Human Histology - O.D. Lutsyk 2003

Introduction

The term "Histology" (from the Greek "histos" - tissue and "logos" - word, study) was proposed by the German scientist R. Mayer in 1819 to describe the science of multicellular animal and human Tissues. However, the scope and significance of histology today have expanded far beyond the literal Translation of its name. Histology studies not only tissues but also the Cells that compose them, as well as The Structure of Organs and Organ Systems. Accordingly, the subject is divided into several branches: Cytology (the Study of Cells); General Histology, or histology proper (The Study of tissues); and Special Histology (the STUDY OF THE structure of organs and organ systems). Embryology, the science of embryonic development, is also closely linked to histology, as bodily structures are studied in The process of their origin and development. Like cytology, embryology has now branched off from histology to become an independent science; however, in the curriculum of medical higher education institutions, they are combined with histology into a single subject. Thus, the full name of the course is Histology, Cytology, and Embryology.

The subject of human histology encompasses the study of the fine (microscopic) and ultra-fine (submicroscopic) structure of human body components, their development, and their changes under various physiological conditions. The Use of light or Electron Cell/15.html">Microscopy to extend the capabilities of the human eye is what distinguishes histology from anatomy. Unlike biochemistry, which studies chemical compounds and their transformations, histology characterizes the Structural Organization of macromolecules into specific microscopic objects. Physiology integrates all the aforementioned levels of knowledge by investigating the mechanisms underlying the functioning of organs and organ systems. Thus, anatomy, histology, biochemistry, and physiology, by successively studying smaller and smaller objects and their functional interrelations, collectively provide a holistic understanding of The Human Body, forming the THEORETICAL FOUNDATIONS OF medicine. It is worth noting that the application of histological Research Methods has revealed microscopic Structural and functional elements, cells, and their aggregates within all Organs of the human body. These elements, repeatedly duplicated, enable organs to perform their specialized Functions.

A brief outline of The history of histology. Histological science is rich in facts. The earliest concepts of the fine structure and development of animal organisms date back to antiquity. From the texts of the Bhagavad Gita and Ayurveda, we learn that the ancient Indians knew of the amnion and believed that the embryo was nourished through vessels that carried Blood to it from the mother. We owe the Discovery of the incubation of bird eggs to the ancient Egyptians. This method later played a crucial role in The Development of embryology.

Hippocrates noted that as the embryo grows, its Water content decreases. The Hellenic scholar pointed out that the umbilical cord serves for fetal Respiration and observed similarities between the development of a chick and a human embryo, thereby pioneering comparative embryology.

Aristotle studied the development of chick embryos, investigating the Origin of the Heart and other organs. He concluded that organs in the embryo do not appear all at once but gradually, one after another, from an unstructured mass. This theory was later called The Theory of epigenesis. Aristotle also correctly identified the Functions of the Placenta and umbilical cord, and established the difference between Primary and secondary sexual characteristics.

Claudius Galen described the umbilical vessels, allantois, and amnion. He was aware of the presence of the foramen ovale in The Heart of the embryo and fetus, which connects the atria, as well as The connection between the pulmonary trunk and the aorta.

Hippocrates, Aristotle, Galen, and Avicenna attempted in their works to identify homogeneous parts (tissues) within the Organism. However, the ancient scholars' knowledge of tissues was based on the physical characteristics and macroscopic structure of organs. Consequently, tissues that are completely different from the standpoint of modern histology were grouped together simply because they shared external similarities.

Histology as an independent science, founded on microscopic technique, began with the invention and application of the Light Microscope. The first microscopes were constructed in the early 17th century almost simultaneously by Galileo Galilei (Italy), Cornelius Drebbel (England), and Hans and Zacharias Janssen (Netherlands). The term "microscope" was proposed in 1625 by Johann Faber. Microscopic studies conducted in the mid-17th century by Francesco Stelluti, Federico Cesi, Robert Hooke, Nehemiah Grew, Jan Swammerdam, and Antonie van Leeuwenhoek immediately brought about significant Changes in the understanding of the structure of living matter. In particular, in 1665, the prominent English scientist Robert Hooke, having improved the microscope's design, first described small compartments in plants, which he called cells (Latin - cellulae, English - cells).

The Dutch scientist Antonie van Leeuwenhoek achieved perhaps the greatest fame as a microscopist. His single-lens microscopes (essentially magnifying glasses) magnified objects more than 200 times. The inventor used a secret lens-making technique known only to him. Antonie van Leeuwenhoek began his scientific research when he was about forty years old. From 1673, the Royal Society of London regularly received letters from him describing the microscopic structure of plants and certain animals. He also holds the distinction of discovering spermatozoa (along with the student Ham), Ciliates, flagellates, and even Bacteria: bacilli, cocci, and spirochetes. For a long time, scientific authorities were reluctant to recognize him as a scholar, but in 1680, he was finally elected a Fellow of the Royal Society of London.

