Using light microscopy in plants. Light microscopy. Setting up lighting and focusing the microscope

20.02.2024

The answers to tasks 1–21 are a sequence of numbers, a number or a word (phrase).

Consider the proposed scheme of evolutionary directions. Write down in your answer the missing term indicated in the diagram with a question mark

Choose two correct answers out of five and write down the numbers under which they are indicated.

Using light microscopy, it is possible to distinguish in a plant cell

1. ribosomes

2. vacuole

3. microtubules

4. cell wall

5. endoplasmic reticulum

How many DNA molecules are contained in the cell nucleus after replication if the diploid set contains 46 DNA molecules? Write down only the corresponding number in your answer.

Answer: ______

All of the signs listed below, except two, are used to describe the processes occurring in interphase. Identify two characteristics that “drop out” from the general list and write down the numbers under which they are indicated in the table.

1. DNA replication

2. ATP synthesis

3. formation of the nuclear envelope

4. synthesis of all types of RNA

5. chromosome spiralization

Establish a correspondence between the characteristics and cell organelles: for each position given in the first column, select the corresponding position from the second column

CHARACTERISTICS

A. closed DNA molecule

B. oxidative enzymes on cristae

B. internal contents - karyoplasm

D. linear chromosomes

D. presence of chromatin in interphase

E. folded inner membrane

ORGANOIDS

2. mitochondrion

How many different phenotypes are produced in the offspring of crossing two heterozygous sweet pea plants with pink flowers (red is incompletely dominant over white)? In your answer, write down only the number of phenotypes.

All of the characteristics below, except two, are used to describe mutational variability. Identify two characteristics that “fall out” from the general list and write down the numbers under which they are indicated in the table

1. formed under the influence of x-rays

2. has a directional modification

3. varies within the normal range of reaction

4. formed as a result of disruption of meiosis

5. occurs suddenly in some individuals

Establish a correspondence between the examples and methods of reproduction: for each position given in the first column, select the corresponding position from the second column.

A. propagation of violets by leaves

B. viviparity in a shark

B. division in two of the ciliate-slipper

G. hydra budding

D. spawning by fish

E. bee parthenogenesis

METHODS OF REPRODUCTION

1. asexual

2. sexual

Choose three correct answers out of six and write down the numbers under which they are indicated in the table.

The following characteristics are characteristic of mushrooms:

2. have limited growth

3. by type of nutrition - heterotrophs

4. have root hairs

5. act as decomposers in the ecosystem

6. are prenuclear organisms

Establish a correspondence between the characteristics and classes of arthropods: for each position given in the first column, select the corresponding position from the second column.

CHARACTERISTICS

A. presence of two pairs of antennae

B. transmission of certain types of diseases dangerous to humans

B. external digestion

D. regulation of insect numbers

D. purification of reservoirs from organic residues

E. the presence of four pairs of limbs

CLASSES OF ARTHROPODA

1. Crustaceans

2. Arachnids

Establish the sequence of systematic taxa, starting with the smallest. Write down the corresponding sequence of numbers in the table

2. Arthropods

3. Diptera

4. Insects

5. Malaria mosquito

6. Animals

Choose three correctly labeled captions for the drawing “Human Skull”. Write down the numbers under which they are indicated in the table.

1. frontal bone

2. occipital bone

3. temporal bone

4. parietal bone

5.mandibular bone

6. zygomatic bone

Establish a correspondence between human organs and the body cavities in which these organs are located: for each position given in the first column, select the corresponding position from the second column.

HUMAN ORGANS

A. heart

V. lungs

G. trachea

D. liver

E. spleen

BODY CAVITIES

1. chest

2. abdominal

Establish the sequence of signals passing through the sensory visual system. Write down the corresponding sequence of numbers in the table.

1. cornea

2. visual cortex

3. vitreous body

4. optic nerve

5. lens

6. retina

Read the text. Select three sentences that describe the ecological criterion of the plant species Pemphigus vulgare. Write down the numbers under which they are indicated.

(1) Pemphigus vulgaris is mainly found in the Mediterranean region of Europe and Africa. (2) Common bladderwort grows in ditches, ponds, standing and slow-flowing reservoirs, and swamps. (3) Plant leaves are dissected into numerous thread-like lobes; leaves and stems are equipped with vesicles. (4) Pemphigus blooms from June to September. (5) The flowers are yellow, 5-10 per peduncle. (6) Common bladderwort is an insectivorous plant.

Establish a correspondence between the characteristics and ways to achieve biological progress: for each position given in the first column, select the corresponding position from the second column

CHARACTERISTICS

A. private adaptations to living conditions

B. the emergence of animal classes

B. formation of genera within families

D. increasing the level of organization of organisms

D. emergence of plant divisions

WAYS TO ACHIEVE BIOLOGICAL PROGRESS

1. aromorphosis

2. idioadaptation

Choose three correct answers out of six and write down the numbers under which they are indicated. Natural biogeocenoses include

1. oak grove

6. pasture

Establish a correspondence between the characteristics and ecosystems: for each position given in the first column, select the corresponding position from the second column.

SIGNS

A. low self-regulation

B. diversity of producers

B. dominance of monoculture

D. short food chains

D. extensive power networks

E. species diversity of animals

ECOSYSTEMS

1. wheat field

2. feather grass steppe

Establish the sequence of stages of development of the liver fluke, starting with the release of eggs by the definitive host into the external environment. Write down the corresponding sequence of numbers.

1. cyst formation

2. introduction of the larva into the body of the small pond snail

3. larval reproduction

4. emergence of larvae from eggs in water

5. Attachment of the tailed larva to aquatic objects

6. exit of the larva from the body of the small pond snail

Look at the picture depicting the phase of the cardiac cycle. Determine the name of this phase, its duration and direction of blood movement. Fill in the blank cells of the table using the terms and processes given in the list. For each cell indicated by a letter, select the appropriate term or process from the list provided.

List of terms and processes:

1. blood flow from the atrium to the ventricle

2. blood flow from the ventricle to the artery

3. blood flow from the veins to the atrium

4. atrial systole

6. ventricular systole

Analyze the table “Time required to recognize a test image.” The subjects were shown numbers of different colors and black and white images of varying complexity. The time required for the subject to recognize and name the object was recorded.

Select statements that can be formulated based on the analysis of the data presented.

1. The simpler the object, the less light is needed to recognize it

2. The time it takes to recognize numbers does not depend on their color.

3. Black objects are recognized faster than colored objects

4. Colored numbers are recognized faster than complex images

5. At dusk, color object recognition becomes weaker.

Part 2.

First write down the task number (22, 23, etc.), then the detailed solution. Write down your answers clearly and legibly.

The fruits of some plant varieties (oranges, tangerines) do not have seeds. What classical breeding methods are used to obtain such varieties and how are these plants propagated?

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Response elements:

1. Classical breeding methods - to obtain plant varieties without seeds, artificial mutagenesis is used with subsequent plant hybridization.

2. Seedless varieties reproduce vegetatively. For example, vegetative propagation of these varieties is possible by grafting buds (cuttings) treated with mutagens into the crown of non-mutant plants.

