensity of illumination is controlled by the aperture. Most microscopes use a standard two-lens Abbe condenser.

It is worth noting that in an optical microscope one of two main methods of illumination can be used: illumination of transmitted light and illumination of reflected light. In the first case, the light flux passes through the object, as a result of which an image is formed. In the second - light is reflected from the surface of the object.

As for the optical system as a whole, depending on its structure, it is customary to distinguish direct microscopes (objectives, attachment, eyepieces are located above the object), inverted microscopes (the entire optical system is located under the object), stereoscopic microscopes (binocular microscopes, essentially consisting of two microscopes located at an angle to each other and forming a three-dimensional image).

Now let's move on to mechanical part of the microscope .

tube. The tube is the tube that holds the eyepiece. The tube must be strong enough, it must not deform, which will worsen the optical properties, therefore only in the cheapest models the tube is made of plastic, but aluminum, stainless steel or special alloys are more often used. To eliminate "glare", the inside of the tube, as a rule, is covered with black light-absorbing paint.
Base. Usually it is quite massive, made of metal casting, to ensure the stability of the microscope during operation. A tube holder, a tube, a condenser holder, focus knobs, a revolving device and a nozzle with eyepieces are attached to this base.
Turret for quick lens changes. As a rule, in cheap models with only one lens, this element is absent. The presence of a revolving head allows you to quickly adjust the magnification, changing lenses by simply turning it.
Subject table on which the test specimens are placed. These are either thin sections on glass slides - for "transmitted light" microscopes, or volumetric objects for "reflected light" microscopes.
Mounts used to fix the slides on the slide table.
Coarse focus screw. Allows, by changing the distance from the lens to the test sample, to achieve the clearest image.
Fine focus screw. The same, only with a smaller pitch and less "travel" of the thread for the most accurate adjustment.

Electrical part of the microscope

Unlike a magnifier, a microscope has at least two levels of magnification. The functional and structural-technological parts of the microscope are designed to ensure the operation of the microscope and obtain a stable, most accurate, magnified image of the object. Here we will look at the structure of the microscope and try to describe the main parts of the microscope.

Functionally, the microscope device is divided into 3 parts:

1. Lighting part

The lighting part of the microscope design includes a light source (a lamp and an electric power supply) and an optical-mechanical system (collector, condenser, field and aperture adjustable/iris diaphragms).

2. Playback part

Designed to reproduce an object in the image plane with the image quality and magnification required for research (i.e., to build such an image that reproduces the object as accurately as possible and in all details with the resolution, magnification, contrast and color reproduction corresponding to the microscope optics).
The reproducing part provides the first stage of magnification and is located after the object to the image plane of the microscope.
The reproducing part includes a lens and an intermediate optical system.

Modern microscopes of the latest generation are based on optical systems of lenses corrected for infinity. This additionally requires the use of so-called tube systems, which “collect” parallel beams of light coming out of the objective in the image plane of the microscope.

3. Visualizing part

Designed to obtain a real image of an object on the retina, photographic film or plate, on the screen of a television or computer monitor with additional magnification (the second stage of magnification).
The imaging part is located between the image plane of the lens and the eyes of the observer (digital camera).
The imaging part includes a monocular, binocular or trinocular visual attachment with an observational system (eyepieces that work like a magnifying glass).
In addition, this part includes systems of additional magnification (systems of a wholesaler / change of magnification); projection nozzles, including discussion nozzles for two or more observers; drawing devices; image analysis and documentation systems with appropriate adapters for digital cameras.

Layout of the main elements of an optical microscope

From a constructive and technological point of view, the microscope consists of the following parts:

mechanical;
optical;
electric.

1. The mechanical part of the microscope

Microscope device turns on tripod, which is the main structural and mechanical unit of the microscope. The tripod includes the following main blocks: base and tube holder.

Base is a block on which the entire microscope is mounted and is one of the main parts of the microscope. In simple microscopes, illuminating 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;
illumination according to Kohler.

a lens change unit with the following design options - a revolving device, a threaded device for screwing in the lens, a “sled” for threadless lens mounting using special guides;
focusing mechanism for coarse and fine adjustment of the microscope for sharpness - a mechanism for focusing movement of lenses or tables;
attachment point for interchangeable object tables;
attachment point for focusing and centering movement of the condenser;
attachment point for interchangeable nozzles (visual, photographic, television, various transmitting devices).

Microscopes may use racks to mount nodes (for example, the focusing mechanism in stereo microscopes or the illuminator mount in some models of inverted microscopes).