William Harvey summarized his embryological research in his work "On the Generation of Animals" (1651). The scientist proved that the actual embryo in a chicken egg is only a small part of it, the so-called blastoderm (germinal disc). Continuing Aristotle's teachings, he advocated the theory of epigenesis, demonstrating the gradual appearance and development of organ primordia in the embryo. William Harvey made an important generalization, showing that during embryonic development, every animal passes through different stages of organization, "becoming in turn an egg, a worm, and an embryo, approaching perfection in each phase."

In the 18th century, Ukrainian names began to appear among prominent histologists. Aleksandr Shumlansky, a native of Poltava and a graduate of the Kyiv-Mohyla Academy, described the "membrane" (later known as "Bowman's capsule") in his dissertation "On the Structure of Kidneys" (1783), predating the English anatomist William Bowman. He was the first to use the term "renal glomerulus" and described the Loop of Henle before the German anatomist Jakob Henle. Aleksandr Shumlansky was also the first to prove the existence of a direct connection between arterial and venous vessels in the kidneys, demonstrating that the renal Circulatory system is closed.

It should be noted that observations of the microscopic structure of organisms for over 100 years could not significantly challenge the natural philosophical views dominant in science at the time. This was because they relied on random observations made with imperfect microscopes. In the late 18th and early 19th centuries, achromatic microscopes were developed, which eliminated spherical and chromatic aberrations and significantly improved the quality of microscopic observations, making them far more reliable. This led to the systematization of data on the microscopic organization and development of cells, tissues, and organs.

Caspar Friedrich Wolff (1733-1794), in his dissertation "Theoria Generationis" (1759), was one of the first to boldly challenge natural philosophical views on embryonic development. His dissertation and subsequent works marked the Introduction of METABOLISM/2.html">THE CONCEPT OF organismal development from simple to complex into biology. He succeeded in proving the fallacy of the preformation theory dominant at the time, which interpreted development as a mere increase in the size of organs already present (preformed) in the embryo. The scientist substantiated the theory of epigenesis, which became the foundation of modern embryology. In fact, Caspar Friedrich Wolff is the founder of modern embryology; his works laid the groundwork for the entire doctrine of development in biology.

While studying the Development of Plants and animals, Caspar Friedrich Wolff attempted to find commonalities in their structure. He was the first to suggest the crucial role of cells in the process of organismal development. The view that the structures of both the animal and plant kingdoms must share a common structural element was expressed by Lorenz Oken, Alexander Monro, Felice Fontana, Pavel Goryaninov, Jan Purkinje, and Matthias Schleiden. Building on the work of his predecessors and his own research, the prominent German scientist Theodor Schwann (1839) formulated the core tenets of the Cell Theory.

In the last quarter of the 19th century, Eduard Strasburger, Petro Peremezhko, and Walther Flemming discovered the process of indirect Cell Division—mitosis—and described its phases in detail.

Advances in the study of the microscopic structure of cells led to the Separation of cytology into a distinct branch of histology. This was facilitated by improvements in microscopic techniques: the use of immersion objectives, microtomes, new fixatives, cell and tissue culture methods, and darkfield microscopy. The method of silver impregnation of histological specimens, developed by Camillo Golgi and Santiago Ramón y Cajal, proved exceptionally fruitful. This allowed for a fundamental study of the microscopic structure of The Nervous system, laying the foundations of neurohistology and establishing the neuron doctrine. In recognition of their achievements, the Italian and Spanish scientists were awarded the Nobel Prize in 1906.

At the end of the 19th century, Christian Heinrich Pander and Karl Ernst von Baer, continuing the research of Caspar Wolff, made significant progress in understanding the embryonic development of animals: Pander described The formation of germ layers, while Baer traced their subsequent development and the formation of individual organs from them. Karl Ernst von Baer proved that during embryonic development, the General characteristics of the phylum appear first, followed by those of the Class, species, and only at the very end, the individual CHARACTERISTICS OF THE organism. Baer is also credited with the discovery of the mammalian and human oocyte.

Since the 1950s, histological science has transitioned to a methodologically new, higher level of research, enriching itself with new data on the STRUCTURE OF THE organism. This was primarily due to the application of the Electron microscope in biology and medicine. The latter made it possible to accumulate substantial information regarding the ultrastructure of cells and their structural components in a relatively short period. The use of histochemical methods, morphometry, cytochemistry, and cytospectrophotometry has contributed, and continues to contribute, to a deeper understanding of cell physiology and their macromolecular level of organization, refining concepts of differentiation, regeneration, embryonic, and Postnatal development.