Determine the type and phase of division of the original diploid cell shown in the diagram. Give a reasoned answer.

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Response elements:

1. Type of division: Meiosis.

2. Division phase: Metaphase of meiosis II.

3. The diagram shows meiosis - metaphase II of meiosis, since the chromosomes have two chromatids, but are represented by one pair (there is no homologous pair). The diagram shows metaphase, so the chromosomes are arranged in one line at the equator of the cell.

Find three errors in the given text. Indicate the numbers of the sentences in which errors were made and correct them.

(1) Fish are inhabitants of the aquatic environment. (2) Based on their origin and structural features, fish are divided into two classes: Cartilaginous fish and Bony fish. (3) The head, pointed at the front, is fused with the body, which starts from the free edge of the gill covers and ends with the caudal region. (4) In all fish, the gills open from the outside of the body into gill slits. (5) All fish have a swim bladder. (6) The most ancient of the bony fishes are lobe-finned fish. (7) They are characterized by fleshy, scale-covered fins, a developed notochord in adult fish, a poorly developed swim bladder and other features

Show answer

Response elements:

Errors were made in sentences 3, 4, 5.

(3) The head, pointed at the front, is fused with the body, which starts from the free edge of the gill covers and ends at the anal fin (or anus).

(4) Not all fish have gills that open outside the body with gill slits; in bony and osteochondral fish they are covered with gill covers.

(5) Not all fish have a swim bladder.

What structural features of a joint make it strong, mobile and reduce friction between bones? List four features. Explain your answer.

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Response elements:

1. The joint is covered with an articular capsule, which consists of connective tissue and gives it strength.

2. The articular head corresponds to the articular cavity, this ensures the mobility of the joint.

3. Joints are strengthened by ligaments.

4. Fluid is released inside the joint capsule, which reduces friction.

As a result of long-term use of pesticides, outbreaks of pest growth may be observed in fields. Explain why such bursts of population growth may occur. Give at least four reasons

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Response elements:

1. As a result of the use of pesticides, predators that fed on pests died, since high concentrations of pesticides accumulate at the end of the food chain.

2. As a result of hereditary variability (mutation) and natural selection, pests have acquired resistance to pesticides and do not die from them.

3. Due to the high rate of reproduction, insects pass on these characteristics to the next generations.

4. Insects that have acquired resistance to pesticides are in very good conditions (abundance of food, absence of competitors and predators), so their numbers sharply increase.

It is known that all types of RNA are synthesized on a DNA template. The fragment of the DNA molecule on which the region of the central loop of tRNA is synthesized has the following nucleotide sequence: GAAGCTTGTTCGGACT. Establish the nucleotide sequence of the tRNA region that is synthesized on this fragment and the amino acid that this tRNA will carry during protein biosynthesis if the third triplet corresponds to the tRNA anticodon. Justify the sequence of your actions. To solve the task, use the genetic code table.

Genetic code (mRNA)

Rules for using the table

The first nucleotide in the triplet is taken from the left vertical row; the second - from the top horizontal row and the third - from the right vertical row. Where the lines coming from all three nucleotides intersect, the desired amino acid is located

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The problem solution scheme includes:

1. Using the principle of complementarity based on DNA, we find the nucleotide sequence of tRNA, the nucleotide sequence of the tRNA region TSUU-TsGA-CAA-GCC-UGA.

2. The nucleotide sequence of the CAA anticodon (third triplet) corresponds to the codon on the GUU mRNA.

3. According to the table of the genetic code, this codon corresponds to the amino acid VAL (valine), which this tRNA will carry.

Note. In this type of assignment, the key words are: “all types of RNA are synthesized on a DNA template.” That is, we need to find exactly tRNA - molecules consisting of 70-90 nucleotides, which are folded in a certain way and resemble a clover leaf in shape and carry amino acids in protein biosynthesis.

Therefore, first we determine the tRNA region on DNA according to the principle of complementarity. Then we find the triplet that is central, we translate it into mRNA according to the principle of complementarity, and only now we find the amino acid using the table of the genetic code.

When crossing sweet pea plants with tendrils on shoots and bright flowers and plants without tendrils on shoots with pale flowers, all F 1 hybrids were obtained with tendrils and bright flowers. In the analytical crossing of F 1 hybrids, the following plants were obtained: 323 with tendrils and bright flowers, 311 without tendrils and with pale flowers, 99 with tendrils and pale flowers, 101 without tendrils and with bright flowers. Make crossbreeding schemes. Determine the genotypes of parents and offspring in two crosses. Explain the formation of four phenotypic groups in the offspring.

Show answer

A, a - alleles that determine, respectively, the presence and absence of antennae;

B, c are alleles that determine, respectively, the presence of bright and pale flowers.

P1 ♀ AABB - with tendrils on shoots and bright flowers; ♂ aavv - without tendrils on shoots with pale flowers

F1 A?B? - with tendrils and bright flowers.

Hybrid from the first crossing - A?B? - with tendrils and bright flowers; aavv - without tendrils on shoots with pale flowers - because An analysis cross is a cross with a recessive dihomozygote.

323 with tendrils and bright flowers,

311 without tendrils and with pale flowers,

99 with tendrils and pale flowers,

101 without tendrils and with bright flowers.

The problem solution scheme includes:

1) P1 ♀ AABB x ♂ aabb (so there was no splitting in the first generation).

Gametes ♀ AB ♂ ab

100% diheterozygous with antennae and bright colors.

2) Analyzing crossing. Because in the offspring, the 1:1:1:1 split is disrupted, which means the AB/ab/ genes are linked - we determine this by the number of non-crossover individuals (there should be more than 323 and 311).

Р2 ♀ AаBв × ♂ аавв

Gametes ♀AB/, ♀Aw, ♀aB, ♀av/ and ♂av/

F2 AB//av (323 with tendrils and bright flowers), av//av (311 without tendrils and with pale flowers), Aavv (99 with tendrils and pale flowers), Aavv (101 without tendrils and with bright flowers)

Thus, the few offspring 99 with antennae and pale flowers, 101 without antennae and with bright flowers appeared as a result of crossing over.

Genotypes of the parents of the first cross: AABB, aavv.

Genotype of the offspring of the first cross: AaBv.

Genotypes of the parents of the second cross: AB//av, ab//av.

Genotypes of the offspring of the second cross: AB//av (323 with tendrils and bright flowers), ab//av (311 without tendrils and with pale flowers), Aavv (99 with tendrils and pale flowers), Aavv (101 without tendrils and with bright flowers) flowers).

The formation of four phenotypic groups in the offspring is explained by the fact that the characters with antennae - bright flowers and without antennae - pale flowers are linked, but the linkage is incomplete and the AaBb individual undergoes the process of crossing over.

Confocal microscope and images made with its help: anther cracking, xylem vessels, chloroplasts in stigma cells.

Light microscopy

One of the main methods of cytology today remains microscopy, designed to study the structure of the cell; it is widely used in fundamental and applied research. The invention of the microscope is associated with the names of Galileo Galilei (Italian) and the Jansen brothers (Dutch) in 1609-1611. The term "microscope" was coined by Faber (German) in 1625.

