1. All living organisms on Earth consist of cells that are similar in structure, chemical composition and functioning. This speaks of the kinship (common origin) of all living organisms on Earth (the unity of the organic world).

2. The cage is:

• structural unit (organisms are made up of cells)
• functional unit (body functions are performed due to the work of cells)
• genetic unit (the cell contains hereditary information)
• unit of growth (an organism grows due to the multiplication of its cells)
• unit of reproduction (reproduction occurs due to germ cells)
• unit of vital activity (processes of plastic and energy metabolism occur in the cell), etc.

3. All new daughter cells are formed from existing mother cells through division.

4. The growth and development of a multicellular organism occurs due to the growth and reproduction (through mitosis) of one or more original cells.

Guys

Hook opened the cells.

Leeuwenhoek discovered living cells (sperm, red blood cells, ciliates, bacteria).

Brown opened the core.

Schleiden And Schwann developed the first cell theory (“All living organisms on Earth consist of cells that are similar in structure”).

Methods

1. Light microscope increases up to 2000 times (regular school - from 100 to 500 times). The nucleus, chloroplasts, and vacuole are visible. You can study the processes occurring in a living cell (mitosis, movement of organelles, etc.).

2. Electron microscope increases up to 10 7 times, which makes it possible to study the microstructure of organelles. The method does not work with living objects.

3. Ultracentrifuge. The cells are destroyed and placed in a centrifuge. Cell components are separated according to density (the heaviest parts are collected at the bottom of the tube, the lightest on the surface). The method allows selective isolation and study of organelles.

Choose two correct answers out of five and write down the numbers under which they are indicated. Specify the formulation of one of the provisions of the cell theory
1) The fungal cell wall consists of carbohydrates
2) Animal cells lack a cell wall
3) The cells of all organisms contain a nucleus
4) Cells of organisms are similar in chemical composition
5) New cells are formed by dividing the original mother cell

Answer

Choose three options. What provisions does the cell theory contain?
1) New cells are formed as a result of division of the mother cell
2) Sex cells contain a haploid set of chromosomes
3) Cells are similar in chemical composition
4) The cell is the unit of development of all organisms
5) The tissue cells of all plants and animals are the same in structure
6) All cells contain DNA molecules

Answer

1) biogenic migration of atoms
2) relatedness of organisms

4) the appearance of life on Earth about 4.5 billion years ago

6) relationships between living and inanimate nature

Answer

Choose one, the most correct option. Which method allows you to selectively isolate and study cell organelles?
1) coloring
2) centrifugation
3) microscopy
4) chemical analysis

Answer

Choose one, the most correct option. Due to the fact that nutrition, respiration, and the formation of waste products occur in any cell, it is considered a unit
1) growth and development
2) functional
3) genetic
4) body structure

Answer

Choose three options. The basic principles of cell theory allow us to draw conclusions about
1) the influence of the environment on fitness
2) relatedness of organisms
3) the origin of plants and animals from a common ancestor
4) the development of organisms from simple to complex
5) similar structure of cells of all organisms
6) the possibility of spontaneous generation of life from inanimate matter

Answer

Choose three options. Similar structure of plant and animal cells - proof
1) their relationship
2) common origin of organisms of all kingdoms
3) the origin of plants from animals
4) complications of organisms in the process of evolution
5) unity of the organic world
6) diversity of organisms

Answer

Choose one, the most correct option. The cell is considered the unit of growth and development of organisms, since
1) it has a complex structure
2) the body consists of tissues
3) the number of cells increases in the body through mitosis
4) gametes participate in sexual reproduction

Answer

Choose one, the most correct option. A cell is a unit of growth and development of an organism, since
1) it has a core
2) it stores hereditary information
3) it is capable of division
4) tissues are made up of cells

Answer

1. Choose two correct answers out of five and write down the numbers under which they are indicated. Using light microscopy in a plant cell one can distinguish:
1) endoplasmic reticulum
2) microtubules
3) vacuole
4) cell wall
5) ribosomes

Answer

2. Choose two correct answers out of five and write down the numbers under which they are indicated. You can see with a light microscope
1) cell division
2) DNA replication
3) transcription
4) photolysis of water
5) chloroplasts