The purely mechanical part of the microscope is object table, intended for fastening or fixing in a certain position of the object of observation. Tables are fixed, coordinate and rotating (centered and non-centered).

2. Optics of the microscope (optical part)

Optical components and accessories provide the main function of the microscope - the creation of an enlarged image of an object with a sufficient degree of reliability in terms of shape, size ratio of the constituent elements and color. In addition, the optics must provide an image quality that meets the objectives of the study and the requirements of the analysis methods.
The main optical elements of a microscope are the optical elements that form the illuminating (including the condenser), observational (eyepieces) and reproducing (including lenses) systems of the microscope.

microscope objectives

- are optical systems designed to build a microscopic image in the image plane with the appropriate magnification, resolution of the elements, fidelity in the shape and color of the object of study. Objectives are one of the main parts of a microscope. They have a complex optical-mechanical design, which includes several single lenses and components glued from 2 or 3 lenses.
The number of lenses is determined by the range of tasks solved by the lens. The higher the image quality that the lens gives, the more complex its optical design. The total number of lenses in a compound lens can be up to 14 (for example, this could be a plan apochromat lens with a magnification of 100x and a numerical aperture of 1.40).

The lens consists of frontal and subsequent parts. The front lens (or lens system) is facing the preparation and is the main one in constructing an image of the appropriate quality, determines the working distance and the numerical aperture of the lens. The subsequent part in combination with the front provides the required magnification, focal length and image quality, and also determines the height of the objective and the length of the microscope tube.

Lens classification

The classification of lenses is much more complicated than the classification of microscopes. Lenses are divided according to the principle of calculated image quality, parametric and constructive-technological features, as well as research and contrast methods.

According to the principle of calculated image quality lenses can be:

achromatic;
apochromatic;
flat field lenses (plan).

Achromatic objectives.

Achromatic lenses are designed for use in the spectral range 486-656 nm. Correction of any aberration (achromatization) is performed for two wavelengths. These lenses eliminate spherical aberration, position chromatic aberration, coma, astigmatism, and partially spherochromatic aberration. The image of the object has a slightly bluish-reddish tint.

Apochromatic objectives.

Apochromatic objectives have an extended spectral region and achromatization is performed for three wavelengths. At the same time, in addition to position chromatism, spherical aberration, coma and astigmatism, the secondary spectrum and spherochromatic aberration are also corrected quite well, thanks to the introduction of lenses made of crystals and special glasses into the scheme. Compared to achromats, these lenses typically have larger numerical apertures, produce sharper images, and accurately reproduce the color of an object.

Semi-apochromats or microfluaries.

Modern lenses with intermediate image quality.

plan lenses.

In plan lenses, the curvature of the image along the field has been corrected, which provides a sharp image of the object over the entire field of observation. Plan lenses are usually used for photography, and the use of plan apochromats is most effective.

The need for this type of lenses is growing, but they are quite expensive due to the optical design that implements a flat image field and the optical media used. Therefore, routine and working microscopes are equipped with so-called economic objectives. These include lenses with improved image quality across the field: achrostigmata (LEICA), СР-achromats and achroplanes (CARL ZEISS), stigmachromats (LOMO).

By parametric features lenses are divided as follows:

objectives with a finite tube length (for example, 160 mm) and objectives corrected for the length of the tube "infinity" (for example, with an additional tube system having a microscope focal length of 160 mm);
small lenses (up to 10x); medium (up to 50x) and large (more than 50x) magnifications, as well as lenses with extra high magnification (over 100x);
objectives of small (up to 0.25), medium (up to 0.65) and large (more than 0.65) numerical apertures, as well as objectives with increased (compared to conventional) numerical apertures (for example, apochromatic correction objectives, as well as special objectives for fluorescent microscopes);
objectives with increased (compared to conventional) working distances, as well as with large and extra long working distances (objectives for work in inverted microscopes). The working distance is the free distance between the object (the plane of the coverslip) and the lower edge of the frame (lens if it protrudes) of the frontal lens component;
lenses providing observation within a normal linear field (up to 18 mm); wide-field lenses (up to 22.5 mm); ultra-wide-field lenses (more than 22.5 mm);
lenses are standard (45 mm, 33 mm) and non-standard in height.

Height - the distance from the reference plane of the lens (the plane of contact of the screwed-in lens with the revolving device) to the plane of the object with a focused microscope, is a constant value and ensures the parfocality of a set of lenses of different magnifications, similar in height, installed in the revolving device. In other words, if a sharp image of an object is obtained using a lens of one magnification, then when moving to subsequent magnifications, the image of the object remains sharp within the depth of field of the lens.

