The value of the microscope in biological research. A Brief History of the Development of Biology. The latest types of microscopes

Everyone knows that biology is the science of life. At present, it represents the totality of the sciences of living nature. Biology studies all manifestations of life: the structure, functions, development and origin of living organisms, their relationships in natural communities with the environment and with other living organisms.
Since man began to realize his difference from the animal world, he began to study the world around him. At first, his life depended on it. Primitive people needed to know which living organisms can be eaten, used as medicines, for making clothes and dwellings, and which of them are poisonous or dangerous.
With the development of civilization, a person could afford such a luxury as doing science for educational purposes.
Studies of the culture of ancient peoples have shown that they had extensive knowledge about plants and animals and widely applied them in everyday life.?

Modern biology is a complex science, which is characterized by the interpenetration of ideas and methods of various biological disciplines, as well as other sciences, primarily physics, chemistry, and mathematics.

The main directions of development of modern biology. Currently, three directions in biology can be conditionally distinguished.
First, it is classical biology. It is represented by natural scientists who study the diversity of wildlife. They objectively observe and analyze everything that happens in wildlife, study living organisms and classify them. It is wrong to think that in classical biology all discoveries have already been made. In the second half of the XX century. not only many new species have been described, but also large taxa have been discovered, up to kingdoms (Pogonophores) and even superkingdoms (Archaebacteria, or Archaea). These discoveries forced scientists to take a fresh look at the entire history of the development of wildlife. For true natural scientists, nature is a value in itself. Every corner of our planet is unique for them. That is why they are always among those who acutely feel the danger to the nature around us and actively advocate for it.
The second direction is evolutionary biology. In the 19th century, the author of the theory of natural selection, Charles Darwin, began as an ordinary naturalist: he collected, observed, described, traveled, revealing the secrets of wildlife. However, the main result of his work, which made him a famous scientist, was a theory explaining organic diversity.

Currently, the study of the evolution of living organisms is actively continuing. The synthesis of genetics and evolutionary theory led to the creation of the so-called synthetic theory of evolution. But even now there are still many unresolved questions that evolutionary scientists are looking for answers to.

Created at the beginning of the 20th century. by our outstanding biologist Alexander Ivanovich Oparin, the first scientific theory of the origin of life was purely theoretical. Currently, experimental studies of this problem are being actively conducted, and thanks to the use of advanced physicochemical methods, important discoveries have already been made and new interesting results can be expected.
New discoveries made it possible to supplement the theory of anthropogenesis. But the transition from the animal world to man still remains one of the biggest mysteries of biology.
The third direction is physicochemical biology, which studies the structure of living objects using modern physical and chemical methods. This is a rapidly developing area of ​​biology, important both in theoretical and practical terms. We can say with confidence that new discoveries are waiting for us in physical and chemical biology, which will allow us to solve many problems facing humanity,

The development of biology as a science. Modern biology is rooted in antiquity and is associated with the development of civilization in the Mediterranean countries. We know the names of many outstanding scientists who contributed to the development of biology. Let's name just a few of them.

Hippocrates (460 - c. 370 BC) gave the first relatively detailed description of the structure of man and animals, pointed out the role of the environment and heredity in the occurrence of diseases. He is considered the founder of medicine.
Aristotle (384-322 BC) divided the surrounding world into four kingdoms: the inanimate world of earth, water and air; plant world; the animal world and the human world. He described many animals, laid the foundation for taxonomy. The four biological treatises written by him contained almost all the information about animals known by that time. The merits of Aristotle are so great that he is considered the founder of zoology.
Theophrastus (372-287 BC) studied plants. He described more than 500 plant species, gave information about the structure and reproduction of many of them, introduced many botanical terms. He is considered the founder of botany.
Gaius Pliny the Elder (23-79) collected information about living organisms known by that time and wrote 37 volumes of the encyclopedia Natural History. Almost until the Middle Ages, this encyclopedia was the main source of knowledge about nature.

Claudius Galen made extensive use of dissections of mammals in his scientific research. He was the first to make comparative

anatomical description of man and monkey. Studied the central and peripheral nervous system. Historians of science consider him the last great biologist of antiquity.
In the Middle Ages, religion was the dominant ideology. Like other sciences, biology during this period had not yet emerged as an independent field and existed in the general mainstream of religious and philosophical views. And although the accumulation of knowledge about living organisms continued, one can speak of biology as a science at that time only conditionally.
The Renaissance is a transitional period from the culture of the Middle Ages to the culture of modern times. The fundamental socio-economic transformations of that time were accompanied by new discoveries in science.
The most famous scientist of this era, Leonardo da Vinci (1452-1519), made a certain contribution to the development of biology.

He studied the flight of birds, described many plants, ways of connecting bones in the joints, the activity of the heart and the visual function of the eye, the similarity of human and animal bones.

In the second half of the XV century. natural sciences begin to develop rapidly. This was facilitated by geographical discoveries, which made it possible to significantly expand information about animals and plants. Rapid accumulation of scientific knowledge about living organisms
led to the division of biology into separate sciences.
In the XVI-XVII centuries. Botany and zoology began to develop rapidly.
The invention of the microscope (early 17th century) made it possible to study the microscopic structure of plants and animals. Microscopically small living organisms, bacteria and protozoa, invisible to the naked eye, were discovered.
A great contribution to the development of biology was made by Carl Linnaeus, who proposed a classification system for animals and plants.
Karl Maksimovich Baer (1792-1876) in his works formulated the main provisions of the theory of homologous organs and the law of germline similarity, which laid the scientific foundations of embryology.

In 1808, in his Philosophy of Zoology, Jean-Baptiste Lamarck raised the question of the causes and mechanisms of evolutionary transformations and outlined the first theory of evolution in time.

The cell theory played a huge role in the development of biology, which scientifically confirmed the unity of the living world and served as one of the prerequisites for the emergence of Charles Darwin's theory of evolution. The zoologist Theodor Schwann (1818-1882) and the botanist Matthias Jakob Schleiden (1804-1881) are considered the authors of the cell theory.

Based on numerous observations, Charles Darwin published in 1859 his main work "On the Origin of Species by Means of Natural Selection, or the Preservation of Favored Breeds in the Struggle for Life." In it, he formulated the main provisions of the theory of evolution, proposed the mechanisms of evolution and the ways of evolutionary transformations of organisms.