At the moment, there are two main types of microscopy - light and electron. The differences between them lie in the principle of considering the object. In the first case, the object is considered in the flow of the visible part of electromagnetic radiation (wavelength = 400-750 nm), in the second case - in the flow of electrons. These two methods have different resolutions. Resolution or resolution limit is the minimum distance between two points at which they are visible separately. The resolution limit of the microscope is set by the wavelength of the radiation flux in which the object is being studied. Therefore, radiation of a given wavelength can be used to study only such structures, the minimum dimensions of which are comparable to the wavelength of the radiation itself. The resolution limit of light microscopy was reached by microscope designers at the end of the 19th century and amounted to 0.2 microns. This means that two objects, if separated by less than 0.2 microns, will appear as one, even if we greatly enlarge the image, for example by projecting it onto a screen. Therefore, with the help of a light microscope it is not possible to examine two centrioles in the cell center; they look like one point (it must be said that in modern commercially produced microscopes, the maximum resolution is not realized). Due to the limited resolution of a light microscope, it can be used to study a limited number of intracellular structures, including: the nucleus, plastids, large vacuoles, and the plant cell wall. The smallest objects clearly visible in a light microscope are bacteria and mitochondria, the size of which is about 500 nm (0.5 microns), smaller objects are not clearly visible, increasing the precision of lens processing cannot overcome this limitation, which is set by the wave nature of light.

Resolution depends not only on the wavelength of the light source, but also on the refractive index of the medium through which the object is observed, as well as on the angle at which the light rays enter the lens. The standard set of microscope lenses consists of: low magnification lenses (x8) with an aperture of A = 0.2 and high magnification lenses (x20) with A = 0.40 and - (x40) with A = 0.65. These lenses are called “dry”, since the object is viewed through the air (refractive index n=1). But most microscopes are also equipped with special immersion objectives, which require a special immersion environment (n=1). Such a medium can be water; the x40 VI lens has an aperture of 0.75. The most common is oil immersion (n=1.51), at x90 the lens aperture value is A=1.25. If immersion is used, the resolution of the light microscope improves. However, high-resolution lenses have disadvantages: shallow depth of field and low contrast.

The most common method of light microscopy is the brightfield method, in which light rays from an illuminator pass through the object and enter the lens. Fixed and stained cells are studied in this way. The discovery of basic cellular structures involves the development and use of a set of dyes that selectively stain cell components and provide contrast for their observation. There is a wide variety of dyes. Some of them are extracted from plants and animals; there are still no synthetic analogues. For example, the widely used hematoxylin is an extract of the tropical logwood tree, and carmine is the pigment of the fat body of some types of aphids. These are all so-called nuclear dyes that color structures containing nucleic acids. The use of a non-nuclear dye, silver nitrate, allowed Camillo Golgi in 1898 to observe and describe what was later called the Golgi apparatus.

Staining a living cell is possible only in rare cases, so other methods are used to study them. Unlike the bright field method, when observing objects using the dark field method, the illuminator rays do not enter the lens and the image is created only by scattered rays coming from the object. In this case, against a dark background you can see luminous particles, which are smaller in size than the resolution of the lens, although the size and shape of the particles are difficult to determine. Transmitted light dark-field microscopy is used to study transparent objects normally invisible in bright field and, especially, to view living cells. Living and dying cells look completely different against a dark background. The protoplast of dying cells glows brighter, there is no explanation for this fact. This method was invented by Zsigmondy (Austria) in 1912. In a light microscope, one can distinguish objects that change the amplitude of the illumination rays, but living cells are transparent to visible light and the rays, passing through the cell, practically do not change the amplitude. The human eye is not able to perceive the phase shift of rays without changing the amplitude. Therefore, the methods of phase contrast (invented by Zernike (Dutch) in 1934) and interference microscopy (invented by Lebedev in 1932) are used specifically to study living cells. In such systems, the passage of light through a living cell is accompanied by a change in the phase of the light wave. Light is delayed when passing through thick areas of the cell, such as the nucleus. A recombination of two sets of waves occurs, which create an image of cellular structures.

To study objects that are birefringent (starch grains, plant fibers, crystals), polarization microscopy is used, the foundations of which were laid by Ebner in 1882. In this method, a special polarizer device is used, which converts multidirectional light waves and they acquire one direction.

In fluorescence microscopy, an object is viewed in the light emitted by itself. The first fluorescent microscope was designed by Keller and Zindentokf in 1908. This method is based on the ability of a number of substances to glow when illuminated with short-wavelength rays (violet or ultraviolet). Fluorescence microscopy is often used to identify specific proteins, antibodies, and Koons was the first to use fluorochromes to bind to antibodies, and this reaction was named after him. In cytoembryological studies, this method is used to study structures containing the carbohydrate callose. For this method, a special optical system with a mercury lamp connected to a light microscope is used.

Recently, the capabilities of light microscopy have increased significantly due to the use of sensitive video systems. The image created by a light microscope is processed in a video camera. It is cleared of “noise,” converted into digital signals and sent to a computer, where it undergoes additional processing to extract hidden information. Computer interference microscopy allows you to achieve high contrast and analyze transparent objects and living cells.

Electron microscopy

Long continuous efforts to improve research methods brought the desired results at the end of the Second World War. It was then, thanks to an amazing coincidence of circumstances, almost at the same time, that scientists were enriched with a number of new powerful tools and research methods. In morphology, such a tool was the electron microscope. Created back in the 30s of the 20th century, it had sufficient resolution to penetrate the cell, down to nanometer-sized structures. At the same time, the electron beam had weak penetrating power, and this required the preparation of very thin samples of the material and a high vacuum. Such stringent requirements created serious difficulties, but in a surprisingly short time it was possible to develop methods for preparing tissue samples and construct devices for obtaining thin sections from them. The quality of the objects steadily improved and by the early 1960s many previously unknown cellular structures had been described.

So, the resolution of an electron microscope is much higher than that of a light microscope. Theoretically, at a voltage of 100,000 V, its resolution is 0.002 nm, but due to the correction of electronic lenses, it decreases and in reality is 0.1 nm in modern electron microscopes. Significant difficulties in observing biological objects further reduce the normal resolution; it does not exceed 2 nm. However, this is 100 times greater than that of a light microscope, which is why electron microscopy is called ultramicroscopic.

The general design of a transmission electron microscope resembles that of a light microscope. It is significantly larger than the light one and seems to be upside down. The source of radiation in an electron microscope is a cathode filament that emits electrons (electron gun). The electrons are emitted from the top of a cylindrical column about two meters high. To ensure that there are no obstacles to the movement of electrons, this happens in a vacuum, the electrons are accelerated by the anode and penetrate through a tiny hole into the lower part of the column with a narrow electron beam. The electron beam is focused by ring magnets along the column, which act like the glass lenses of a light microscope. The sample is placed in the path of the electron beam. At the moment of its passage through the sample, part of the electrons is scattered in accordance with the density of the substance, the rest of the electrons are focused, forming an image on a photographic plate or on a screen.