By constructive and technological features there is the following division:

lenses with and without a spring-loaded frame (starting with a numerical aperture of 0.50);
lenses having an iris diaphragm inside to change the numerical aperture (for example, in lenses with an increased numerical aperture, in transmitted light lenses for implementing the dark field method, in polarized reflected light lenses);
lenses with a corrective (control) frame that provides movement of optical elements inside the lens (for example, to correct the image quality of the lens when working with different thicknesses of the coverslip or with different immersion liquids; as well as to change the magnification during a smooth - pancratic - change of magnification) and without her.

To provide methods of research and contrasting Lenses can be divided as follows:

objectives working with and without cover glass;
lenses of transmitted and reflected light (reflexless); luminescent lenses (with a minimum of intrinsic luminescence); polarizing lenses (without glass tension in optical elements, i.e., not introducing their own depolarization); phase lenses (having a phase element - a translucent ring inside the lens); lenses DIC (DIC), working on the method of differential interference contrast (polarizing with a prism element); epi-objectives (reflected light objectives designed to provide bright and dark field methods have specially designed lighting epi-mirrors in their design);
immersion and non-immersion lenses.

Immersion ( from lat. immersio - immersion) is a liquid that fills the space between the object of observation and a special immersion objective (condenser and glass slide). Three types of immersion liquids are mainly used: oil immersion (MI/Oil), water immersion (VI/W) and glycerol immersion (GI/Glyc), the latter being mainly used in ultraviolet microscopy.
Immersion is used in cases where it is required to increase the resolution of the microscope or its application is required by the technological process of microscopy. When this happens:

increased visibility by increasing the difference between the refractive index of the medium and the object;
increase in the depth of the viewed layer, which depends on the refractive index of the medium.

In addition, the immersion liquid can reduce the amount of stray light by eliminating glare from the object. This eliminates the inevitable loss of light when it enters the lens.

immersion lenses. The image quality, parameters and optical design of immersion objectives are calculated and selected taking into account the thickness of the immersion layer, which is considered as an additional lens with an appropriate refractive index. The immersion liquid placed between the object and the front lens component increases the angle at which the object is viewed (aperture angle). The numerical aperture of the immersion-free (dry) objective does not exceed 1.0 (resolution is about 0.3 µm for the main wavelength); immersion - reaches 1.40, depending on the refractive index of immersion and the technological capabilities of manufacturing the front lens (the resolution of such a lens is about 0.12 microns).
High magnification immersion lenses have a short focal length of 1.5-2.5 mm with a free working distance of 0.1-0.3 mm (the distance from the preparation plane to the frame of the front lens of the objective).

Lens markings.

Data about each lens is marked on its body with the following parameters:

magnification ("x"-fold, times): 8x, 40x, 90x;
numerical aperture: 0.20; 0.65, example: 40/0.65 or 40x/0.65;
additional letter marking if the lens is used for various methods of examination and contrast: phase - Ф (Рп2 - the number corresponds to the marking on a special condenser or insert), polarizing - P (Pol), luminescent - L (L), phase-luminescent - FL ( PhL), EPI (Epi, HD) - epi-objective for working in reflected light using the dark field method, differential interference contrast - DIC (DIC), example: 40x / 0.65 F or Ph2 40x / 0.65;
optical correction type marking: apochromat - APO (APO), planachromat - PLAN (PL, Plan), planachromat - PLAN-APO (Plan-Aro), improved achromat, semi-plan - CX - stigmachromat (Achrostigmat, CP-achromat, Achroplan), microfluar (semi-plan-semi-apochromat) - SF or M-FLUAR (MICROFLUAR, NEOFLUAR, NPL, FLUOTAR).

Eyepieces

Optical systems designed to build a microscopic image on the retina of the observer's eye. In general, eyepieces consist of two groups of lenses: the eye lens, which is closest to the observer's eye, and the field lens, which is closest to the plane in which the lens builds an image of the object in question.

Eyepieces are classified according to the same groups of features as lenses:

eyepieces of compensatory (K - compensate for the chromatic difference in the magnification of the lenses over 0.8%) and non-compensated action;
regular and flat field eyepieces;
wide-angle eyepieces (with an ocular number - the product of the eyepiece magnification and its linear field - more than 180); ultra wide-angle (with an eyepiece number of more than 225);
eyepieces with extended pupil for work with and without glasses;
observation eyepieces, projection eyepieces, photo eyepieces, gamals;
eyepieces with internal aiming (with the help of a movable element inside the eyepiece, adjustment is made to a sharp image of the grid or the image plane of the microscope; as well as a smooth, pancratic change in the eyepiece magnification) and without it.