The 20th century began with the rediscovery of Gregor Mendel's laws, which marked the beginning of the development of genetics as a science.
In the 40-50s of the XX century. ideas and methods of physics, chemistry, mathematics, cybernetics, and other sciences began to be widely used in biology, and microorganisms were used as objects of study. As a result, biophysics, biochemistry, molecular biology, radiation biology, bionics, etc. emerged and rapidly developed as independent sciences. Space exploration contributed to the birth and development of space biology.

In the XX century. a direction of applied research appeared - biotechnology. This trend will undoubtedly develop rapidly in the 21st century. You will learn more about this direction in the development of biology when studying the chapter "Fundamentals of Breeding and Biotechnology".

Currently, biological knowledge is used in all spheres of human activity: in industry and agriculture, medicine and energy.
Ecological research is extremely important. We finally began to realize that the delicate balance that exists on our small planet is easy to destroy. Mankind has faced a daunting task - the preservation of the biosphere in order to maintain the conditions for the existence and development of civilization. It is impossible to solve it without biological knowledge and special studies. Thus, at present, biology has become a real productive force and a rational scientific basis for the relationship between man and nature.

The history and invention of the microscope is due to the fact that since ancient times, people wanted to see much smaller objects than the naked human eye allowed. Although the first use of the lens remains unknown due to the age of time, it is believed that the use of the effect of refraction of light was used more than 2000 years ago. In the 2nd century BC, Claudius Ptolemy described the properties of light in a pool of water and accurately calculated the refractive constant of water.

During the 1st century AD (year 100), glass was invented and the Romans looked through the glass and tested it. They experimented with different shapes of clear glass and one of their designs was thicker in the middle and thinner at the edges. They found that an object would appear larger through such glass.

The word "lens" actually comes from the Latin word for "lentil", they named it because it resembles the shape of the legume plant lentil.

At the same time, the Roman philosopher Seneca describes the actual magnification through a jar of water "...letters, small and indistinct, are seen enlarged and clearer through a glass jar filled with water." Further lenses were not used until the end of the 13th century BC. Then around 1600, it was discovered that optical instruments could be made using a lens.

The first optical instruments

Early simple optical instruments were with magnifying glasses and typically had a magnification of about 6 x - 10 x. In 1590, two Dutch inventors, Hans Jansen and his son Zachary, while polishing lenses by hand, discovered that the combination of two lenses made it possible to enlarge the image of an object several times.

They mounted several lenses into a tube and made a very important discovery - the invention of the microscope..

Their first devices were newer than a scientific instrument, as the maximum magnification was up to 9x. The first microscope made for Dutch royalty had 3 extendable tubes, 50 cm long and 5 cm in diameter. The device was stated to have a magnification of 3x to 9x when fully deployed.

Leeuwenhoek's microscope

Another Dutch scientist Anthony van Leeuwenhoek (1632-1723), considered one of the pioneers of microscopy, at the end of the 17th century became the first person to actually use the invention of the microscope in practice.

Van Leeuwenhoek achieved more success than his predecessors by developing a method of making lenses by grinding and polishing. He achieved magnification up to 270x, the best known at the time. This magnification makes it possible to view objects one millionth of a meter in size.

Anthony Leeuwenhoek became more involved in science with his new invention of the microscope. He could see things no one had ever seen before. He first saw bacteria floating in a drop of water. He noted plant and animal tissues, sperm and blood cells, minerals, fossils, and more. He also discovered nematodes and rotifers (microscopic animals) and discovered bacteria by looking at plaque samples from his own teeth.

People began to realize that magnification could reveal structures that had never been seen before - the hypothesis that everything is made of tiny components invisible to the naked eye was not yet considered.

The work of Anthony Leeuwenhoek was further developed by the English scientist Robert Hooke, who published the results of microscopic studies "Micrography" in 1665. Robert Hooke has described detailed research in the field of microbiology.

The Englishman Robert Hooke discovered the microscopic milestone and the basic unit of all life - the cell. In the mid-17th century, Hooke saw structural cells while studying a specimen that reminded him of small cloister rooms. Hooke is also credited with being the first to use the three primary lens configuration as is used today after the invention of the microscope.

In the 18th and 19th centuries, not many changes in the design of the basic microscope were introduced. Lenses have been developed using cleaner glass and various shapes to address issues such as color distortion and poor image resolution. In the late 1800s, German optical physicist Ernst Abbe discovered that oil-coated lenses prevent light distortion at high resolution. The invention of the microscope helped the great Russian scientist-encyclopedist Lomonosov in the middle of the 18th century to carry out his experiments to move Russian science.

Modern development of microscopy

In 1931, German scientists began working on the invention of the electron microscope. This kind of device focuses electrons on a sample and forms an image that can be captured by an electronically sensitive element. This model allows scientists to view very fine details with magnification up to one million times. The only drawback is that living cells cannot be observed with an electron microscope. However, digital and other new technologies have created a new instrument for microbiologists.

The Germans, Ernst Ruska and Dr. Max Knoll, first created the "lens" of the magnetic field and electric current. By 1933, scientists had built an electron microscope that surpassed the magnification limits of an optical microscope at the time.

Ernst received the Nobel Prize in Physics in 1986 for his work. An electron microscope can achieve much higher resolution because the wavelength of an electron is shorter than the wavelength of visible light, especially when the electron is accelerated in a vacuum.

Light and electron microscopy advancing in the 20th century. Today, magnifiers use fluorescent labels or polarizing filters to view samples. More modern ones are used to capture and analyze images that are not visible to the human eye.

The invention of the microscope in the 16th century made it possible to create already reflective, phase, contrast, confocal and even ultraviolet devices..

Modern electronic devices can give an image of even a single atom.