The first electron microscope was created by Siemens in 1939. It made it possible to see many amazing structures in the cell. But for this, completely new methods of preparing drugs had to be invented, which began to be used since 1952. Cells are fixed in this case with glutaraldehyde, which covalently binds proteins, and then with osmic acid, which stabilizes the protein and lipid layers. The sample is dehydrated and impregnated with resins, which form a solid block after polymerization. Sections for electron microscopy should be approximately 1:200 the thickness of a single cell. To make such sections, an ultramicrotome (1953) was created, which uses glass or diamond knives. The resulting sections are placed on a special copper mesh. The electron microscope image depends on electron scattering, which is determined by the atomic number of the substance. Biological objects consist mainly of carbon, oxygen and hydrogen, which have a low atomic number. To enhance the contrast, they are impregnated with heavy metals such as osmium, uranium, and lead. Thin sections with transmission electron microscopy do not allow one to judge the three-dimensional structure of the cell; this deficiency can be compensated for by a series of sections from which the cell is reconstructed. It's a long process.

There is also a direct method for studying the three-dimensional structure of biological objects - scanning electron microscopy - it was created in 1965. In this case, electrons scattered or emitted by the surface of the object, which must be fixed, dried and covered with a film of heavy metal, are used to obtain an image. This method is only applicable for studying surfaces and its resolution is low - about 10 nm.

Electron microscope

Transmission, probe and scanning electron microscopes. Electron microscopic image of the surface of an anther and pollen grain

Chemical methods for studying cells

A classic light microscope has low resolution, which does not allow studying the details of the structure of a cell smaller than 0.25 microns in size. The second stage of studying the cell dates back to the time when microscopists worked to improve their instruments. At the same time - the end of the 18th century. - French scientist Antoine de Lavoisier and Englishman Joseph Priestley create a new science - chemistry. Unlike morphology, which progresses from complex to simple, chemistry progresses from simple to complex. Chemistry began with the identification of elements, atoms, and then moved along the path of studying some of their simplest combinations - molecules.

The synthesis of the biological molecule urea, first carried out in 1828 by the German scientist Friedrich Wöhler, helped cross the border between inorganic and organic chemistry and allow penetration into the living world of chemistry. This marked the beginning of the use of a chemical approach to the study of cells. Over the next hundred years, amino acids, sugars, fats, purines, pyrimidines, and other small molecules were discovered, purified, structurally studied, and synthesized. Scientists were able to get an idea of the metabolism of these substances in the body and the ways in which basic biological molecules are formed from them: proteins, polysaccharides and nucleic acids. But again insurmountable obstacles arose on the path of progress: classical chemistry was powerless in the face of the complexities of the structural complexity of these large molecules. For a long time, cells were studied mainly by observing them. But as the experimental method developed in the natural sciences, it began to be used in the study of living organisms. This was facilitated by powerful biomedical research carried out in the second half of the 19th century. At the beginning of the 20th century. American Ross Garrison and Frenchman Alexis Carrel found that animal cells can be cultured in vitro, similar to how they do with single-celled organisms. Thus, they demonstrated the ability of cells to live independently and created a cultivation method, which is now one of the most relevant.

But all these methods, essentially revolutionary, were still indirect; the cell remained a closed black box. The huge gap between the smallest particle visible in a light microscope and the largest molecule accessible to chemical study remained unknown. In this unknown space, important concepts and concepts were hidden, the functions of the described cellular structures, their connection with known biomolecules remained unknown - without all this, the life of the cell remained unsolved.

In turn, biochemistry has also been enriched with a number of fundamentally new devices and methods. Of particular interest was chromatography, based on a very simple phenomenon - the formation of a rim or halo around a stain (what we see when we try to remove a stain with a special solution). This phenomenon is based on differences in the speed of movement of different paints in the flow of spreading liquid. At the beginning of the 20th century, Russian physiologist and biochemist Mikhail Semenovich Tsvet was the first to use this phenomenon. By passing the extract from the leaves through a vertical tube filled with an adsorbent powder, he was able to separate the main leaf pigments - green and orange - and obtain them in the form of individual colored stripes or rings along the tube. He called his method chromatography (Greek khroma - color, graphein - to record). Color died relatively young and the potential of his method remained unexploited until the early 40s. There are now many variations of chromatography - applicable to all substances that can be identified chemically.

Close to chromatography is gel electrophoresis, in which it is not the flow of solvent, but the electromotive force that promotes the movement and separation of electrically charged components. These methods revolutionized the field of chemical analysis. Now analysis can be carried out on trace amounts of a mixture of almost any composition.

The second method that radically changed the chemical study of living cells was the method of isotope labeling. Isotopes are varieties of the same chemical element that differ in atomic mass. Some isotopes exist in nature, many can be produced artificially through nuclear reactions. Isotopes are used to specifically label certain molecules so that such molecules can be distinguished from related ones without disturbing the overall structure. This method is used in the analysis of biosynthetic processes that could not be studied in any other way. For example, with the production of labeled amino acids, it became possible to study their combination into proteins in a living organism or under experimental conditions, even despite the infinitesimal amount of newly formed protein, due to its radioactivity. This method became widespread with the creation of nuclear reactors and the production of a wide range of radioisotopes. Without the tagged atom method, advances in cellular and molecular biology would have been impossible.

Thus, both morphology and biochemistry, enriched by new methods, were constantly improved, the gap between their knowledge became less and less and disappeared completely when it became possible to divide the cell into parts in such a way that each part could be independently studied.

The methods used for such fractionation are based primarily on centrifugation. This method uses differences in the physical properties, in particular the size and density, of certain components of the cell to separate them from each other. This made it possible to study a large part of the cell and combine morphological and biochemical knowledge.

However, one part of the cell—its crucial central part, the nucleus—remained largely inaccessible until another event occurred. It began with an attempt to analyze, using genetics, the characteristics of some simple viruses that infect bacteria and are called bacteriophages or bacteria eaters. This research turned out to be the right approach to solving the problem of genetic organization, which even in the simplest noncellular organisms was unusually complex. For a long time, the new discipline known today as molecular biology was limited to the study of viruses and bacteria, but then it literally burst into the eukaryotic cell, making it possible to study the regulation of cell activity.