The development of systematic histological research in Ukraine began in the 19th century, associated with the establishment of independent departments of histology within the medical faculties of Kharkiv, Kyiv, and Lviv universities. The heads of these departments and the pioneers of Ukrainian histology were prominent scientists such as Nikanor Khrzhonshchevsky, Volodymyr Betz, Vladyslav Shymonovych, and others. The further development of histological science in Ukraine in the 20th century was linked to the transformation of university medical faculties into independent higher education institutions and the opening of new medical institutes where departments of histology were established. Today, departments of histology and embryology operate in 17 medical higher education institutions of Ukraine: in Vinnytsia, Dnipropetrovsk, Donetsk, Zaporizhzhia, Ivano-Frankivsk, Kyiv (two departments), Luhansk, Lviv, Odesa, Poltava, Simferopol, Sumy, Ternopil, Uzhhorod, Kharkiv, and Chernivtsi.

In 1867, Nikanor Khrzhonshchevsky organized and headed Ukraine's first department of histology at the medical faculty of Kharkiv University. He enriched global science with classic works on the structure of the Adrenal Glands, Lungs, Liver, and renal blood supply. He also proposed an original method for the vital injection of Dyes into Cells and Tissues.

A student of Professor Khrzhonshchevsky, Nikolai Kulchitsky, chaired the Department of Histology and Embryology of the medical faculty at Kharkiv University from 1891 to 1911. His manual on microscopic technique (1885) was highly popular among morphologists. Furthermore, Professor Kulchitsky authored the fundamental textbook "Basics of Animal and Human Histology" (Kharkiv, 1900), which went through five editions. The scientist's research was focused on studying the Fine Structure of the Central Nervous System, nerve fibers, and nerve endings. His proposed modification of myelin nerve fiber staining is widely known. While studying the mucosa of the digestive tract, he discovered enterochromaffin cells, which became known in world literature as Kulchitsky cells. This marked the beginning of the study of endocrine cells scattered throughout the Internal Organs of animals and humans.

Volodymyr Rubashkin chaired the Department of Histology at the Kharkiv Medical Institute from 1923 to 1932. He authored a textbook published in 1933 in Ukrainian ("Elements of Histology", Parts I and II) and Russian ("Fundamentals of Human Histology and Histogenesis", Parts I and II). Professor Rubashkin conducted pioneering research on mitochondrial Morphology in the cells of various organs, studied the fine structure of neuroglia, and performed a detailed Analysis of the secretory process in pancreatic exocrine cells.

Borys Aloshyn chaired the Department of Histology at the Kharkiv Medical Institute from 1937 to 1974. He became widely known following a series of scientific studies dedicated to elucidating the morphological basis of Neurohumoral regulation of bodily functions and the histophysiology of the hypothalamo-hypophyseal system. The scientist investigated the interaction of nervous and endocrine factors in bodily integration. Thanks to the work of Aloshyn and his students, understanding of the regulatory influences of the Hypothalamus on Endocrine glands was expanded, The Significance of afferent influences of peripheral endocrine glands on hypothalamic activity was clarified, as was The Role of sympathetic impulses in regulating hormonopoiesis in the Pituitary Gland and hypothalamus.

From 1974 to 1995, the Department of Histology at the Kharkiv Medical Institute was headed by Professor Yevhen Pankov, whose scientific interests focused on the study of Bone tissue morphogenesis and the Development of the concept of structural and functional units of organs.

The ESTABLISHMENT OF THE Department of Histology and Embryology at Kyiv Saint Volodymyr University is closely linked to the activities of the HEAD of the Department of Anatomy, Volodymyr Betz—a world-renowned scientist and the discoverer of the giant pyramidal Neurons of the Cerebral Cortex, known in world literature as Betz cells. In studying the structure of the adrenal glands, bone development, and cerebral cytoarchitectonics, he utilized microscopic research methods and, like no other, understood the value of histological knowledge for future physicians. Professor Betz brilliantly delivered lectures and conducted Practical Classes in histology. He rightly believed that histology bridges anatomy and physiology, referring to histology as "higher anatomy." Betz was one of the initiators of establishing the Department of Histology within the medical faculty and is rightfully considered its "godfather." The outstanding anatomist donated a significant portion of the microscopes at his disposal to the newly formed department. The teaching of histology and embryology as an independent subject at a separate department of Saint Volodymyr University in Kyiv began in 1868. Petro Peremezhko became the first head of the department.

Petro Peremezhko was a graduate of the Medical Faculty of Kyiv University. From the very first years of his scientific career, he proved to be a talented and brilliant scientist. In his doctoral dissertation, he described "Muscle nuclei" and clarified their role in the development and regeneration of striated muscle fibers. In fact, he discovered myosatellite cells, and only the limited resolution of the light microscope prevented him from drawing the correct Conclusion. Myosatellite cells were "rediscovered" using an electron microscope only in 1961. Professor Peremezhko authored classical works on the microscopic structure and Embryogenesis OF THE Spleen, Thyroid Gland, and pituitary gland. As mentioned above, Petro Peremezhko was one of the first scientists to describe mitosis. He observed the mitotic division of cells in the epidermis, Connective Tissue, endothelium, and leukocytes, and described the sequence, duration, and Specific features of individual mitotic phases.