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Abstract on the topic:

Modern methods of microscopic research

Completed by a student

2nd year 12 groups

Schukina Serafima Sergeevna

Introduction

1. Types of microscopy

1.1 Light microscopy

1.2 Phase contrast microscopy

1.3 Interference microscopy

1.4 Polarizing microscopy

1.5 Fluorescence microscopy

1.6 Ultraviolet microscopy

1.7 Infrared microscopy

1.8 Stereoscopic microscopy

1.9 Electron microscopy

2. Some types of modern microscopes

2.1 Historical background

2.2 The main components of the microscope

2.3 Microscope types

Conclusion

List of used literature

Introduction

Microscopic research methods - ways to study various objects using a microscope. In biology and medicine, these methods make it possible to study the structure of microscopic objects whose dimensions lie beyond the resolution of the human eye. The basis of microscopic research methods (M.m.i.) is light and electron microscopy. In practical and scientific activities, doctors of various specialties - virologists, microbiologists, cytologists, morphologists, hematologists, etc., in addition to conventional light microscopy, use phase-contrast, interference, luminescent, polarization, stereoscopic, ultraviolet, infrared microscopy. These methods are based on various properties of light. In electron microscopy, the image of the objects of study arises due to the directed flow of electrons.

microscopy polarizing ultraviolet

1. Types of microscopy

1.1 Light microscopy

For light microscopy and other M.m.i. In addition to the resolution of the microscope, the determining factor is the nature and direction of the light beam, as well as the features of the object under study, which can be transparent and opaque. Depending on the properties of the object, the physical properties of light change - its color and brightness associated with the wavelength and amplitude, phase, plane and direction of wave propagation. On the use of these properties of light, various M. m. and are built. For light microscopy, biological objects are usually stained in order to reveal one or another of their properties ( rice. one ). In this case, the tissues must be fixed, since staining reveals certain structures of only killed cells. In a living cell, the dye is isolated in the cytoplasm in the form of a vacuole and does not stain its structure. However, living biological objects can also be studied in a light microscope using the method of vital microscopy. In this case, a dark-field condenser is used, which is built into the microscope.

Rice. Fig. 1. Myocardial micropreparation in case of sudden death from acute coronary insufficiency: Lee staining allows to reveal contracture overcontractions of myofibrils (areas of red color); Ch250.

1.2 Phase contrast microscopy

Phase-contrast microscopy is also used to study living and unstained biological objects. It is based on the diffraction of a beam of light depending on the characteristics of the radiation object. This changes the length and phase of the light wave. The objective of a special phase-contrast microscope contains a translucent phase plate. Living microscopic objects or fixed, but not colored, microorganisms and cells, due to their transparency, practically do not change the amplitude and color of the light beam passing through them, causing only a shift in the phase of its wave. However, after passing through the object under study, the light rays deviate from the translucent phase plate. As a result, a difference in wavelength arises between the rays that have passed through the object and the rays of the light background. If this difference is at least 1/4 of the wavelength, then a visual effect appears, in which a dark object is clearly visible against a light background, or vice versa, depending on the features of the phase plate.

1.3 interference microscopy

Interference microscopy solves the same problems as phase-contrast microscopy. But if the latter allows us to observe only the contours of the objects of study, then with the help of interference microscopy it is possible to study the details of a transparent object and carry out their quantitative analysis. This is achieved by bifurcating a beam of light in a microscope: one of the beams passes through the particle of the observed object, and the other passes by it. In the eyepiece of a microscope, both beams are connected and interfere with each other. The resulting phase difference can be measured by determining thus. many different cellular structures. Sequential measurement of the phase difference of light with known refractive indices makes it possible to determine the thickness of living objects and non-fixed tissues, the concentration of water and dry matter in them, the content of proteins, etc. Based on interference microscopy data, one can indirectly judge the permeability of membranes, enzyme activity, cellular metabolism of the objects of study.

1.4 Polarizing microscopy

Polarizing microscopy makes it possible to study objects of study in light formed by two beams polarized in mutually perpendicular planes, i.e., in polarized light. To do this, filmy polaroids or Nicol prisms are used, which are placed in a microscope between the light source and the preparation. Polarization changes during the passage (or reflection) of light rays through various structural components of cells and tissues, the properties of which are inhomogeneous. In the so-called isotropic structures, the propagation velocity of polarized light does not depend on the plane of polarization; in anisotropic structures, its propagation velocity varies depending on the direction of the light along the longitudinal or bath light in the norm.

Rice. 2a). Micropreparation of the myocardium in the polarization of the transverse axis of the object.

If the refractive index of light along the structure is greater than in the transverse direction, positive birefringence occurs, with reverse relationships - negative birefringence. Many biological objects have a strict molecular orientation, are anisotropic and have positive double refraction of light. Myofibrils, cilia of the ciliated epithelium, neurofibrils, collagen fibers, etc. have such properties. fig.2 ). Polarizing microscopy is one of the histological research methods, a method of microbiological diagnostics, is used in cytological studies, etc. At the same time, both stained and unstained and non-fixed, so-called native preparations of tissue sections, can be examined in polarized light.

Rice. 2b). A micropreparation of the myocardium in polarized light with sudden death from acute coronary insufficiency - areas are identified in which there is no characteristic transverse striation of cardiomyocytes; Ch400.

1.5 Fluorescent microscopy

Fluorescent microscopy is widely used. It is based on the property of some substances to give luminescence - luminescence in UV rays or in the blue-violet part of the spectrum. Many biological substances, such as simple proteins, coenzymes, some vitamins and drugs, have their own (primary) luminescence. Other substances begin to glow only when special dyes are added to them - fluorochromes (secondary luminescence). Fluorochromes can be diffusely distributed in a cell or selectively stain individual cell structures or certain chemical compounds of a biological object. This is the basis for the use of luminescent microscopy in cytological and histochemical studies. With the help of immunofluorescence in a fluorescent microscope, viral antigens and their concentration in cells are detected, viruses are identified, antigens and antibodies, hormones, various metabolic products, etc. are determined. ( rice. 3 ). In this regard, luminescent microscopy is used in the laboratory diagnosis of infections such as herpes, mumps, viral hepatitis, influenza, etc., is used in the rapid diagnosis of respiratory viral infections, examining prints from the nasal mucosa of patients, and in the differential diagnosis of various infections. In pathomorphology, using luminescent microscopy, malignant tumors are recognized in histological and cytological preparations, areas of ischemia of the heart muscle are determined in the early stages of myocardial infarction, and amyloid is detected in tissue biopsies.

Rice. 3. Micropreparation of peritoneal macrophage in cell culture, fluorescent microscopy.

1.6 ultraviolet microscopy

Ultraviolet microscopy is based on the ability of certain substances that make up living cells, microorganisms, or fixed, but not stained, transparent tissues in visible light, to absorb UV radiation with a certain wavelength (400-250 nm). High-molecular compounds, such as nucleic acids, proteins, aromatic acids (tyrosine, tryptophan, methylalanine), purine and pyramidine bases, etc., have this property. Using ultraviolet microscopy, the localization and amount of these substances are specified, and in the case of studying living objects, their changes in the process of life.