To study the molecular basis of cell organization, detailed biochemical analysis is required. It requires a significant number of cells of a certain type, so it is impossible to use pieces of tissue, because they contain cells of different types. At the first stage of work, pieces of fabric are turned into a suspension. This can be done by destroying the intercellular substance and intercellular connections. To do this, the tissue is treated with proteolytic enzymes that destroy proteins (trypsin, collagenase). Calcium plays an important role in connecting cells and their adhesion, so chelating substances that bind calcium are also used. The tissues are then subjected to gentle mechanical destruction and separated into individual cells. The second stage is the separation of the suspension into separate fractions. To do this, they use centrifugation, with the help of which large cells are separated from small ones, and light ones from heavy ones, or antibodies are used, and the ability of cells to attach to glass or plastic with different strengths. The third stage is the introduction of isolated cells into culture. The first experiments were carried out in 1907 by Harrison, who cultivated the spinal cord of amphibians in a plasma clot. Culture media have a rather complex composition. The standard medium was developed in the early 70s, it contains a set of 13 amino acids, 8 vitamins, and mineral salts. In addition, the medium may include glucose, penicillin, streptomycin, horse or calf serum. As Hayflick and Moorhead showed in 1961, most mammalian cells die in culture after a certain number of divisions. Human skin cells divide in culture 50-100 times. However, mutant cells sometimes appear in culture and can multiply indefinitely, forming a cell line. In 1952, a continuous cell line was isolated from cervical cancer known as the HeLa line. Such lines are stored at a temperature of -70 C; after defrosting, they retain the ability to divide. The method of culturing plant cells was developed by 1964. Using it, it was possible to grow a whole carrot plant in vitro from root cells.

Light microscopy is the most ancient and at the same time one of the most common methods for studying and studying plant and animal cells. It is assumed that the beginning of the study of cells was precisely with the invention of the light optical microscope. The main characteristic of a light microscope is the resolution of the light microscope, which is determined by the wavelength of the light. The resolution limit of a light microscope is determined by the wavelength of light; an optical microscope is used to study structures that have minimal dimensions equal to the wavelength of light radiation. Many constituent cells are similar in optical density and require pre-treatment before microcopying, otherwise they are practically invisible under a conventional light microscope. In order to make them visible, various dyes with a certain selectivity are used. Using selective dyes, it becomes possible to study the internal structure of the cell in more detail.

For example:

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.”

When conducting microscopic examinations, 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.

During the preparation of the preparation for microcopying, thin sections are made on a microtome (Appendix 1, Fig. 1). This device uses the bread slicer principle. Slightly thicker sections are made for plant tissues than for animal tissues because plant cells are relatively larger. Thickness of plant tissue sections for - 10 microns - 20 microns. Some tissues are too soft to cut straight away. Therefore, after fixation, they are poured into molten paraffin or special resin, which saturates the entire fabric. After cooling, a solid block is formed, which is then cut using a microtome. This is explained by the fact that plant cells have strong cell walls that make up the tissue framework. Lignified shells are especially strong.

When using the filling during preparation, the cut runs the risk of damaging the cell structure; to prevent this, use the method of quick freezing. When using this method, you can do without fixing and filling. Frozen tissue is cut using a special microtome - cryotome (Appendix 1, Fig. 2).

Frozen sections better preserve natural structural features. However, they are more difficult to cook and the presence of ice crystals ruins some of the details.

phase-contrast (Appendix 1, Fig. 3) and interference microscopes (Appendix 1, Fig. 4) allow you to examine living cells under a microscope with a clear manifestation of the details of their structure. 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.

Light microscopy has several varieties.

Exercise 1.

Consider the proposed scheme of evolutionary directions. Write down the missing term in your answer, indicated by a question mark in the diagram.

Explanation: the overlooked direction of biological progress is idioadaptation. Idiomatic adaptation- a private change in the body that does not lead to an increase in the level of organization (hairiness of leaves, change in color, etc.).

The correct answer is idioadaptation.

Task 2.

Choose two correct answers out of five and write down the numbers under which they are indicated.

Can be distinguished in a plant cell using light microscopy.

1. Endoplasmic reticulum

2. Microtubules

3. Vacuole

4. Cell wall

5. Ribosomes

Explanation: Using light microscopy, only large parts of the cell can be distinguished, such as the cell wall and the vacuole (in old cells, the vacuole occupies almost the entire intracellular space). Smaller organelles (microtubules, endoplasmic reticulum and ribosomes) can only be seen with an electron microscope.

The correct answer is 34.

Task 3.

How many DNA molecules are contained in the cell nucleus after replication if the diploid set contains 46 DNA molecules? Write down only the corresponding number in your answer.

Explanation: Replication is the doubling of DNA molecules, which means that 46 molecules after doubling turn into 92 molecules.

The correct answer is 92.

Task 4.

All of the characteristics listed below, except two, are used to describe the structure and functions of the endoplasmic reticulum. Identify two characteristics that “drop out” from the general list.

1. Protein breakdown

2. Transport of substances

3. Oxidative phosphorylation

4. Protein synthesis on ribosomes

5. Division of the cytoplasm into compartments

Explanation: The endoplasmic reticulum surrounds the nucleus, thereby dividing the cytoplasm into compartments and carrying out intracellular transport of substances. EPS can be smooth or rough. The rough ER carries out protein synthesis using ribosomes, which are located on the membranes of the network.

The correct answer is 13.

Task 5.

Establish a correspondence between the processes and phases of mitosis.

Processes

A. The nuclear membrane is formed

B. Sister chromosomes diverge

B. The spindle finally disappears

D. Chromosomes despiral

D. Centromeres of chromosomes separate

Phases of mitosis

1. Anaphase

2. Telophase

Explanation: anaphase is the fastest phase of division, as chromosomes diverge to the poles of the cell (and chromosome centromeres separate). All other processes occur after chromosome divergence - in telophase.

The correct answer is 21221.

Task 6.

How many different phenotypes are produced when crossing two heterozygous sweet pea plants with pink flowers (red color is incompletely dominant over white). In your answer, write down only the number of phenotypes.

Explanation: with incomplete dominance, the combination of red (A) and white (a) color genes gives the pink color (A). Crossing two roseate plants:

R: Aa x Aa

G: A, a x A, a

F1: we get splitting by genotype - 1AA:2Aa:1aa

Phenotypic split: 1: 2: 1 (25% red, 50% pink, 25% white flowers).

The correct answer is 3.

Task 7.

All of the following characteristics, except two, characterize modification variability. Identify two characteristics that “drop out” from the general list and write down the numbers under which they are indicated.

1. Different shapes of underwater and above-water arrowhead leaves

2. Brown and blue eye colors among members of the same family

3. Variation in the size of tubers of one potato plant

4. The difference in the length of birch leaves on the north and south sides

5. Having children with Down syndrome

Explanation: Modification variability- variability of a particular organism (or group of organisms) depending on environmental conditions within the normal range of reaction. Such variability affects the phenotype of the organism, but does not affect the genotype, which means such modifications are not inherited. Therefore, examples of this type of variability cannot be genetic traits - different eye colors and Down syndrome.

The correct answer is 25.

Task 8.

Establish correspondence between processes and plant departments.

Processes

A. Formation of endosperm

B. Formation of a green shoot

B. Fusion of immobile gametes

D. Pollen tube development

D. Reproduction and dispersal by spores

Plant departments

2. Ferns

Explanation: Ferns form a green outgrowth (from spores), and also reproduce and disperse through spores. Their male reproductive cells are mobile and fertilization occurs only in water.

The correct answer is 12112.

Task 9.

What signs are characteristic of the organism shown in the figure.