Peremezhko's student Fedir Lominsky chaired the Department of Histology at the Medical Faculty of Kyiv University from 1906 to 1924. He is one of the founders of the histophysiological direction in morphology; he was the first to describe the mitotic division of neuroblasts, noting that this phenomenon occurs only during the Embryonic period of development. To this day, his works on the morphological remodeling of neurons under The Influence of chemical substances and mechanical trauma, as well as the STUDY OF MORPHOLOGICAL markers of nerve Cell Differentiation in higher and lower vertebrates, remain highly relevant. Professor Lominsky conducted original research on the MICROSTRUCTURE OF THE lens, physiological degeneration of striated muscle fibers, the connection of Muscles with tendons, and reactive changes in pancreatic exocrine and endocrine cells. His descriptions of cytoplasmic canaliculi, which later became known as Holmgren's canals and represent dilated regions of The Endoplasmic reticulum, are of great interest.

Oleksandr Cherniakhivsky chaired the Department of Histology of the Kyiv Medical Institute from 1924 to 1929. He authored a series of fundamental studies on the microscopic structure, reactive changes, and embryogenesis of the Autonomic nervous system and neuroglia. He collaborated with neurohistologists from Italy, Germany, and Spain, including Nobel laureate Ramón y Cajal, who highly esteemed the Ukrainian scientist's research. Professor Cherniakhivsky translated the most famous European textbooks on histology and embryology into Ukrainian, initiated the development of Ukrainian histological and embryological nomenclature, and actively participated in the medical section of the All-Ukrainian Academy of Sciences.

The legacy of studying the nervous system at the Kyiv Medical Institute was continued by Mykola Zazybin, who chaired the Department of Histology from 1954 to 1975. Together with his numerous students (K. Kabak, A. Kolomiytsev, H. Konstantynovsky, V. Karupu, V. Yatsenko, and others), Professor Zazybin studied age-related and reactive changes in the Peripheral Nervous System. He authored the fundamental monograph "Embryogenesis of the Peripheral Nervous System", in which, contrary to many scientific authorities, he convincingly proved that the development of the peripheral nervous system occurs not through the mere complication of the initial material, but As a result of the growth, degeneration, and remodeling of various PARTS OF THE nervous system.

Kostiantyn Kabak chaired the Department of Histology and Embryology from 1976 to 1992. Under his leadership, the department's staff continued studying the nervous system, a research direction that remains active today. They investigated the characteristics of neuro-tissue interactions in fetuses, newborns, adults, and the elderly, demonstrating the unique nature of neurotrophic support to tissues during these age periods. Morphometry and applied research occupy a significant place in the department's scientific endeavors. In 1982, Andriy Kolomiytsev and Valentyn Yatsenko were awarded the State Prize of Ukraine for their study and experimental-morphological testing of medical polymers. In 1996, Yuriy Chaykovsky and Valentyn Yatsenko received the same prize for their Participation in the development and Implementation of new methods for diagnosing and treating peripheral nervous system injuries.

Mykola Zazybin's students successfully continued their research on the nervous system at various medical universities in Ukraine. Professor M. Zaitsev, who headed the Department of Histology at the Ivano-Frankivsk Medical Institute from 1950 to 1957 and the Department of Histology at the Odesa Medical Institute from 1958 to 1976, investigated the reactivity of the peripheral nervous system. Professor O. Kimbarovska, heading the Department of Histology at the Donetsk Medical Institute from 1967 to 1990, studied the central and peripheral nervous systems under normal and pathological conditions. Professor I. Zhutaiev, heading the Department of Histology at the Poltava Medical Institute from 1975 to 1986, investigated the regenerative properties of peripheral nerves, Skin, and certain internal organs under experimental peroxidation syndrome and its pharmacological correction.

The Lviv histological school traces its origins back to February 1896, when a histological laboratory was established at the Department of Anatomy of Lviv University through the efforts of its head, Professor Henryk Kadyi. In February 1897, Władysław Szymonowicz, a graduate of the Jagiellonian University in Kraków and a native of the Ternopil region, was elected Professor of Histology and Embryology in Lviv, heading the Department of Histology and Embryology for the next 40 years. Professor Szymonowicz possessed an exceptional mastery of histological techniques. Using his own illustrative material, he authored a textbook on human histology that went through 11 editions in five languages (German, English, Polish, Italian, and Spanish), gaining immense popularity among students and being widely regarded as one of the best in Europe. W. Szymonowicz's scientific works focused on elucidating the histophysiology of the adrenal glands and the fine structure of nerve endings. His findings were summarized in a monograph on the comparative histology of nerve endings in several dozen mammalian species.