1.7 infrared microscopy

Infrared microscopy makes it possible to study objects that are opaque to visible light and UV radiation by absorbing light with a wavelength of 750–1200 nm by their structures. Infrared microscopy does not require prior chem. drug processing. This type of M. m. and. most often used in zoology, anthropology, and other branches of biology. In medicine, infrared microscopy is mainly used in neuromorphology and ophthalmology.

1.8 stereoscopic microscopy

Stereoscopic microscopy is used to study volumetric objects. The design of stereoscopic microscopes allows you to see the object of study with the right and left eyes from different angles. Explore opaque objects at relatively low magnification (up to 120 times). Stereoscopic microscopy finds application in microsurgery, in pathomorphology with a special study of biopsy, surgical and sectional material, in forensic laboratory research.

1.9 electron microscopy

Electron microscopy is used to study the structure of cells, tissues of microorganisms and viruses at the subcellular and macromolecular levels. This M. m. and. allowed to move to a qualitatively new level of study of matter. It has found wide application in morphology, microbiology, virology, biochemistry, oncology, genetics, and immunology. A sharp increase in the resolution of an electron microscope is provided by the flow of electrons passing in vacuum through electromagnetic fields created by electromagnetic lenses. Electrons can pass through the structures of the object under study (transmission electron microscopy) or be reflected from them (scanning electron microscopy), deviating at different angles, resulting in an image on the luminescent screen of the microscope. With transmission (transmission) electron microscopy, a planar image of structures is obtained ( rice. 4 ), with scanning - volumetric ( rice. five ). The combination of electron microscopy with other methods, for example, autoradiography, histochemical, immunological research methods, allows for electron radioautographic, electron histochemical, electron immunological studies.

Rice. 4. Electron diffraction pattern of a cardiomyocyte obtained by transmission (transmission) electron microscopy: subcellular structures are clearly visible; Ch22000.

Electron microscopy requires special preparation of objects of study, in particular chemical or physical fixation of tissues and microorganisms. Biopsy material and sectional material after fixation are dehydrated, poured into epoxy resins, cut with glass or diamond knives on special ultratomes, which make it possible to obtain ultrathin tissue sections with a thickness of 30–50 nm. They are contrasted and then examined under an electron microscope. In a scanning (raster) electron microscope, the surface of various objects is studied by depositing electron-dense substances on them in a vacuum chamber, and examining the so-called. replicas that follow the contours of the sample.

Rice. 5. Electron diffraction pattern of a leukocyte and a bacterium phagocytosed by it obtained by scanning electron microscopy; CH20000.

2. Some types of modern microscopes

Phase contrast microscope(anoptral microscope) is used to study transparent objects that are not visible in a bright field and are not subject to staining due to the occurrence of anomalies in the samples under study.

interference microscope makes it possible to study objects with low refractive indices and extremely small thicknesses.

Ultraviolet and infrared microscopes designed to study objects in the ultraviolet or infrared part of the light spectrum. They are equipped with a fluorescent screen on which an image of the test preparation is formed, a camera with photographic material sensitive to these radiations, or an electron-optical converter for forming an image on the oscilloscope screen. The wavelength of the ultraviolet part of the spectrum is 400-250 nm, therefore, a higher resolution can be obtained in an ultraviolet microscope than in a light microscope, where illumination is carried out by visible light radiation with a wavelength of 700-400 nm. The advantage of this M. is also that objects invisible in a conventional light microscope become visible, since they absorb UV radiation. In an infrared microscope, objects are observed on the screen of an electron-optical converter or photographed. Infrared microscopy is used to study the internal structure of opaque objects.

polarizing microscope allows you to identify heterogeneities (anisotropy) of the structure when studying the structure of tissues and formations in the body in polarized light. Illumination of the preparation in a polarizing microscope is carried out through a polarizer plate, which ensures the passage of light in a certain plane of wave propagation. When polarized light, interacting with structures, changes, the structures contrast sharply, which is widely used in biomedical research when studying blood products, histological preparations, sections of teeth, bones, etc.

Fluorescent microscope(ML-2, ML-3) is designed to study luminescent objects, which is achieved by illuminating the latter with UV radiation. By observing or photographing preparations in the light of their visible excited fluorescence (i.e., in reflected light), one can judge the structure of the test sample, which is used in histochemistry, histology, microbiology, and immunological studies. Direct staining with luminescent dyes makes it possible to more clearly identify cell structures that are difficult to see in a light microscope.

X-ray microscope used to study objects in X-rays, therefore, such microscopes are equipped with a microfocus X-ray source of radiation, an X-ray image-to-visible converter - an electron-optical converter that forms a visible image on an oscilloscope tube or on photographic film. X-ray microscopes have a linear resolution of up to 0.1 µm, which makes it possible to study the fine structures of living matter.

Electron microscope designed to study ultrafine structures that are indistinguishable in light microscopes. Unlike light, in an electron microscope, resolution is determined not only by diffraction phenomena, but also by various aberrations of electronic lenses, which are almost impossible to correct. The aiming of the microscope is mainly carried out by diaphragming due to the use of small apertures of electron beams.

2.1 Historical background

The property of a system of two lenses to give enlarged images of objects was already known in the 16th century. in the Netherlands and northern Italy to craftsmen who made spectacle lenses. There is evidence that around 1590 an instrument of the M type was built by Z. Jansen (Netherlands). The rapid spread of M. and their improvement, mainly by optician artisans, begins from 1609–10, when G. Galileo, studying the telescope he designed (see. Spotting Scope), used it as M., changing the distance between the lens and eyepiece. The first brilliant successes in the use of M. in scientific research are associated with the names of R. Hooke (circa 1665; in particular, he established that animal and plant tissues have a cellular structure) and especially A. Leeuwenhoek, who discovered microorganisms with the help of M. (1673-- 77). At the beginning of the 18th century M. appeared in Russia: here L. Euler (1762; Dioptrics, 1770–71) developed methods for calculating the optical units of M. In 1827, J. B. Amici was the first to use an immersion lens in M.. In 1850, the English optician G. Sorby created the first microscope for observing objects in polarized light.