Answer

3. Choose two correct answers out of five and write down the numbers under which they are indicated. When studying a plant cell under a light microscope, you can see
1) cell membrane and Golgi apparatus
2) membrane and cytoplasm
3) nucleus and chloroplasts
4) ribosomes and mitochondria
5) endoplasmic reticulum and lysosomes

Answer

Choose two correct answers out of five and write down the numbers under which they are indicated. The following people contributed to the development of cell theory:
1) Oparin
2) Vernadsky
3) Schleiden and Schwann
4) Mendel
5) Virchow

Answer

Choose two correct answers out of five and write down the numbers under which they are indicated. The centrifugation method allows
1) determine the qualitative and quantitative composition of substances in the cell
2) determine the spatial configuration and some physical properties of macromolecules
3) purify macromolecules removed from the cell
4) obtain a three-dimensional image of the cell
5) divide cell organelles

Answer

Choose two correct answers out of five and write down the numbers under which they are indicated. What is the advantage of using electron microscopy over light microscopy?
1) higher resolution
2) the ability to observe living objects
3) the high cost of the method
4) the complexity of preparing the drug
5) the ability to study macromolecular structures

Answer

Choose two correct answers out of five and write down the numbers under which they are indicated. What organelles were discovered in the cell using an electron microscope?
1) ribosomes
2) kernels
3) chloroplasts
4) microtubules
5) vacuoles

Answer

Identify two characteristics that “drop out” from the general list, and write down the numbers under which they are indicated in your answer. The basic principles of cell theory allow us to conclude that
1) biogenic migration of atoms
2) relatedness of organisms
3) the origin of plants and animals from a common ancestor
4) the appearance of life on Earth about 4.5 billion years ago
5) similar structure of cells of all organisms

Answer

1. Select two correct answers out of five and write down the numbers under which they are indicated in the table. Methods used in cytology
1) hybridological
2) genealogical
3) centrifugation
4) microscopy
5) monitoring

Answer

© D.V. Pozdnyakov, 2009-2019

By the end of the 19th century. most of the structures that can be seen from using a light microscope(i.e., a microscope that uses visible light to illuminate an object) was already discovered. The cell was then imagined as something like a small lump of living protoplasm, always surrounded by a plasma membrane, and sometimes, as, for example, in plants, by a non-living cell wall. The most prominent structure in the cell was the nucleus, which contains an easily stained material - chromatin (the word translated means “colored material”).

Chromatin is a despiralized form chromosomes. Before cell division, chromosomes look like long, thin threads. Chromosomes contain DNA, the genetic material. DNA regulates the life of the cell and has the ability to replicate, i.e. it provides formation of new cells.

The figures show generalized animals and plant cells as they appear under a light microscope. (The “generalized” cell shows all the typical structures found in any cell.)

Single cell structures, which are shown here and which by the end of the 19th century. have not yet been discovered - these are lysosomes. The pictures show microphotographs of some animal and plant cells.

Live cell contents, filling the space between its nucleus and the plasma membrane is called cytoplasm. The cytoplasm contains many different organelles. An organelle is a cellular structure of a specific structure that performs a specific function. The only structure found in animal cells that is absent in plant cells is the centriole. In general, plant cells are very similar to animal cells, but they contain more different structures. Unlike animal cells, plant cells have:

1) relatively rigid cell wall, covering the outside of the plasma membrane; thin threads, the so-called plasmodesmata, pass through the pores in the cell wall, which connect the cytoplasm of neighboring cells into a single whole;
2) chloroplast, in which photosynthesis occurs;
3) large central vacuole; in animal cells there are only small vacuoles, with the help of which, for example, .

About how to use light microscope the reader will find out in the corresponding article.

Prokaryotes and eukaryotes

In the previous article we already talked about two types of cells - prokaryotes cultural and eukaryotes ical, - the differences between which are fundamental. In prokaryotic cells, DNA lies free in the cytoplasm, in an area called the nucleoid; This is not a real core. In eukaryotic cells, DNA is located in the nucleus, surrounded by a nuclear envelope consisting of two membranes. DNA combines with protein to form chromosomes. The differences between prokaryotic and eukaryotic cells are discussed in more detail in the corresponding article.

Many methods have been developed and used to study cells, the capabilities of which determine the level of our knowledge in this area. Advances in the study of cell biology, including the most outstanding achievements of recent years, are usually associated with the use of new methods. Therefore, for a more complete understanding of cell biology, it is necessary to have at least some understanding of the appropriate methods for studying cells.