From 1947 to 1963, the Department of Histology and Embryology in Lviv was headed by Andriy Dyban, a graduate of the Kyiv Medical Institute. The scientific focus during this period was the study of normal and pathological embryogenesis, as well as the histophysiology of the Endocrine System. Professor Dyban developed a method for the differential detection of pituitary basophils; in 1951, he published the book "Some Issues of the Pathology of Early stages of Human Embryogenesis", and in 1959, the monograph "Essays on Human Pathological Embryology". From 1964 to 1988, the Department of Histology and Embryology in Lviv was chaired by Professor Yevdokiya Detyuk. The department's research focus during this period was the experimental study of The impact of maternal thyroid pathology on reproductive function, as well as the embryonic and postnatal development of offspring. Under the supervision of Professor Ye. Detyuk, 6 doctoral and 25 candidate dissertations were completed and defended.

In 1988, Antonina Ivanova-Sohomonyan took head of the Department of Histology and Embryology in Lviv. She authored the first list of Ukrainian histological terms in the book "International Histological Nomenclature" (Kyiv, 1980), which, after refinement and The addition of Ukrainian embryological terminology, was republished in 1993, and further expanded with Ukrainian cytophysiological terms in 2001. Together with Professors Kabak and Lutsyk, Antonina Ivanova-Sohomonyan published the first textbook on human histology in independent Ukraine, which was awarded the State Prize in 1994.

Volodymyr Karpov was the founder of the Department of Histology and Embryology of the Katerynoslav (now Dnipro) State Medical Academy and an internationally recognized expert in the field of vital cell imaging. Professor Karpov authored the textbook "Introductory Course in Histology", which went through several editions. While working in Vienna in the laboratory of the prominent German optical physicist Ernst Abbe, Professor Karpov wrote the book "An Outline of the General Theory of the Microscope in Its Historical Development".

From 1962 to 1989, the Department of Histology at the Dnipropetrovsk Medical Institute was headed by Volodymyr Arkhypenko, a student of Professor Alyoshyn. The main works of this scientist were dedicated to studying the Mechanism of hormone Action.

A significant contribution to the development of the Odesa histological school was made by prominent scientists: Ilya Mechnikov, Volodymyr Pidvysotsky, and Oleksandr Bohomolets. From 1870 to 1882, Ilya Mechnikov served as a professor at the Department of Zoology and Comparative Anatomy of the Novorossiysk (now Odesa) University. He became famous for his works on comparative embryology. While studying intracellular Digestion in lower animals, he discovered The phenomenon of phagocytosis, which he interpreted as a protective response of the organism. In recognition of his achievements, Mechnikov, along with Paul Ehrlich, was awarded the Nobel Prize in 1908 for the development of immunology.

During his student years, under the supervision of Professor Peremezhko, Volodymyr Pidvysotsky carried out a brilliant study on the fine structure of the Pancreas. His doctoral dissertation, dedicated to the regeneration of liver parenchyma, was highly praised by Khrzhonschevsky and Betz. From 1900 to 1905, Professor Pidvysotsky worked in Odesa, where, as head of the Department of General Pathology, he also taught a course in histology. Under Professor Pidvysotsky's guidance in Odesa, Oleksandr Bohomolets completed his first scientific works, which focused on the fine structure of the duodenal glands and the adrenal glands. Later, Professor Bohomolets studied the normal and pathological states of connective tissue, as well as the endocrine and autonomic nervous systems. He developed the Concept of the physiological system of connective tissue.

Ukrainian science has given the world outstanding embryologists. Mykola Kashchenko laid the foundation for pathological embryology by studying the structure of atrophic human embryos. In his dissertation on the structure of the human chorion, he was the first to describe large rounded cells in the connective tissue core of the chorionic villi, which later became known as Hofbauer cells. The scientist also established that mesenchymal cells originate not only from the mesoderm but also from other germ layers.

From 1869 to 1873, Oleksandr Kovalevsky worked at Kyiv University, and from 1873 to 1890, at Novorossiysk (later Odesa) University. Through his works, this scientist laid the foundations of experimental and evolutionary embryology.

Semen Shakhov, a student of Professor Rubashkin, chaired the Department of Histology at the Kyiv Medical Institute from 1930 to 1953, and at the Odesa Medical Institute from 1954 to 1958. His main scientific works were dedicated to the study of human embryogenesis in health and disease. Professor Shakhov described the Asymmetry of several embryonic organ primordia in humans, clarified aspects of the development of The Thyroid Gland and Thymus, and described Developmental anomalies of the human embryo. The results of these studies were systematized in his fundamental monograph "Anomalies of Human embryonic development".

A significant contribution to the development of embryology was made by scientists of the Crimean Medical Institute. The staff of the Department of Histology, headed by Borys Khvatov, Yuriy Shapovalov, Arkadiy Brusylovsky, and Borys Trotsenko, studied the patterns of embryonic Histo- and Organogenesis in humans and animals, the Functional Morphology of the placenta, and the histophysiology of the maternal-fetal system. A unique collection of early human embryos, fetuses, and placentas has been gathered at the department.