Wide development of methods of microscopic researches and improvement of various types of M. in 2nd half of 19 and in 20 centuries. The scientific activity of E. Abbe, who developed (1872–73) the classical theory of the formation of images of non-luminous objects in M., contributed greatly to the scientific activity. In 1893, the English scientist J. Sirks laid the foundation for interference microscopy. In 1903, the Austrian researchers R. Zigmondy and G. Siedentopf created the so-called. ultramicroscope. In 1935, F. Zernike proposed the phase contrast method for observing transparent objects that weakly scatter light in M.. A great contribution to the theory and practice of microscopy was made by owls. scientists - L. I. Mandelstam, D. S. Rozhdestvensky, A. A. Lebedev, V. P. Linnik.

2.2 The main components of the microscope

In most types of M. (with the exception of inverted ones, see below), a device for attaching lenses is located above the object table on which the preparation is fixed, and a condenser is installed under the table. Any M. has a tube (tube) in which eyepieces are installed; Mechanisms for coarse and fine focusing (carried out by changing the relative position of the preparation, objective, and eyepiece) are also an obligatory accessory of M.. All these nodes are mounted on a tripod or M body.

The type of condenser used depends on the choice of observation method. Bright-field condensers and condensers for observation by the method of phase or interference contrast are two- or three-lens systems that differ greatly from one another. For bright-field condensers, the numerical aperture can reach 1.4; they include an aperture iris diaphragm, which can sometimes be shifted to the side to obtain oblique illumination of the preparation. Phase-contrast condensers are equipped with annular diaphragms. Complex systems of lenses and mirrors are dark-field condensers. A separate group is made up of epicondensers, which are necessary when observing by the method of a dark field in reflected light, a system of annular lenses and mirrors installed around the lens. In UV microscopy, special mirror-lens and lens condensers are used, which are transparent to ultraviolet rays.

The lenses in most modern microscopes are interchangeable and are selected depending on the specific conditions of observation. Often several lenses are fixed in one rotating (so-called revolving) head; lens change in this case is carried out by simply turning the head. According to the degree of correction of chromatic aberration (see Chromatic aberration), micro lenses are distinguished Achromats and apochromats (see Achromat). The first are the simplest in design; chromatic aberration in them is corrected for only two wavelengths, and the image remains slightly colored when the object is illuminated with white light. In apochromats, this aberration is corrected for three wavelengths, and they give colorless images. The image plane of achromats and apochromats is somewhat curved (see Curvature of the field). The accommodation of the eye and the ability to view the entire field of view with the help of refocusing M. partly compensate for this shortcoming in visual observation, but it greatly affects microphotography - the extreme parts of the image are blurred. Therefore, microobjectives with additional field curvature correction are widely used - planachromats and planapochromats. In combination with conventional lenses, special projection systems are used - gomals, inserted instead of eyepieces and correcting the curvature of the image surface (they are unsuitable for visual observation).

In addition, microobjectives differ: a) in terms of spectral characteristics - for lenses for the visible region of the spectrum and for UV and IR microscopy (lens or mirror-lens); b) according to the length of the tube for which they are designed (depending on the design of the M.), - for lenses for a tube of 160 mm, for a tube of 190 mm and for the so-called. "the length of the tube is infinity" (the latter create an image "at infinity" and are used in conjunction with an additional - the so-called tube - lens, which translates the image into the focal plane of the eyepiece); c) according to the medium between the lens and the preparation - into dry and immersion; d) according to the method of observation - into ordinary, phase-contrast, interference, etc.; e) by type of preparations - for preparations with and without a cover slip. A separate type are epi lenses (a combination of a conventional lens with an epicondenser). The variety of lenses is due to the variety of methods of microscopic observation and the design of microscopes, as well as differences in the requirements for correcting aberrations under different working conditions. Therefore, each lens can only be used in the conditions for which it was designed. For example, a lens designed for a 160 mm tube cannot be used in an M. with a tube length of 190 mm; With a cover slip slide lens, slides without a cover slip cannot be observed. It is especially important to observe the design conditions when working with dry lenses of large apertures (A > 0.6), which are very sensitive to any deviations from the norm. The thickness of the coverslips when working with these objectives should be equal to 0.17 mm. An immersion lens can only be used with the immersion for which it was designed.

The type of eyepiece used for this method of observation is determined by the choice of the M objective. compensation eyepieces calculated so that their residual chromatic aberration is of a different sign than that of lenses, which improves image quality. In addition, there are special photo eyepieces and projection eyepieces that project an image onto a screen or photographic plate (this also includes the gomals mentioned above). A separate group consists of quartz eyepieces that are transparent to UV rays.

Various accessories to M. allow to improve conditions of supervision and to expand possibilities of researches. Illuminators of various types are designed to create the best lighting conditions; ocular micrometers (see Ocular micrometer) are used to measure the size of objects; binocular tubes make it possible to observe the drug simultaneously with both eyes; microphoto attachments and microphoto setups are used for microphotography; drawing devices make it possible to sketch images. For quantitative studies, special devices are used (for example, microspectrophotometric nozzles).

2.3 Types of microscopes

The design of an M., its equipment, and the characteristics of its main units are determined either by the field of application, the range of problems, and the nature of the objects for which it is intended, or by the method (methods) of observation for which it is designed, or by both. All this led to the creation of various types of specialized metrics, which make it possible to study strictly defined classes of objects (or even only some of their specific properties) with high accuracy. On the other hand, there are so-called. universal M., with the help of which it is possible to observe various objects by various methods.

Biological M. are among the most common. They are used for botanical, histological, cytological, microbiological, and medical research, as well as in areas not directly related to biology—to observe transparent objects in chemistry, physics, and so on. There are many models of biological M. that differ in their constructive design and accessories that significantly expand the range of objects under study. These accessories include: replaceable illuminators for transmitted and reflected light; replaceable condensers for work on methods of bright and dark fields; phase contrast devices; ocular micrometers; microphoto attachments; sets of light filters and polarizing devices, which make it possible to use the technique of luminescent and polarizing microscopy in ordinary (non-specialized) M.. In auxiliary equipment for biological M., a particularly important role is played by the means of microscopic technology (see Microscopic technology), designed to prepare preparations and perform various operations with them, including directly during the observation process (see Micromanipulator, Microtome).

Biological research microscopes are equipped with a set of interchangeable lenses for various conditions and methods of observation and types of specimens, including epi-objectives for reflected light and often phase-contrast lenses. A set of objectives corresponds to a set of eyepieces for visual observation and microphotography. Usually such M. have binocular tubes for observation with two eyes.

In addition to general-purpose M., various M., specialized in the method of observation, are also widely used in biology (see below).