Light microscopy

The oldest and, at the same time, the most common method of studying cells is microscopy. We can say that the beginning of the study of cells was laid by the invention of the light optical microscope.

The naked human eye has a resolution of about 1/10 mm. This means that if you look at two lines that are less than 0.1mm apart, they merge into one. To distinguish structures located more closely, optical instruments, such as a microscope, are used.

But the possibilities of a light microscope are not limitless. The resolution limit of a light microscope is set by the wavelength of light, that is, an optical microscope can only be used to study structures whose minimum dimensions are comparable to the wavelength of light radiation. The best light microscope has a resolving power of about 0.2 microns (or 200 nm), which is about 500 times better than the human eye. It is theoretically impossible to build a light microscope with high resolution.

Many components of the cell are similar in their optical density and, without special treatment, are practically invisible in a conventional light microscope. In order to make them visible, various dyes with a certain selectivity are used.

At the beginning of the 19th century. There was a need for dyes for dyeing textile fabrics, which in turn caused the accelerated development of organic chemistry. It turned out that some of these dyes also stain biological tissues and, quite unexpectedly, often preferentially bind to certain components of the cell. The use of such selective dyes makes it possible to more accurately study the internal structure of the cell. Here are just a few examples:

· hematoxylin dye colors some components of the nucleus blue or violet;

· after treatment sequentially with phloroglucinol and then with hydrochloric acid, the lignified cell membranes become cherry red;

· Sudan III dye stains suberized cell membranes pink;

A weak solution of iodine in potassium iodide turns starch grains blue.

For microscopic examination, most tissues are fixed before staining. Once fixed, the cells become permeable to dyes and the cell structure is stabilized. One of the most common fixatives in botany is ethyl alcohol.

Fixation and staining are not the only procedures used to prepare preparations. Most tissues are too thick to be immediately observed at high resolution. Therefore, thin sections are performed using a microtome. This device uses the bread slicer principle. Plant tissues are cut into slightly thicker sections than animal tissues because plant cells are typically larger. The thickness of plant tissue sections for light microscopy is about 10 microns - 20 microns. Some tissues are too soft to be cut straight away. Therefore, after fixation, they are poured into molten paraffin or special resin, which saturate the entire fabric. After cooling, a solid block is formed, which is then cut using a microtome. True, filling is used much less frequently for plant tissues than for animals. This is explained by the fact that plant cells have strong cell walls that make up the tissue frame. Lignified shells are especially strong.

However, pouring can disrupt the structure of the cell, so another method is used where this danger is reduced? quick freezing. Here you can do without fixing and filling. Frozen tissue is cut using a special microtome (cryotome).

Frozen sections prepared in this manner have the distinct advantage of better preserving the natural structural features. However, they are more difficult to cook, and the presence of ice crystals still ruins some of the details.

Microscopists have always been concerned about the possibility of loss and distortion of some cell components during the fixation and staining process. Therefore, the results obtained are verified by other methods.

The opportunity to study living cells under a microscope seemed very tempting, but in such a way that the details of their structure would appear more clearly. This opportunity is provided by special optical systems: phase-contrast and interference microscopes. It is well known that light waves, like water waves, can interfere with each other, increasing or decreasing the amplitude of the resulting waves. In a conventional microscope, as light waves pass through individual components of a cell, they change their phase, although the human eye cannot detect these differences. But due to interference, the waves can be converted, and then the different components of the cell can be distinguished from each other under a microscope, without resorting to staining. These microscopes use 2 beams of light waves that interact (superpose) on each other, increasing or decreasing the amplitude of the waves entering the eye from different components of the cell.

In order to be able to see a small object, it is necessary to enlarge it. Magnification is achieved using a system of lenses located between the examiner's eye and the object. Contrast and resolution are of great importance for microscopic observations, allowing one to clearly distinguish an object from the background and separately see very close details of the image. Depending on the principle of image creation, microscopy is divided into light, electron and laser.