Lighting system

The lighting system is an important part microscope designs and is a system of lenses, diaphragms and mirrors (the latter are used if necessary), which provides uniform illumination of the object and complete filling of the lens aperture.
The illumination system of a transmitted light microscope consists of two parts, a collector and a condenser.

Collector.
With a built-in transmitted light illumination system, the collector part is located near the light source at the base of the microscope and is designed to increase the size of the luminous body. To ensure tuning, the collector can be made movable and move along the optical axis. Near the collector is the field diaphragm of the microscope.

Condenser.
The optical system of the condenser is designed to increase the amount of light entering the microscope. The condenser is located between the object (subject table) and the illuminator (light source).
Most often, in educational and simple microscopes, the condenser can be made non-removable and motionless. In other cases, the condenser is a removable part and, when adjusting the illumination, has a focusing movement along the optical axis and a centering movement perpendicular to the optical axis.
The condenser always has an illuminating aperture iris diaphragm.

The condenser is one of the main elements that ensure the operation of the microscope in various methods of illumination and contrast:

oblique illumination (diaphragm from the edge to the center and displacement of the illumination aperture diaphragm relative to the optical axis of the microscope);
dark field (maximum aperture from the center to the edge of the illumination aperture);
phase contrast (annular illumination of the object, while the image of the light ring fits into the phase ring of the lens).

Classification of condensers close in groups of features to the lenses:

condensers according to image quality and type of optical correction are divided into non-achromatic, achromatic, aplanatic and achromatic-aplanatic;
condensers of small numerical aperture (up to 0.30), medium numerical aperture (up to 0.75), large numerical aperture (over 0.75);
conventional, long and extra long working distance condensers;
conventional and special condensers for various research and contrasting methods;
the condenser design is single, with a folding element (frontal component or large-field lens), with a screwed-in frontal element.

Abbe condenser- a condenser not corrected for image quality, consisting of 2 non-achromatic lenses: one is biconvex, the other is plano-convex, facing the object of observation (the flat side of this lens is directed upwards). Condenser aperture, A= 1.20. Has an iris diaphragm.

Aplanatic condenser- a condenser consisting of three lenses arranged as follows: the upper lens is plano-convex (the flat side is directed towards the lens), followed by concave-convex and biconvex lenses. Corrected for spherical aberration and coma. Condenser aperture, A = 1.40. Has an iris diaphragm.

Achromatic condenser- condenser fully corrected for chromatic and spherical aberration.

Dark field condenser- a condenser designed to obtain the effect of a dark field. It can be special or converted from a conventional bright-field condenser by installing an opaque disk of a certain size in the plane of the iris diaphragm of the condenser.

Condenser marking.
On the front of the condenser, the marking of the numerical aperture (illumination) is applied.

3. Electrical part of the microscope

In modern microscopes, instead of mirrors, various light sources are used, powered by an electrical network. It can be both conventional incandescent lamps, and halogen, and xenon, and mercury lamps. LED lights are also becoming more and more popular. They have significant advantages over conventional lamps, such as durability, lower power consumption, etc. To power the light source, various power supplies, ignition units and other devices are used that convert current from the electrical network to a suitable one for powering a particular light source. It can also be rechargeable batteries, which allows you to use microscopes in the field in the absence of a connection point.

The study of microorganism cells invisible to the naked eye is possible only with the help of microscopes. These devices make it possible to obtain an image 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, since it provides the ability to study an object in transmitted light in a bright and dark field of view.

The main elements of modern light microscopes are the mechanical and optical parts (Fig. 1).

The mechanical part includes a tripod, a tube, a turret, a micromechanism box, an object stage, macrometric and micrometric screws.

Tripod consists of two parts: a base and a tube holder (column). Base Rectangular-shaped microscope has four support platforms at the bottom, which ensures a stable position of the microscope on the surface of the desktop. tube holder connects to the base and can be moved in a vertical plane with macro and micrometer screws. Turning the screws clockwise lowers the tube holder, while turning it counterclockwise raises it away from the preparation. At the top of the tube holder is reinforced head with a socket for a monocular (or binocular) nozzle and a guide for a revolving nozzle. 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 objective. 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 sockets into which 3 – 4 lenses. By rotating the turret, you can quickly set any lens to its working position under the opening of the tube.