In this brief outline, it is impossible to characterize the work of all prominent histologists, their scientific achievements, and discoveries. This subject is discussed in greater detail in the books "Outstanding Histologists: A Biographical Directory" (2001) by O. Deltsova, Yu. Chaykovsky, S. Herashchenko et al.; "Embryological Dictionary" (2001) by Yu. Chaykovsky, M. Akimchenkov, O. Deltsova, S. Herashchenko; "Eponyms in Morphology" (1989) by R.P. Samusev and N.I. Goncharov, in other chapters of this book, as well as in more specialized publications.

METHODS OF HISTOLOGICAL Research. Modern histology possesses a wide arsenal of diverse research methods. All these methods share the requirement of using a special instrument—the microscope—and are therefore all microscopic methods. Depending on the state of the object under study, these methods are divided into vital (or supravital) methods, which study living cells, tissues, organs, and even whole organisms, and post-vital methods, which examine dead, fixed specimens.

The development of the post-vital method, or the method of preparing permanent histological slides, occurred in parallel with the development of the science of histology itself In the second half of the 19th century. It is also referred to as the method of classical histology. This method, known as histological or microscopic technique, requires quite complex preparation of the specimen. The latter is the subject of specialized, rather extensive manuals. A student beginning to study histology needs to become familiar with the basics of slide preparation techniques in order to better understand these slides and learn to analyze or "read" them, as permanent histological slides are widely used in both the educational process and scientific research.

The first step in slide preparation is obtaining the material. At this stage, as well as in all subsequent ones, unnecessary trauma to the specimen must be avoided. Therefore, when cutting a piece of an organ or tissue, one should use sharp scissors or a blade and avoid squeezing the tissue with forceps. The pieces should be small—about 1 cm³ (ideally 7x7x3 mm). The material must be fresh and should be harvested as quickly as possible after sacrificing the experimental animal or after human death.

The next step is tissue fixation, which is performed by immersing the harvested specimen in a fixing fluid. The goal of this stage is to preserve histological structures and macromolecules in the exact Location and state they were in the living organism. Although fixatives usually cause certain alterations to the vital state of structures, these changes can be minimized by selecting specific fixing agents. Fixatives include alcohols (ethyl, methyl), formalin solutions, heavy metal salts, and acids (acetic, picric, osmic). More commonly, various complex fixing mixtures are used, which include these components in different ratios.

The third step is the dehydration of the fixed material. For this purpose, alcohols of gradually increasing concentrations, from 50-70 to 100 degrees, are used. Dehydration is necessary for the next stage—embedding, which is performed using paraffin, celloidin, or synthetic resins. Since the vast majority of these substances do not mix with water, to infiltrate the material with them, water must be thoroughly removed from the tissue, which is then cleared with xylene (toluene, benzene)—a substance that dissolves paraffin well and mixes with 100-degree ethyl alcohol. After infiltrating the specimen with liquid paraffin at a Temperature of 55-5б°C, it is allowed to solidify at room temperature along with the paraffin in special Molds. This produces a paraffin block. This Procedure is called embedding. Rapid hardening can be achieved by freezing tissue pieces with dry ice (carbon dioxide) or liquid nitrogen, though the structure of the studied histological specimens is preserved less effectively in this case.

The embedding of the material makes it possible to cut thin (5-7 µm thick) or semi-thin (0.5-1 µm) sections used for light microscopy; for electron microscopy, ultrathin sections (0.05-0.2 µm) are used. Sectioning is performed using special instruments—microtomes (for light microscopy) and ultramicrotomes (for electron microscopy). Thin, semi-thin, or ultrathin sections are transparent to light rays or electron beams, enabling their study under the respective microscopes. To distinguish the structural details of the specimen, most of which lack natural contrast, the obtained section must be stained (for light microscopy) or contrasted (for electron microscopy).

In histology, There are many methods of staining slides, and many different dyes are used depending on the purpose of the study. Histological stains are classified by origin into plant-derived, animal-derived, and synthetic (aniline) dyes. An example of a plant dye is hematoxylin, obtained from the bark of the logwood tree native to Central America, while an animal dye is carmine, obtained from cochineal insects. The vast majority of dyes are synthetic, such as eosin, fuchsin, azure, etc.

The Classification of histological dyes by their chemical properties is of paramount importance, as it forms the basis for a range of concepts and terms encountered throughout the course. Accordingly, based on their chemical properties, histological dyes are classified into acidic, basic, and neutral. The properties of acidic dyes are determined by -СООН, -HSО₃, and -Н₂РО₃ groups; these are known as anionic dyes. Acidic dyes stain the Cell Cytoplasm and are therefore called cytoplasmic dyes. Examples of such dyes include eosin (which yields a bright pink color) and light green (which yields a green color). Histological structures capable of being stained by acidic dyes are termed oxyphilic (acidophilic, eosinophilic). These include, for instance, the cytoplasmic granules of eosinophilic leukocytes, Collagen fibers, and others.