Modern light microscopes are complex and have three lens systems (Fig. 2.1). The condenser system is responsible for properly illuminating the field of view and is located between the light source and the object. With an external light source, the rays are directed into the condenser by a mirror. Many modern microscopes have a built-in light source and do not have a mirror. The image of the objective lens system facing the object and the eyepiece in contact with the researcher’s eye are magnified. Total magnification is defined as the product of the objective magnification and the eyepiece magnification. The resolving power of a microscope depends on the wavelength of the light used, the optical properties of the lenses, and the refractive index of the medium in contact with the outer objective lens.

Rice. 2.1.

The simplest technique to increase the resolution of a microscope is the use of immersion. A drop of liquid whose refractive index exceeds the refractive index of air is placed between the outer lens of the lens and the object. A special immersion lens is used for each liquid. The most common are water (white ring) and oil (black ring) lenses. Modifications of conventional bright-field microscopy are ultraviolet, dark-field, and phase-contrast microscopy.

The use of shorter wavelength ultraviolet rays also improves the resolution of the microscope. However, the use of special light sources and quartz optics lead to a significant increase in the cost of microscopic studies.

In dark-field microscopy, the object is illuminated only by oblique side beams using a special dark-field condenser. With this lighting, the field of view remains dark, and small particles glow with reflected light. Dark-field microscopy allows one to discern the contours of objects that lie beyond the visibility of a conventional microscope, such as prokaryotic flagella. However, with this method of observation it is impossible to examine the internal structure of the object.

When using a phase-contrast device, you can observe living transparent objects that practically do not differ in density from the surrounding background. The color and brightness of the rays passing through such objects almost do not change, but a phase shift occurs that is not registered by the human eye. A phase contrast device, used as an attachment to a conventional microscope, converts phase differences in light waves into changes in their color and brightness. Transparent objects become clearer, and even individual structures and inclusions can be observed in the cells of large microorganisms.

Lecture 13. Microscopy as a method for studying cells and tissues.

1. Light microscopy.

2. Electron microscopy.

Modern cytology has numerous and varied research methods, without which it would be impossible to accumulate and improve knowledge about the structure and functions of cells. In this chapter we will get acquainted only with the basic, most important research methods.

The modern light microscope is a very advanced device, which is still of paramount importance in the study of cells and their organelles. Using a light microscope, magnification of 2000-2500 times is achieved. The magnification of a microscope depends on its resolution, i.e., the smallest distance between two points that are visible separately.

The smaller the particle visible through a microscope, the greater its resolution. The latter, in turn, is determined by the lens aperture (aperture is the actual opening of the optical system, determined by the size of the lenses or diaphragms) and the wavelength of the light.

The resolution of a microscope is determined using the formula: a = 0.6, where a is the minimum distance between two points; -- wavelength of light; n is the refractive index of the medium located between the preparation and the first, i.e., frontal, objective lens; a is the angle between the optical axis of the lens and the most strongly deviating ray entering the lens, or the angle of diffraction of the rays.

The value indicated in the denominator of the fraction (n sin a) is constant for each lens and is called its numerical aperture. The numerical aperture as well as the magnification are engraved on the lens barrel. The relationship between the numerical aperture and the minimum resolvable distance is as follows: the larger the numerical aperture, the smaller this distance, i.e., the higher the resolution of the microscope.

Increasing the resolution of the microscope, which is absolutely necessary for studying the details of the cell structure, is achieved in two ways:

1) increasing the numerical aperture of the lens;

2) reducing the wavelength of light that illuminates the drug.

In order to increase the numerical aperture, immersion objectives are used. The liquids used are: water (r = 1.33), glycerin (r = 1.45), cedar oil (/1 = 1.51) compared to air n equal to 1.

Since the refractive index of immersion liquids is greater than 1, the numerical aperture of the lens increases and rays that make a larger angle with the optical axis of the lens can enter it than in the case when there is air between the front lens of the lens and the specimen.

The second way to increase the resolution of a microscope is to use ultraviolet rays, the wavelength of which is shorter than the wavelength of visible light rays.

However, the resolution of a microscope can only be increased to a certain limit, limited by the wavelength of light. The smallest particles that are clearly visible in a modern light microscope must have a value greater than 1/3 wavelength of light. This means that when using the visible part of daylight with a wavelength from 0.004 to 0.0007 mm, particles of at least 0 will be visible in the microscope .0002-0.0003 mm. Consequently, with the help of modern microscopes it is possible to examine those details of the cell structure that have a size of at least 0.2-0.3 microns.