Inverted microscopes are distinguished by the fact that the lens in them is located under the observed object, and the condenser is on top. The direction of the rays passing from top to bottom through the lens is changed by a system of mirrors, and they fall into the eye of the observer, as usual, from bottom to top ( rice. 8). M. of this type are intended for the study of bulky objects that are difficult or impossible to place on the object tables of conventional M. In biology, with the help of such M., tissue cultures in a nutrient medium are studied, which are placed in a thermostatic chamber to maintain a given temperature. Inverted meters are also used to study chemical reactions, determine the melting points of materials, and in other cases when cumbersome auxiliary equipment is required to carry out the observed processes. Inverted microscopes are equipped with special devices and cameras for microphotography and film microfilming.

The scheme of an inverted microscope is especially convenient for observing the structures of various surfaces in reflected light. Therefore, it is used in most metallographic M. In them, the sample (section of metal, alloy or mineral) is installed on the table with the polished surface down, and the rest of it can have an arbitrary shape and does not require any processing. There are also metallographic M., in which the object is placed from below, fixing it on a special plate; the mutual position of nodes in such meters is the same as in ordinary (non-inverted) meters. The surface under study is often preliminarily etched, so that the grains of its structure become sharply distinguishable from each other. In M. of this type, you can use the bright field method with direct and oblique illumination, the dark field method, and observation in polarized light. When working in a bright field, the lens simultaneously serves as a condenser. For dark-field illumination mirror parabolic epicondensers are used. The introduction of a special auxiliary device makes it possible to carry out phase contrast in metallographic M. with a conventional lens ( rice. nine).

Luminescent microscopes are equipped with a set of interchangeable light filters, by selecting which it is possible to isolate in the illuminator's radiation a part of the spectrum that excites the luminescence of a particular object under study. A light filter is also selected that transmits only luminescence light from the object. The glow of many objects is excited by UV rays or the short-wavelength part of the visible spectrum; therefore, the sources of light in luminescent lamps are ultrahigh-pressure mercury lamps that give just such (and very bright) radiation (see Gas-discharge light sources). In addition to special models of luminescent lamps, there are luminescent devices used in conjunction with conventional lamps; they contain an illuminator with a mercury lamp, a set of light filters, etc. opaque illuminator for illumination of preparations from above.

Ultraviolet and infrared microscopes are used for research in regions of the spectrum invisible to the eye. Their fundamental optical schemes are similar to those of conventional MMs. Because of the great difficulty in correcting aberrations in the UV and IR regions, the condenser and objective in such MMs often represent mirror-lens systems in which chromatic aberration is significantly reduced or completely absent. Lenses are made from materials that are transparent to UV (quartz, fluorite) or IR (silicon, germanium, fluorite, lithium fluoride) radiation. Ultraviolet and infrared M. are supplied with cameras in which the invisible image is fixed; visual observation through an eyepiece in ordinary (visible) light serves, when possible, only for preliminary focusing and orientation of the object in the field of view of the M. As a rule, these M. have electron-optical converters that convert an invisible image into a visible one.

Polarizing meters are designed to study (with the help of optical compensators) changes in the polarization of light that has passed through an object or reflected from it, which opens up possibilities for quantitative or semi-quantitative determination of various characteristics of optically active objects. The nodes of such M. are usually made in such a way as to facilitate accurate measurements: the eyepieces are supplied with a crosshair, a micrometer scale or a grid; a rotating object table -- with a goniometric limb for measuring the angle of rotation; often a Fedorov table is attached to the object table (see Fedorov table), which makes it possible to arbitrarily rotate and tilt the specimen to find the crystallographic and crystal-optical axes. The lenses of polarizing lenses are specially selected so that there are no internal stresses in their lenses that lead to the depolarization of light. M. of this type usually has an auxiliary lens (the so-called Bertrand lens) that can be switched on and off, which is used for observations in transmitted light; it allows one to consider interference patterns (see Crystal optics) formed by light in the rear focal plane of the objective after passing through the crystal under study.

With the help of interference microscopes, transparent objects are observed using the method of interference contrast; many of them are structurally similar to conventional M., differing only in the presence of a special condenser, objective and measuring unit. If the observation is made in polarized light, then such microscopes are supplied with a polarizer and an analyzer. By area of ​​application (mainly biological research), these M. can be attributed to specialized biological M. Interferometric M. often also include microinterferometers - M. of a special type used to study the microrelief of the surfaces of machined metal parts.

Stereomicroscopes. The binocular tubes used in conventional microscopes, despite the convenience of observing with two eyes, do not produce a stereoscopic effect: in this case, the same rays enter both eyes at the same angles, only they are divided into two beams by a prism system. Stereomicroscopes, which provide a truly three-dimensional perception of a microobject, are in fact two microscopes made in the form of a single structure so that the right and left eyes observe the object at different angles ( rice. 10). Such M. are most widely used where it is required to perform any operations with an object in the course of observation (biological research, surgical operations on blood vessels, the brain, in the eye - Micrurgy, the assembly of miniature devices, such as Transistors), - stereoscopic perception facilitates these operations. Convenience of orientation in the field of view of M. is also included in its optical scheme of prisms that play the role of turning systems (see Turning system); the image in such M. is straight, not inverted. So how is the angle between the optical axes of lenses in stereo microscopes usually? 12°, their numerical aperture, as a rule, does not exceed 0.12. Therefore, a useful increase in such M. is no more than 120.

Comparison lenses consist of two structurally combined ordinary lenses with a single ocular system. The observer sees images of two objects at once in two halves of the field of view of such a lens, which makes it possible to directly compare them in terms of color, structure, distribution of elements, and other characteristics. Comparison markers are widely used in assessing the quality of surface treatment, determining grade (comparison with a reference sample), etc. Special markers of this type are used in criminology, in particular, to identify the weapon from which the bullet under study was fired.

In television M., working according to the microprojection scheme, the image of the drug is converted into a sequence of electrical signals, which then reproduce this image on an enlarged scale on the screen of a cathode ray tube (see. Cathode ray tube) (kinescope). In such M., it is possible, by purely electronic means, by changing the parameters of the electrical circuit through which the signals pass, to change the contrast of the image and to adjust its brightness. Electrical amplification of signals allows images to be projected onto a large screen, while conventional micro-projection requires extremely strong illumination, often harmful to microscopic objects. The great advantage of television meters is that they can be used to remotely study objects whose proximity is dangerous for the observer (for example, radioactive).