Basic dyes are cationic, with the vast majority containing positively charged nitrogen atoms within their molecular structure. These dyes selectively stain cell nuclei and are thus referred to as nuclear dyes. Examples include hematoxylin (staining blue-violet), carmine (light red), safranin (dark red), and azure II (blue). Histological structures capable of being stained by basic dyes are termed basophilic. These include granules in the cytoplasm of basophilic leukocytes, cell nuclei, and others.

Neutral dyes are formed by combining aqueous solutions of acidic and basic dyes, such as eosin-methylene blue. In addition, a distinction should be made for neutral dye mixtures, where both basic and acidic dyes are simultaneously present in the solution. Structures that readily take up both basic and acidic dyes are called neutrophilic or polychromatophilic. Examples include the granules of neutrophilic leukocytes, the cytoplasm of polychromatophilic erythroblasts, and others. The ability of histological structures to alter the color of a basic dye is termed metachromasia. Metachromatically stained structures include the granules of basophilic leukocytes, the Extracellular matrix of Cartilage tissue, and others. Specimens are typically stained by combining one acidic and one basic dye, which allows for the visualization of The Nucleus, cytoplasm, and all basophilic and oxyphilic structures. One of the most commonly used combinations is hematoxylin and eosin.

In addition to acidic, basic, and neutral dyes, there are special stains used to detect specific substances or structures. For example, Sudan III stains Lipids orange, while orcein stains elastic fibers brown.

Stained specimens are typically dehydrated in graded alcohols, cleared in xylene, mounted in a thin layer of Canada balsam, and covered with a coverslip. Once the balsam dries, a permanent slide is obtained, which can be used for a long time.

For electron microscopy, sections obtained using ultramicrotomes are placed on special grids, contrasted with uranium or lead salts, examined under a microscope, and photographed. The resulting electron micrographs are studied alongside histological specimens.

In addition to the thin sections described above, there are Other types of histological preparations used much less frequently, only in specific cases. These include smears (blood, Bone Marrow, saliva, etc.), imprints (liver, thymus, Urinary Bladder mucosa), stretch preparations or films (connective tissue, Pleura, Peritoneum, pia mater), and whole mounts (early-stage embryos, Germ Cells).

Vital (live) Methods of cell or tissue examination provide information on their life processes, allowing researchers to track cell movement, division, growth, interaction, and response to various factors. Live imaging methods are an essential Complement to the data obtained by classical histology regarding cell and tissue structure. Live studies are performed within a living organism, i.e., in vivo. To study living cells, vital and supravital staining methods are used. These methods employ special dyes that are non-toxic to living tissues. In vital staining, the dye is introduced into a living animal, where it selectively stains specific cells. This approach is used to study Cells of the macrophage system by administering trypan blue or lithium carmine. Supravital staining refers to the staining of living cells isolated from the organism. This technique is used to visualize Lysosomes (using neutral red dye), Mitochondria (Janus green), and blood reticulocytes (brilliant cresyl blue).

For vital, supravital, and post-vital examinations of unstained histological specimens, several specialized light microscopy techniques are used, including phase-contrast, darkfield, and Fluorescence Microscopy.

The phase-contrast method provides the necessary contrast for studying unstained structures using a special annular Diaphragm located in the condenser and a phase plate located in the objective. This optical design of the light microscope converts phase shifts of light passing through the specimen into amplitude changes, which the eye perceives as variations in brightness. As a result, structures with different refractive indices can be distinguished.

The darkfield microscopy method allows the visualization of unstained structures by using a special darkfield condenser. Consequently, the silvery outlines of the objects are visible against a dark Background.

Luminescent (or fluorescence) microscopy is based on the phenomenon of luminescence, which is the ability of living structures to emit light upon absorbing rays from the short-wavelength (ultraviolet, violet, or blue) part of the spectrum. In this case, the emission wavelength of fluorescence is always longer than the excitation wavelength. All living cells exhibit fluorescence, known as intrinsic or primary fluorescence. Since it is weak, secondary fluorescence is more commonly used, where specimens are pretreated with special dyes called fluorochromes. Among these, acridine orange is most frequently employed. When used, cell nuclei containing DNA emit a bright green glow, while the cytoplasm, due to the presence of RNA, glows bright red.

In recent decades, methods of histochemistry, autoradiography, immunomorphology, and cytospectrophotometry have become widely adopted. The histochemical method makes it possible to determine the localization of specific chemical substances within various Structural components of cells and tissues. During histochemical studies, substances within the cells react with chemical Reagents to form colored reaction products, which can be used to determine both the localization and, to some extent, the quantitative content of substances within particular structures.

The autoradiographic method is based on the use of radioactive isotopes and labeled compounds. These compounds are introduced into the experimental animal, and the radioactive substances are subsequently detected in histological sections using a photographic emulsion that is coated onto the slide and developed. In areas where the emulsion contacts the radioactive substance, exposed regions—tracks—remain. This method can be used to study iodine metabolism in the thyroid gland, the synthesis of Nucleic Acids, Proteins, and more.