1. Closed circulatory system

2. Division of the body into head, chest and abdomen

3. Ventral nerve cord

4. Four pairs of legs

5. One pair of antennae

6. Breathing with the help of pulmonary sacs and tracheas

Explanation: arachnids have four pairs of legs, an open circulatory system, body parts: the cephalothorax and abdomen, there is an abdominal nerve cord, they breathe with the help of pulmonary sacs and tracheas. There are no antennae.

The correct answer is 346.

Task 10.

Establish a correspondence between the characteristics of organisms and the kingdoms for which they are characteristic.

Signs of organisms

A. Heterotrophic type of nutrition

B. Presence of chitin in the exoskeleton

B. Availability of educational fabric

D. Regulation of life activity only with the help of chemicals

D. Formation of urea during metabolism

E. Presence of a rigid cell wall made of polysaccharides

Kingdoms

1. Plants

2. Animals

Explanation: The characteristics of animals include a heterotrophic type of nutrition, the presence of chitin in the exoskeleton and the formation of urea in the process of protein metabolism.

The presence of educational tissue, regulation of life activity with the help of chemicals and the presence of a cell wall are considered to be plant characteristics.

Plants are autotrophs because they consume inorganic substances and convert them into organic substances. The exoskeleton is present only in animals (arthropods), animals have only nervous, epithelial, muscle and connective tissue, and plants have educational, mechanical, integumentary, basal and conductive tissue. Animals regulate internal processes with the help of nervous and humoral regulation, and plants only with the help of chemicals. Urea is formed in animals. A cell wall (made of cellulose) is present in plants and absent in animals.

The correct answer is 221121.

Task 11.

Establish the sequence of systematic taxa, starting with the largest.

1. Plants

2. Bush cherry

3. Rosaceae

4. Dicotyledons

5. Angiosperms

6. Cherry

Explanation: We arrange the taxa starting with the largest.

Kingdom - Plants

Department - Angiosperms

Class - Dicotyledons

Family - Rosaceae

Rod - Cherry

Type - Bush cherry

The correct answer is 154362.

Task 12.

Choose three correct answers out of six and write down the numbers under which they are indicated.

1. Narrowing of the pulmonary arteries

2. Increased breathing

3. Evaporation of water through sweat glands

4. Changes in the rate of blood clotting

5. Dilatation of skin capillaries

6. Lower blood pressure

Explanation: When heat is lost, the pulmonary arteries narrow (due to increased pressure), water evaporates through the sweat glands and the skin capillaries dilate (the skin turns red).

The correct answer is 135.

Task 13.

Establish a correspondence between the structures of the ear and the sections in which they are located.

Structure

A. Auricle

B. Oval window

V. Snail

G. Stremechko

D. Eustachian tube

E. Hammer

Departments

1. Outer ear

2. Middle ear

3. Inner ear

Explanation: Let's look at the picture.

The inner ear includes the pinna, the middle ear includes the auditory ossicles (hammer stapes), and the inner ear includes the oval window, cochlea, and eustachian tube.

The correct answer is 133232.

Task 14.

Arrange the subordination of systems of different levels in the correct order, starting with the largest.

1. Shaped elements

2. Red blood cell

3. Hemoglobin

4. Iron ion

5. Connective tissue

6. Blood

Explanation: We arrange the structures, starting from the largest: connective tissue - blood - formed elements - erythrocyte - hemoglobin - iron ion. Iron is part of the hemoglobin protein, which carries oxygen and is located on the red blood cell - a formed element of blood. Blood is one of the types of connective tissue.

The correct answer is 561234.

Task 15.

Read the text. Select three sentences that describe the ecological criterion of the plant species Pemphigus vulgare. Write down the numbers under which they are indicated.

1. Pemphigus vulgaris is mainly found in the Mediterranean region of Europe and Africa. 2. Common bladderwort grows in ditches, ponds, standing and slow-flowing reservoirs, and swamps. 3. Plant leaves are dissected into numerous thread-like lobes; leaves and stems are equipped with vesicles. 4. Pemphigus blooms from June to September. 5. The flowers are painted yellow, 5-10 per peduncle. 6. Common bladderwort is an insectivorous plant.

Explanation: The ecological criterion describes the lifestyle of a species and its relationship with other organisms. Sentence 2 - describes the features of the habitat (not specific places, but in general).

Sentence 4 - flowering time (which means pollination).

Suggestion 6 - nutritional features.

The correct answer is 246.

Task 16.

Match examples with evidence of evolution.

Examples

A. Fossil transitional forms

B. Homologous organs

V. Rudiments

G. Fossils

D. Atavisms

E. Unified body plan

Evidence of evolution

1. Paleontological

2. Comparative anatomical

Explanation: paleontological evidence includes what scientists find—fossil transitional forms, fossils. Everything else is comparative anatomical evidence - homologous organs, rudiments, atavisms, a single structural plan.

Atavism- the appearance of signs in the body characteristic of distant ancestors (hair, multiple nipples, etc.).

Rudiments- organs that have lost their function (wisdom teeth, appendix, tailbone, third eyelid, etc.).

The correct answer is 122122.

Task 17.

Choose three correct answers out of six and write down the numbers under which they are indicated.

Consumers in the ecosystem include

2. Rotting bacteria

3. Green plants

4. Artiodactyls

5. Predators

6. Cyanobacteria

The correct answer is 145.

Task 18.

Establish a correspondence between features and ecosystems.

Signs

A. Extensive power networks

B. Short food chains

B. Low self-regulation

D. Diversity of producers

D. Species diversity of animals

E. Dominance of monocultures

Ecosystems

1. Feather grass steppe

2. Wheat field

Explanation: Essentially, the task requires distinguishing a natural ecosystem (feather grass steppe) from an agroecosystem (wheat field).

The agroecosystem is characterized by short food chains, low self-regulation and dominance of monocultures. Everything else is signs of a stable natural ecosystem.

The correct answer is 122112.

Task 19.

Establish the sequence of appearance and development of ecosystems on bare rocks.

1. Scale lichens and bacteria

2. Herbaceous and shrub community

3. Forest community

4. Herbaceous flowering plants

5. Mosses and fruticose lichens

Explanation: on bare rocks, a plant community is formed in the same way as the development of plant life on Earth. That is, crustose lichens and bacteria, then mosses and fruticose lichens, then herbaceous flowering plants, the herbaceous-shrub community and, finally, the forest community.

The correct answer is 15423.

Task 20.

List of terms and processes:

1. Ventricular systole

2. Atrial systole

3. Blood flow from the ventricles to the arteries

4. 0.1 s

5. 0.8 s

6. Blood flow from the atrium to the ventricle

7. Blood flow from the veins into the atrium

8. 0.3 s

Explanation: The figure shows the phase of atrial contraction (atrial systole). In this case, blood from the atrium enters the ventricle. The process happens very quickly and takes 0.1 s.

The correct answer is 246.

Task 21.

Images	Average recognition time (ms)
Simple	25,0
Medium difficulty	37,5
Complex	70,0
Black numbers	27,5
Red numbers	37,5
Blue numbers	62,5
Green numbers	45,0
Yellow numbers	67,5

Select statements that can be formulated based on the analysis of the data presented.