Currently, many different models of light microscopes have been created. They provide the opportunity for a multifaceted study of cellular structures and their functions.

Biological microscope. The biological microscope (MBI-1, MBI-2, MBI-3, MBR, etc.) is intended for studying preparations illuminated by transmitted light. It is this type of microscope that is most widely used for studying the structure of cells and other objects.

However, with the help of a biological microscope it is possible to study in detail mainly fixed and stained cell preparations. Most living, unstained cells are colorless and transparent in transmitted light (they do not absorb light), and they cannot be examined in detail.

Phase contrast microscopy. A phase contrast device provides a contrast image of preparations of living cells, almost invisible when observing them in a biological microscope).

The phase contrast method is based on the fact that individual areas of a transparent sample differ from the surrounding environment in refractive index. Therefore, light passing through them travels at different speeds, i.e., it experiences a phase shift, which is expressed in a change in brightness. Phase changes of light waves are converted into light vibrations of different amplitudes, and a contrast image of the preparation is perceived by the eye, in which the distribution of illumination corresponds to the distribution of wide opportunities for studying living cells, their organelles and inclusions in an intact state. This circumstance plays an important role, since fixing and staining cells, as a rule, damages cellular structures.

A phase contrast device for a biological microscope consists of a set of phase lenses that differ from conventional ones in the presence of an annular phase plate, a condenser with a set of annular diaphragms, and an auxiliary microscope that magnifies the image of the annular diaphragm and the phase plate when they are combined.

Interference microscopy. The interference contrast method is close to the phase contrast microscopy method and makes it possible to obtain contrast images of unstained transparent living cells, as well as to calculate the dry weight of cells. A special interference microscope used for these purposes is designed in such a way that a beam of parallel light rays coming from a light source is divided into two parallel branches - upper and lower.

The lower branch passes through the preparation, and the phase of its light vibration changes, while the upper wave remains unchanged. For the drug, i.e. in the lens prisms, both branches reconnect and interfere with each other. As a result of interference, areas of the drug that have different thicknesses or unequal refractive indices are painted in different colors and become contrasting and clearly visible.

Fluorescence microscopy. Like phase contrast, fluorescence (or luminescence) microscopy makes it possible to study a living cell. Fluorescence is the glow of an object, excited by light energy absorbed by it. Fluorescence can be excited by ultraviolet, blue and violet rays.

A number of structures and substances contained in cells have their own (or primary) fluorescence. For example, the green pigment chlorophyll, found in the chloroplasts of plant cells, has a characteristic bright red fluorescence. A rather bright glow is produced by vitamins A and B and some pigments of bacterial cells; this makes it possible to recognize individual types of bacteria.

However, most substances contained in cells do not have their own fluorescence. Such substances begin to glow, revealing a variety of colors, only after pre-treatment with luminescent dyes (secondary fluorescence). These dyes are called fluorochromes. These include fluorescein, acridine orange, berberine sulfate, phloxin, etc. Fluorochromes are usually used in very weak concentrations (for example, 1:10000, 1:100000) and do not damage a living cell. Many of the fluorochromes selectively stain individual cellular structures and substances in a specific light. Thus, acridine orange, under certain conditions, colors deoxyribonucleic acid (DNA) green and ribonucleic acid (RNA) orange. Therefore, secondary fluorescence with acridine orange is now one of the important methods for studying the localization of nucleic acids in the cells of various organisms.

In addition, the use of fluorochromes makes it possible to obtain contrasting, easy-to-observe preparations in which the desired structures can be easily found, bacterial cells can be recognized and counted. The fluorescence microscopy method also makes it possible to study changes in cells and individual intracellular structures under different functional states, and makes it possible to distinguish between living and dead cells.

When blue and violet light rays are used as a source of fluorescence, the equipment consists of a conventional biological microscope, a low-voltage lamp (for a microscope) with a blue light filter that transmits rays that excite fluorescence, and a yellow light filter that removes excess blue rays. The use of ultraviolet rays as a source of fluorescence requires a special fluorescent microscope with quartz optics that transmits ultraviolet rays.