In many studies, it is necessary to count microscopic particles (for example, bacteria in colonies, aerosols, particles in colloidal solutions, blood cells, etc.), determine the areas occupied by grains of the same kind in thin sections of an alloy, and produce other similar measurements. The transformation of images in television meters into a series of electrical signals (pulses) made it possible to build automatic counters of microparticles that register them by the number of pulses.

The purpose of measuring meters is to accurately measure the linear and angular dimensions of objects (often not at all small). According to the method of measurement, they can be divided into two types. Measuring M. of the 1st type are used only in cases where the measured distance does not exceed the linear dimensions of the field of view of the M. In such M. directly (using a scale or a screw ocular micrometer (see Ocular micrometer)) is measured not the object itself, but its image in the focal plane of the eyepiece, and only then, according to the known value of the lens magnification, the measured distance on the object is calculated. Often, in these microscopes, the images of objects are compared with exemplary profiles printed on the plates of interchangeable eyepiece heads. In the measuring The 2nd type of the subject table with the object and the M.'s body can be moved relative to each other with the help of precise mechanisms (more often - the table relative to the body); by measuring this movement with a micrometric screw or a scale rigidly fastened to the object stage, the distance between the observed elements of the object is determined. There are measuring meters for which measurements are made in only one direction (single-coordinate meters). Much more common are M. with movements of the object table in two perpendicular directions (limits of movement up to 200-500 mm); For special purposes, instruments are used in which measurements (and, consequently, relative displacements of the table and body of the instrument) are possible in three directions, corresponding to three axes of rectangular coordinates. On some M. it is possible to carry out measurements in polar coordinates; for this, the object table is made rotating and equipped with a scale and a Nonius for reading the rotation angles. The most accurate measuring instruments of the second type use glass scales, and readings on them are carried out using an auxiliary (so-called reading) microscope (see below). The accuracy of measurements in M. of the 2nd type is much higher compared to M. of the 1st type. In the best models, the accuracy of linear measurements is usually of the order of 0.001 mm, the accuracy of measuring angles is of the order of 1 ". Measuring meters of the 2nd type are widely used in industry (especially in mechanical engineering) for measuring and controlling the dimensions of machine parts, tools, etc.

In devices for especially precise measurements (for example, geodetic, astronomical, etc.), readings on linear scales and divided circles of goniometric instruments are made using special reading meters - scale meters and micrometers. The first has an auxiliary glass scale. By adjusting the magnification of the objective lens, its image is made equal to the observed interval between divisions of the main scale (or circle), after which, by counting the position of the observed division between the strokes of the auxiliary scale, it can be directly determined with an accuracy of about 0.01 of the interval between divisions. The accuracy of readings (on the order of 0.0001 mm) is even higher in M. micrometers, in the ocular part of which a thread or spiral micrometer is placed. The magnification of the lens is adjusted so that the movement of the thread between the images of the strokes of the measured scale corresponds to an integer number of turns (or half turns) of the micrometer screw.

In addition to those described above, there are a significant number of still more narrowly specialized types of thermometers, for example, thermometers for counting and analyzing traces of elementary particles and nuclear fission fragments in nuclear photographic emulsions (see Nuclear photographic emulsion), high-temperature meters for studying objects heated to temperatures of the order of 2000 ° C, contact lenses for studying the surfaces of living organs of animals and humans (the lens in them is pressed close to the surface under study, and the lens is focused by a special built-in system).

Conclusion

What can we expect from the microscopy of tomorrow? What problems can be expected to be solved? First of all - distribution to more and more new objects. The achievement of atomic resolution is certainly the greatest achievement of scientific and technical thought. However, let's not forget that this achievement extends only to a limited range of objects, which are also placed in very specific, unusual and highly influencing conditions. Therefore, it is necessary to strive to extend atomic resolution to a wide range of objects.

Over time, we can expect other charged particles to “work” in microscopes. It is clear, however, that this must be preceded by the search for and development of powerful sources of such particles; in addition, the creation of a new type of microscope will be determined by the emergence of specific scientific problems, to the solution of which these new particles will make a decisive contribution.

Microscopic studies of processes in dynamics will be improved, i.e. occurring directly in the microscope or in devices articulated with it. Such processes include testing samples in a microscope (heating, stretching, etc.) directly during the analysis of their microstructure. Here, success will be due, first of all, to the development of high-speed photography and the increase in the temporal resolution of detectors (screens) of microscopes, as well as the use of powerful modern computers.

List of used literature

1. Small medical encyclopedia. -- M.: Medical Encyclopedia. 1991--96

2. First aid. -- M.: Great Russian Encyclopedia. 1994

3. Encyclopedic dictionary of medical terms. -- M.: Soviet Encyclopedia. -- 1982--1984

4. http://dic.academic.ru/

5. http://ru.wikipedia.org/

6. www.golkom.ru

7. www.avicenna.ru

8. www.bionet.nsc.ru

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Microscopy technology has opened up new possibilities in medical and laboratory practice. Today, neither diagnostic studies nor surgical interventions can do without special optics. The most significant role of microscopes in dentistry, ophthalmology, microsurgery. This is not just about improving visibility and facilitating work, but about a fundamentally new approach to research and operations.

The impact on fine structures at the cellular level means that the patient will more easily endure the intervention, recover faster, and will not suffer damage to healthy tissues and complications. Behind all these advantages of modern medicine is often a microscope - a powerful high-tech device, designed using the latest advances in optics.

Depending on the purpose, microscopes are divided into:

• laboratory;
• dental;
• surgical;
• ophthalmic;
• otolaryngological.

Optical systems for biochemical, hematological, dermatological, cytological studies are functionally different from medical ones. Ophthalmological microscopes are recognized as the most advanced and powerful - with their help, it was possible to make a radical breakthrough in the treatment of cataracts, farsightedness, myopia, astigmatism. Operations at the micron level, performed under 40x magnification, are comparable in invasiveness to an injection, the patient recovers after surgery in a matter of days.

No less interesting are those that allow, under 25x magnification, to accurately treat dental canals and other smallest structures that are not visible to the human eye. Using the latest optics, dentists almost always manage to provide high-quality treatment and save the tooth.

Magnifying devices for microsurgery are characterized by an extended field of view, increased image sharpness, and the possibility of smooth or stepwise adjustment of the magnification. All this provides the best visibility conditions for the surgeon and assistants.