Immunohistochemical methods are based on antigen-antibody reactions. Every cell in the body has a specific antigenic composition, determined mostly by proteins. Through immunization, specific Antibodies corresponding to these Antigens can be obtained. These antibodies are conjugated with fluorochromes or Enzymes. Following the Treatment of the histological specimens, labeled antibody molecules accumulate at the sites of the respective antigens. These are then detected either by fluorescence (fluorescence microscopy) or by the deposition of colored products from a histochemical reaction (light microscopy). Theoretically, this method can identify any cells or substances produced by specific cells, such as Hormones, against which antibodies can be generated.

Cytospectrophotometry is a method for the quantitative measurement of various substances within a cell based on the study of their Light absorption spectra. The method of flow cytometry allows for the analysis of cell characteristics in a suspension as they pass through a focused laser beam. The corresponding instrument is called a flow cytometer. Using this method, one can determine Cell size and shape, assess viability, and sort cells from the initial suspension into subpopulations.

A major step forward in the development of microscopic techniques was the invention and application of the electron microscope. In an electron microscope, a beam of electrons is used to "illuminate" the specimen, which has a much shorter wavelength compared to the visible light used in a light microscope. Consequently, the resolution limit (which is 1/3 of the wavelength at which microscopy is performed) is theoretically 0.2 μm for a light microscope, whereas for an electron microscope, the theoretically calculated resolution limit is 0.002 nm. In practice, the best electron microscopes achieve a resolution of 0.1–0.7 nm.

The latest breakthrough in cell biology is the technique of cryo-electron microscopy, developed by Joachim Frank, which allows for a resolution of 0.1–0.3 nm. In this method, a thin film containing purified functional macromolecular complexes (complexomixes) or, in morphological terms, specific cellular Organelles, is applied to an electron microscope grid.

The film is rapidly frozen at liquid nitrogen temperature. Prolonged exposure to the electron beam destroys the structure of the complexomixes, but even a single pass of the beam, combined with computer analysis (50,000–100,000 images of a given complexomix) and computer graphics, proves sufficient to reconstruct a three-dimensional image of the organelle under study. Thus, in the near future, the number of recognized submicroscopic cellular organelles (complexomixes) may increase to several dozen or even hundreds.

The resolution limit or resolving power of a microscope is the minimum distance between two points on a histological specimen that can be distinguished as two separate, non-merging points. The resolution limit indicates the smallest size of structures that can be resolved using a given microscope. Based on the resolution limit of the light microscope, structures are conventionally divided into microscopic (larger than 0.2 μm) and submicroscopic (smaller than 0.2 μm). The latter can only be visualized under an electron microscope. Today, scanning (or raster) electron microscopes, which provide three-dimensional (volumetric) images of specimens, are increasingly used in research. The key advantages of this type of microscopy include a large depth of field (100 to 1,000 times greater than that of a light microscope), a wide magnification range (from 10x to tens of thousands of times), and high resolution.

The Concept of an Artifact. Despite efforts to preserve the living appearance of the specimen during preparation for microscopy, some changes, however minimal, may occur. An artificial structure that appears in a specimen during its preparation and can lead to false results is called an artifact (from the Latin "artefactum"—artificially made). In histological studies, artifacts can be gross and relatively simple, making them easy to recognize; however, some can be subtle enough that only an experienced histologist can identify them. Examples of simple artifacts include air bubbles trapped under the coverslip during mounting, or fibers from the cloth used to wipe the coverslip. Other examples include dye precipitate that might be mistaken for a nucleus, or knife marks from a nicked microtome blade. More complex artifacts include alterations in cell shape, as well as the formation of artificial spaces or clefts between tissue layers due to tissue shrinkage during fixation, dehydration, and other Processing steps.

Terms to Remember

1. Histology. 2. Cytology. 3. General histology. 4. Special histology. 5. Embryology. 6. Fine (microscopic) structures. 7. Ultrathin (submicroscopic) structures. 8. Microscopic method. 9. Histological (microscopic) technique. 10. Vital (supravital) methods. 11. Post-vital methods. 12. Permanent histological specimen. 13. Tissue sampling. 14. Fixation. 15. Dehydration. 16. Hardening. 17. Embedding. 18. Microtome. 19. Ultramicrotome. 20. Histological section. 21. Section staining. 22. Section contrasting. 23. Histological dyes. 24. Hematoxylin. 25. Eosin. 26. Oxyphilia (eosinophilia, acidophilia). 27. Basophilia. 28. Neutrophilia. 29. Polychromatophilia. 30. Metachromasia. 31. Vital (supravital) staining. 32. Trypan blue. 33. Phase-contrast microscopy. 34. Darkfield microscopy. 35. Fluorescence microscopy. 36. Histochemistry. 37. Autoradiography. 38. Immunomorphology. 39. Cytospectrophotometry. 40. Flow cytometry. 41. Light microscopy. 42. Electron microscopy. 43. Resolution limit (resolving power) of a microscope. 44. Scanning (raster) electron microscopy. 45. Artifact.