1. The time it takes to recognize numbers does not depend on their color

2. Black objects are recognized faster than colored objects

3. The simpler the object, the less light is needed to recognize it

4. Color numbers are recognized faster than complex images

5. At dusk, color object recognition becomes weaker

Explanation: Based on the data given in the table, black objects are recognized faster than colored ones (27.5 ms and 37.5 - 67.5 ms). And color numbers (max - 67.5 ms) are recognized faster than a complex image (70.0 ms). The remaining statements are either false or contain data that is not in the table.

The correct answer is 24.

Task 22.

It is well known that human blood contains proteins and glucose. Why is a single injection of glucose into the blood not dangerous for the body, but the introduction of most proteins is dangerous?

Explanation: With a single injection of glucose into the blood, hormones of carbohydrate metabolism break it down. Glucose is a familiar molecule for human blood (and the main energy molecule), and proteins (non-regulatory) in a normal state should not be in the blood (since they are polymers); monomers of proteins - amino acids - enter the blood from the gastrointestinal tract. Proteins are antigenic in nature and will be perceived by the human body as a foreign molecule.

Task 23.

Name the object shown in the picture. Indicate the name and functions of the structures present in the picture.

Explanation: The picture shows a bacteriophage (bacterial virus). We can distinguish the head (protein capsid - performs a protective function, since it contains nucleic acid - DNA or RNA); tail process with basal plate - through the process the virus injects nucleic acid into the affected cell; fibrils - with the help of them the virus takes root on the cell wall.

Task 24.

Find three errors in the given text. Indicate the numbers of the sentences in which errors were made and correct them.

(1) Fish are inhabitants of the aquatic environment. (2) Based on their origin and structural features, fish are divided into 2 classes: Cartilaginous fish and Bony fish. (3) The head, pointed at the front, is fused with the body, which starts from the free edge of the gill covers and ends with the caudal region. (4) All fish have gills that open from the outside of the body into gill slits. (5) All fish have a swim bladder. (b) The most ancient of the bony fishes are lobe-finned fish. (7) They are characterized by fleshy, scaled fins, a developed notochord in adult fish, a poorly developed swim bladder and other features.

Explanation: sentence 3 - the body ends not with the tail, but with the anus.

Sentence 4 - Not all fish have gills that open from the outside of the body with gill slits. Many fish have gills that are closed by opercula (bony fish).

Sentence 5 - swim bladders are a special organ for adapting to swimming, but not all fish have a swim bladder (salmonids).

Light microscopy provides magnification up to 2-3 thousand times, a color and moving image of a living object, the possibility of micro-filming and long-term observation of the same object, assessment of its dynamics and chemistry.

The main characteristics of any microscope are resolution and contrast. Resolution- this is the minimum distance at which two points are located, demonstrated separately by the microscope. The resolution of the human eye in the best vision mode is 0.2 mm.

Image Contrast is the difference in brightness between the image and the background. If this difference is less than 3 - 4%, then it cannot be caught either by the eye or by a photographic plate; then the image will remain invisible, even if the microscope resolves its details. Contrast is influenced both by the properties of the object, which change the luminous flux compared to the background, and by the ability of the optics to capture the resulting differences in the properties of the beam.

The capabilities of a light microscope are limited by the wave nature of light. The physical properties of light - color (wavelength), brightness (wave amplitude), phase, density and direction of wave propagation change depending on the properties of the object. These differences are used in modern microscopes to create contrast.

Microscope Magnification is defined as the product of the objective magnification and the eyepiece magnification. Typical research microscopes have an eyepiece magnification of 10, and an objective magnification of 10, 45 and 100. Accordingly, the magnification of such a microscope ranges from 100 to 1000. Some microscopes have a magnification of up to 2000. Even higher magnification does not make sense, since resolution does not improve. On the contrary, the image quality deteriorates.

Numerical aperture used to express the resolution of an optical system or the aperture ratio of a lens. Lens aperture-light intensity per unit area of the image is approximately equal to the square of NA. The NA value is approximately 0.95 for a good lens. The microscope is usually sized so that its total magnification is about 1000 NA. If a liquid (oil or, more rarely, distilled water) is introduced between the objective and the sample, an “immersion” objective is obtained with an NA value as high as 1.4 and a corresponding improvement in resolution.

Light microscopy methods

Light microscopy methods(lighting and observation). Microscopy methods are selected (and provided constructively) depending on the nature and properties of the objects being studied, since the latter, as noted above, affect the image contrast.

Bright field method and its varieties

Transmitted light field method used when studying transparent preparations with absorbing (light-absorbing) particles and parts included in them. These can be, for example, thin colored sections of animal and plant tissues, thin sections of minerals, etc. In the absence of a preparation, a beam of light from the condenser, passing through the lens, produces a uniformly illuminated field near the focal plane of the eyepiece. If there is an absorbent element in the preparation, partial absorption and partial scattering of the light incident on it occurs, which causes the appearance of the image. It is also possible to use the method when observing non-absorbing objects, but only if they scatter the illuminating beam so strongly that a significant part of it does not fall into the lens.

Oblique lighting method- a variation of the previous method. The difference between them is that the light is directed at the object at a large angle to the direction of observation. Sometimes this helps to reveal the “relief” of an object due to the formation of shadows.

Bright field method in reflected light used when studying opaque light-reflecting objects, such as thin sections of metals or ores. The preparation is illuminated (from an illuminator and a translucent mirror) from above, through a lens, which simultaneously plays the role of a condenser. In the image created in a plane by the lens together with the tube lens, the structure of the preparation is visible due to the difference in the reflectivity of its elements; In the bright field, inhomogeneities that scatter the light incident on them also stand out.

Dark field method and its variations

Transmitted light dark field method(Dark-field microscopy) used to obtain images of transparent, non-absorbent objects that cannot be seen using the bright field method. Often these are biological objects. Light from the illuminator and mirror is directed onto the preparation by a specially designed condenser - the so-called. dark field condenser. Upon exiting the condenser, the main part of the light rays, which did not change their direction when passing through the transparent preparation, forms a beam in the form of a hollow cone and does not enter the lens (which is located inside this cone). The image in the microscope is formed using only a small part of the rays scattered by microparticles of the drug located on the slide into the cone and passing through the lens. Dark-field microscopy is based on the effect Tyndall(Tyndall effect), a well-known example of which is the detection of dust particles in the air when illuminated by a narrow beam of sunlight. In the field of view against a dark background, light images of the structural elements of the drug are visible, which differ from the surrounding environment in their refractive index. Large particles have only bright edges that scatter light rays. Using this method, it is impossible to determine from the appearance of the image whether the particles are transparent or opaque, or whether they have a higher or lower refractive index compared to the surrounding medium.

Conducting a dark-field study

Slides should be no thicker than 1.1-1.2 mm, coverslips 0.17 mm, without scratches or dirt. When preparing the drug, you should avoid the presence of bubbles and large particles (these defects will be visible with a bright glow and will not allow you to observe the drug). For dark-field, more powerful illuminators and maximum lamp intensity are used.