It is important that the new generation of instruments for microscopy is as convenient as possible to use: working with magnifying optics is simple and does not require much effort or special skills. Due to the built-in lighting system and the convenient shape of the eyepiece, the specialist does not experience fatigue and discomfort even during long continuous work.

A microscope is a fragile instrument that needs to be handled with care. This is especially true for lenses: it is undesirable to touch the optical surfaces with your hands; a special brush and soft wipes soaked in ethyl alcohol are used to clean the device.

Rooms containing microscopes should be maintained at room temperature and low humidity (less than 60%).

Histology as an independent science stood out at the beginning of the 19th century. The prehistory of histology was the results of numerous macroscopic (visual) studies of the constituent parts of various animal and plant organisms. Of decisive importance for the development of histology as a science of tissue structure was the invention of the microscope, the first samples of which were created at the beginning of the 17th century (G. and Z. Jansen, G. Galilei, and others). One of the earliest scientific studies using a microscope of his own design was carried out by the English scientist Robert Hooke (1635-1703). He studied the microscopic structure of many objects. R. Hooke described all the objects studied in the book "Micrography or some physiological descriptions of the smallest bodies made with the help of magnifying glasses ...", published in 1665. From his observations, R. Hooke concluded that bubble-shaped cells, or cells, are widespread, in plant objects and first proposed the term "cell".

In 1671, the English scientist N. Grew (1641-1712) in his book " plant anatomy"Wrote about the cellular structure as a general principle of organization of plant organisms. N. Grew first introduced the term "fabric" to denote plant mass, since the latter resembled clothing fabrics in its microscopic design. In the same year, the Italian J. Malpighi (1628- 1694) gave a systematic and detailed description of the cellular (cellular) structure of various plants. Later, facts gradually accumulated, indicating that not only plant, but also animal organisms are composed of cells. In the second half of the 17th century, A. Leeuwenhoek (1632-1723 ) discovered the world of microscopic animals and for the first time described red blood cells and male sex cells.

Throughout the 18th century there was a gradual accumulation of facts about the cellular structure of plants and animals. Cells of animal tissues were studied and described in detail by the Czech scientist Jan Purkynia (1787-1869) and his students at the beginning of the 19th century.

Of great importance for the development of knowledge about microscopic structure of organisms has further improved microscopes. In the 18th century, microscopes were already being produced in large numbers. They were first brought to Russia from Holland by Peter I. Later, a workshop for the manufacture of microscopes was organized at the Academy of Sciences in St. Petersburg. M.V. did a lot for the development of microscopy in Russia. Lomonosov, who proposed a number of technical improvements in the design of the microscope and its optical system. The second half of the 19th century is notable for the rapid improvement of microscopic technology. New designs of microscopes were created, and, thanks to the invention of immersion lenses (water immersion began to be used from 1850, oil immersion - from 1878), the resolution of optical instruments increased tenfold. In parallel with the improvement of the microscope, the technique of preparing microscopic preparations also developed.

If earlier objects examined under a microscope immediately after their isolation from plants or animals without any preliminary preparation, now they began to resort to various methods of processing them, which made it possible to preserve the structure of biological objects. Various methods of material fixation have been proposed. Chromic, picric, osmic, acetic and other acids, as well as their mixtures, have been used as fixing agents. A simple and in many cases indispensable fixative - formalin - was first used to fix biological objects in 1893.

Manufacturing of drugs, suitable for examination in transmitted light, became possible after the development of methods for pouring pieces into dense media, which made it easier to obtain thin sections. The invention of special structures for cutting - microtomes - in the laboratory of J. Purkins significantly improved the technique for making histological preparations. In Russia, the first microtome was constructed by the Kyiv histologist P.I. Peremezhko. To enhance the contrast of the structures, the sections began to be stained with various dyes. Carmine was the first histological dye that stained cell nuclei and was widely used (beginning in 1858). Another nuclear dye - hematoxylin - has been used since 1865, but for a long time its properties were not fully evaluated. By the second half of the 19th century, aniline dyes were already used, a method was developed for impregnating tissues with silver nitrate (K. Golgi, 1873) and staining of nervous tissue with methylene blue (A.S. Dogel, A.E. Smirnov, 1887).

Due to the fixation of biological material and obtaining the thinnest colored sections from it, researchers of the late 19th century had the opportunity to penetrate much deeper into the secrets of the structure of tissues and cells, on the basis of which a number of the greatest discoveries were made. So, in 1833, R. Brown discovered a permanent component of the cell - the nucleus. In 1861, M. Schultze approved the view of the cell as "a lump of protoplasm with a nucleus lying inside it." The main components of the cell began to be considered the nucleus and cytoplasm. In the 70s of the XIX century, a group of researchers simultaneously and independently discovered an indirect method of cell division - karyokinesis, or mitosis. In the works of I.D. Chistyakov (1874), O. Buchli (1875), E. Strasburger (1875), W. Meisel (1875), P.I. Peremezhko (1878), V. Schleicher (1878), V. Flemming (1879) and others described and illustrated all stages of indirect cell division. This discovery was of great importance for the development of knowledge about the cell. It also served as the basis for a deeper study of such an important biological process as fertilization. The study of mitosis and fertilization attracted particular attention of researchers to the cell nucleus and elucidation of its significance in the process of transferring hereditary properties. In 1884, O. Gertwig and E. Strasburger independently put forward the hypothesis that chromatin is the material carrier of heredity.

The object of close attention of scientists is chromosomes. Along with the study of the cell nucleus, the cytoplasm was also subjected to a thorough analysis.

Advances in microscopic technology have led to opening of organelles in the cytoplasm- its constant and highly differentiated elements, having a certain structure and performing vital functions for the cell. In 1875-76. the German biologist O. Hertwig and the Belgian scientist Van Beneden discovered the cell center, or centrosome; and in 1898 by the Italian scientist K. Golgi - the intracellular reticular apparatus (Golgi complex). In 1897, K. Benda - in animal cells, and in 1904 - F. Mewes - in plant cells described chondriosomes, which later became known as mitochondria.

Thus, by the end of the 19th century, on the basis of the successful development of microscopic technology and analysis of data on the microscopic structure of the cell, a colossal factual material was accumulated, which made it possible to identify a number of important patterns in the structure and development of cells and tissues. At this time, the doctrine of the cell stood out in an independent biological science - cytology.