Polarization microscopy. The method of polarization microscopy is based on the ability of various components of cells and tissues to refract polarized light. Some cellular structures, such as spindle filaments, myofibrils, cilia of the ciliated epithelium, etc., are characterized by a certain orientation of molecules and have the property of birefringence. These are so-called anisotropic structures.

Anisotropic structures are studied using a polarizing microscope. It differs from a conventional biological microscope in that a polarizer is placed in front of the condenser, and a compensator and analyzer are placed behind the specimen and lens, allowing a detailed study of birefringence in the object under consideration. In this case, light or colored structures are usually observed in the cells, the appearance of which depends on the position of the drug relative to the plane of polarization and on the magnitude of birefringence.

A polarizing microscope makes it possible to determine the orientation of particles in cells and other structures, to clearly see structures with birefringence, and with appropriate processing of preparations, observations can be made on the molecular organization of a particular part of the cell.

Dark field microscopy. The study of drugs in the dark is carried out using a special condenser. A dark-field condenser differs from a conventional bright-field condenser in that it transmits only very oblique edge rays from the light source. Because the edge rays are highly inclined, they do not enter the lens, and the field of view of the microscope appears dark, while an object illuminated by scattered light appears light.

Cell preparations usually contain structures of different optical densities. Against a general dark background, these structures are clearly visible due to their different glow, and they glow because they scatter the rays of light falling on them (Tyndall effect).

In a dark field, a variety of living cells can be observed.

Ultraviolet microscopy. Ultraviolet (UV) rays are not perceived by the human eye, making direct study of cells and their structures within them impossible. For the purpose of studying cell preparations in UV rays, E.M. Broomberg (1939) designed the original MUF-1 ultraviolet microscope, and several models of this microscope are currently available. Method E.M. Broomberg is based on the fact that many substances that make up cells have characteristic absorption spectra of UV rays.

When studying various substances in living or fixed unstained cells and tissues in such a microscope, the preparation is photographed three times (on the same plate) in the rays of three different zones of the UV spectrum.

For photography, the UV wavelengths are selected so that in each zone there is an absorption band of any one substance that does not absorb rays in the other two zones. Therefore, the substances that are visible in the photographs turn out to be different in all photographs.

The resulting images are then placed into a special device called a chromoscope. One picture is viewed in blue, the second in green, and the third in red.

Three color images are obtained, which are combined into one in a chromoscope, and in this final image of the object, the various substances of the cell turn out to be painted in different colors.

But an ultraviolet microscope allows not only photography, but also visual observations of tissues and cells, for which it has a special fluorescent screen.

Using this microscope, it is possible to examine particles of slightly smaller sizes than in a conventional biological microscope, due to the fact that UV rays have a much shorter wavelength than ordinary light rays.

Therefore, the resolution of a UV microscope is 0.11 μm, while the resolution of a biological microscope using conventional lighting is 0.2-0.3 μm.

Using an ultraviolet microscope, a quantitative determination of the absorption of UV rays by nucleic acids and other substances contained in cells is carried out, i.e., the amount of these substances in one cell is determined.

Microphotography. Microphotography of various microscopic preparations is carried out in order to obtain their enlarged image - a microphotography. Micrographs are convenient for studying individual structures of cells and other objects; microphotographs represent documents that very accurately reflect all the details of the structure of a microscopic specimen.

Photographing of microscopic preparations is carried out using special microphoto installations or microphoto attachment cameras. The latter are widely used and are suitable for microphotography with a biological and any other microscope. A microphoto camera is a camera in which the lens has been removed and replaced with a microscope.

The optical system of the microscope serves as the lens of this camera. There are several types of microphoto attachments. Microphoto attachments like MFN-8 are very convenient.

There is also a special biological microscope MBI-6 with a permanent camera. MBI-6 allows for routine visual examination of drugs and their photography in transmitted and reflected light, in light and dark fields of view, with phase contrast and in polarized light.

Micro-filming plays an important role in studying the life processes of cells. To study the details of the most important processes occurring in the cell, such as division, phagocytosis, cytoplasmic flow, etc., a time-lapse device is used.

Using this device, it is possible to produce either accelerated filming, which is usually used in rapidly occurring processes, or slow-motion filming of those changes in the cell that are characterized by a slow flow.

Microcine filming is not only a method that allows one to study in detail various structures and processes in a living cell, but also a method for documenting these processes and all the changes that are associated with them.