Setting up darkfield lighting is basically as follows:

Install the light according to Koehler;
Replace the bright-field condenser with a dark-field one;
Immersion oil or distilled water is applied to the upper condenser lens;
Raise the condenser until it touches the bottom surface of the slide;
A low magnification lens is focused on the specimen;
Using centering screws, a light spot (sometimes having a darkened central area) is transferred to the center of the field of view;
By raising and lowering the condenser, the darkened central area disappears and a uniformly illuminated light spot is obtained.

If this cannot be done, then you need to check the thickness of the glass slide (this phenomenon is usually observed when using too thick glass slides - the cone of light is focused in the thickness of the glass).

After setting the light correctly, install a lens of the required magnification and examine the specimen.

At the core ultramicroscopy method The same principle applies: specimens in ultramicroscopes are illuminated perpendicular to the direction of observation. With this method, it is possible to detect (but not literally “observe”) extremely small particles, the sizes of which lie far beyond the resolution of the most powerful microscopes. With the help of immersion ultramicroscopes, it is possible to register the presence in a preparation of particles × particles up to 2 × 10 to the -9th degree m in size. But the shape and exact dimensions of such particles cannot be determined using this method. Their images appear to the observer in the form of diffraction spots, the dimensions of which depend not on the size and shape of the particles themselves, but on the aperture of the lens and the magnification of the microscope. Since such particles scatter very little light, extremely strong light sources, such as a carbon electric arc, are required to illuminate them. Ultramicroscopes are used mainly in colloid chemistry.

Phase contrast method

Phase contrast method and its variety - the so-called. anoptral contrast method are designed to obtain images of transparent and colorless objects that are invisible when observed using the bright field method. These include, for example, living undyed animal tissues. The essence of the method is that even with very small differences in the refractive indices of different elements of the preparation, the light wave passing through them undergoes different changes in phase (acquires the so-called phase relief). Not perceived directly by either the eye or the photographic plate, these phase changes are converted with the help of a special optical device into changes in the amplitude of the light wave, i.e., into changes in brightness (“amplitude relief”), which are already visible to the eye or recorded on the photosensitive layer. In other words, in the resulting visible image, the distribution of brightness (amplitude) reproduces the phase relief. The image obtained in this way is called phase-contrast.

The phase contrast device can be installed on any light microscope and consists of:

A set of lenses with special phase plates;
Condenser with rotating disk. It contains annular diaphragms corresponding to the phase plates in each of the lenses;
An auxiliary telescope for adjusting phase contrast.

The phase contrast setting is as follows:

Replace the lenses and condenser of the microscope with phase ones (indicated by the letters Ph);
Install a low magnification lens. The hole in the condenser disk must be without an annular diaphragm (indicated by the number "0");
Adjust the light according to Koehler;
Select a phase lens of appropriate magnification and focus it on the specimen;
Turn the condenser disk and install the annular diaphragm corresponding to the lens;
Remove the eyepiece from the tube and insert an auxiliary telescope in its place. Adjust it so that the phase plate (in the form of a dark ring) and the annular diaphragm (in the form of a light ring of the same diameter) are clearly visible. Using the adjusting screws on the condenser, these rings are aligned. Remove the auxiliary telescope and reinstall the eyepiece.

Thanks to the use of this method of microscopy, the contrast of living unstained microorganisms increases sharply and they appear dark on a light background (positive phase contrast) or light on a dark background (negative phase contrast).

Phase-contrast microscopy is also used to study tissue culture cells, observe the effects of various viruses on cells, etc. In these cases, biological microscopes with reverse optics - inverted microscopes - are often used. In such microscopes, the objectives are located at the bottom and the condenser is at the top.

is an observation method in polarized light for the microscopic examination of preparations containing optically anisotropic elements (or consisting entirely of such elements). These are many minerals, grains in thin sections of alloys, some animal and plant tissues, etc. The optical properties of anisotropic microobjects are different in different directions and manifest themselves differently depending on the orientation of these objects relative to the direction of observation and the plane of polarization of light incident on them. Observation can be carried out in both transmitted and reflected light. The light emitted by the illuminator is passed through a polarizer. The polarization imparted to it changes with the subsequent passage of light through the preparation (or reflection from it). These changes are studied using an analyzer and various optical compensators. By analyzing such changes, one can judge the main optical characteristics of anisotropic microobjects: the strength of birefringence, the number of optical axes and their orientation, rotation of the plane of polarization, and dichroism.

Interference contrast method (interference microscopy) consists in the fact that each beam bifurcates as it enters the microscope. One of the resulting rays is directed through the observed particle, the other - past it along the same or additional optical branch of the microscope. In the eyepiece part of the microscope, both beams are again connected and interfere with each other. One of the rays, passing through the object, is delayed in phase (acquires a path difference compared to the second ray). The magnitude of this delay is measured by a compensator. We can say that the interference contrast method is similar to the phase contrast method - they are both based on the interference of rays that have passed through and past a microparticle. Like phase contrast microscopy, this method makes it possible to observe transparent and colorless objects, but their images can also be multi-colored (interference colors). Both methods are suitable for studying living tissues and cells and are used in many cases for this purpose. The main difference between interference microscopy and the phase contrast method is the ability to measure path differences introduced by microobjects. The interference contrast method is often used in conjunction with other microscopy methods, in particular with observation in polarized light. Its use in combination with ultraviolet microscopy makes it possible, for example, to determine the content of nucleic acids in the total dry mass of an object. Interference microscopy also includes methods of using microinterferometers.

Research method in the light of luminescence (luminescence microscopy, or fluorescence microscopy) consists of observing under a microscope the green-orange glow of micro-objects, which occurs when they are illuminated with blue-violet light or ultraviolet rays invisible to the eye. Two light filters are introduced into the optical circuit of the microscope. One of them is placed in front of the condenser. It transmits radiation from the illuminator source only at those wavelengths that excite the luminescence of either the object itself (intrinsic luminescence) or special dyes introduced into the preparation and absorbed by its particles (secondary luminescence). The second light filter, which is installed after the lens, transmits only luminescence light to the observer’s eye (or to the photosensitive layer). Fluorescent microscopy uses illumination of preparations both from above (through the lens, which in this case also serves as a condenser) and from below, through a regular condenser. Observation under illumination from above is sometimes called “reflected light luminescence microscopy” (this term is relative - the excitation of the glow of the preparation is not a simple reflection of light). It is often used in conjunction with observation using the phase-contrast method in transmitted light. The method has found wide application in microbiology, virology, histology, cytology, in the food industry, in soil research, in microchemical analysis, and in flaw detection. This variety of applications is explained by the very high color sensitivity of the eye and the high contrast of the image of a self-luminous object on a dark non-luminescent background. In addition, information about the composition and properties of the substances under study, which can be obtained by knowing the intensity and spectral composition of their luminescent radiation, is of great value.

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Part 2.

Light microscopy

Electron microscopy

Electron microscope

Chemical methods for studying cells

Light microscopy methods

Bright field method and its varieties

Dark field method and its variations

Conducting a dark-field study

Phase contrast method