Rice. 1. Microscope device:

1 - base; 2 - tube holder; 3 - tube; 4 - eyepiece; 5 - revolver nozzle; 6 - lens; 7 - subject table; 8 - terminals pressing the preparation; 9 - condenser; 10 – condenser bracket; 11 – handle for moving the condenser; 12 - folding lens; 13 - mirror; 14 - macro screw; 15 - microscrew; 16 - a box with a micrometric focusing mechanism; 17 - head for mounting the tube and turret; 18 - screw for fixing the head

micro-gear 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 focusing mechanism of the microscope, which is a system of gears.

Subject table serves to place a drug or other object of study on it. The table can be square or round, movable or fixed. The movable table moves in a horizontal plane with the help of two side screws, which allows you to view the drug in different fields of view. On a fixed table for examining an object in different fields of view, the drug is moved by hand. In the center of the object table there is a hole for illumination from below by light rays directed from the illuminator. The table has two spring terminals designed to fix the drug.

Some microscope systems are equipped with a slider, which is necessary when examining the surface of a slide or when counting cells. The drug guide allows the movement of the drug in two mutually perpendicular directions. There is a system of rulers - verniers on the preparation master, with the help of which it is possible to assign coordinates to any point of the object under study.

macrometric screw(macro screw) is used for preliminary orientation of the image of the object in question. Turning the macroscrew clockwise lowers the microscope tube, while turning it counterclockwise raises it.

micrometer screw(microscrew) is used to accurately set the image of the 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 in order to roughly set the image in order to prevent the tube from spontaneously lowering. When the microscrew is fully turned, the tube moves 0.1 mm.

The optical part of the microscope consists of the main optical parts (objective and eyepiece) and an auxiliary lighting system (mirror and condenser).

Lenses(from lat. objektum- subject) - 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 facing the preparation with its flat side is called the frontal lens. It is she who provides the increase. The remaining lenses are called corrective lenses and serve to eliminate the shortcomings of the optical image that arise when examining the object under study.

Lenses are dry and immersion, or submersible. Dry a lens is called, in which there is air between the front lens and the object in question. Dry lenses usually have long focal lengths and magnifications of 8x or 40x. Immersion(submersible) is called a lens in which a special liquid medium is located between the front lens and the preparation. Due to the difference between the refractive indices of glass (1.52) and air (1.0), part of the light rays is refracted and does not enter the eye of the observer. As a result, the image is fuzzy, smaller structures remain invisible. It is possible to avoid scattering of the light flux by filling the space between the preparation and the front lens of the objective with a substance whose refractive index is close to that of glass. These substances include glycerin (1.47), cedar (1.51), castor (1.49), linseed (1.49), clove (1.53), anise oil (1.55) and other substances. Immersion lenses have the designations on the frame: I (immersion) – immersion, HI (homogeneous immersion) is a homogeneous immersion, OI (oilimmersion) or MI- oil immersion. At present, as an immersion liquid, synthetic products are more often used, which correspond in optical properties to cedar oil.

Lenses are distinguished by their magnification. The magnification of 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 a magnification of 8x and 40x - 13.8 and 0.6 mm, respectively.

Eyepiece(from lat. ocularis- eye) consists of two lenses - eye (upper) and field (lower), enclosed in a metal frame. The eyepiece is used to magnify the image that the lens gives. The magnification of the eyepiece is indicated on its frame. There are eyepieces with a working magnification from 4x to 15x.

When working with a microscope for a long time, a binocular attachment should be used. The nozzle bodies can move apart within 55–75 mm, depending on the distance between the observer's eyes. Binocular attachments often have their own magnification (about 1.5x) and corrective lenses.

Condenser(from lat. condenso- condense, thicken) consists of two or three short-focus lenses. He collects the rays coming from the mirror and directs them to the object. With the help of a handle located under the object 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 intensity of illumination in the condenser there is an iris (petal) diaphragm, consisting of steel sickle-shaped plates. With a fully open diaphragm, it is recommended to consider stained preparations; with a reduced aperture of the diaphragm, unstained preparations are recommended. Below the condenser is flip lens framed, used when working with low magnification lenses, such as 8x or 9x.

Mirror It has two reflective surfaces - flat and concave. It is hinged at the base of the tripod and can be easily rotated. In artificial light, it is recommended to use the concave side of the mirror, in natural light - flat.

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 case there is a rheostat handle that regulates the incandescence of the lamp and a toggle switch to turn on the illuminator.

In many modern microscopes, the illuminator is built into the base.