Histology And Its Method Of Study PDF

Histology Week 1 (1st sem.): Histology and Its Method of Study Glenn Nathaniel S.D. Valloso, MD, DPSP Anatomic and Clinical Pathologist BulSU College of Medicine Department of Anatomy A.Y. 2024-2025 HISTOLOGY & ITS METHODS OF STUDY: INTRODUCTION Histol...

Histology Week 1 (1st sem.): Histology and Its Method of Study Glenn Nathaniel S.D. Valloso, MD, DPSP Anatomic and Clinical Pathologist BulSU College of Medicine Department of Anatomy A.Y. 2024-2025 HISTOLOGY & ITS METHODS OF STUDY: INTRODUCTION Histology is the study of the tissues of the body and how these tissues are arranged to constitute organs. The Greek root histo can be translated as either "tissue" or "web" and both translations are appropriate because most tissues are webs of interwoven filaments and fibers, both cellular and noncellular, with membranous linings. HISTOLOGY & ITS METHODS OF STUDY: INTRODUCTION Tissues are made of two interacting components: cells and extracellular matrix. The extracellular matrix consists of many kinds of molecules, most of which are highly organized and form complex structures, such as collagen fibrils and basement membranes. LIGHT MICROSCOPY Conventional bright-field microscopy, as well as fluorescence, phase- contrast, differential interference, confocal, and polarizing microscopy are all based on the interaction of light and tissue components and can be used to reveal and study tissue features. Bright-Field Microscopy The microscope is composed of mechanical and optical parts. The optical components consist of three systems of lenses. The condenser collects and focuses light, producing a cone of light that illuminates the object to be observed. The objective lenses enlarge and project the illuminated image of the object in the direction of the eyepiece. The eyepiece or ocular lens further magnifies this image and projects it onto the viewer's retina, photographic film, or (to obtain a digital image) a detector such as a charge-coupled device (CCD) camera. Fluorescence Microscopy In fluorescence microscopy, tissue sections are usually irradiated with ultraviolet (UV) light and the emission is in the visible portion of the spectrum. The fluorescent substances appear brilliant on a dark background. For this method, the microscope has a strong UV light source and special filters that select rays of different wavelengths emitted by the substances. Phase-Contrast Microscopy & Differential Interference Microscopy Phase-contrast microscopy, however, uses a lens system that produces visible images from transparent objects. Phase-contrast microscopy is based on the principle that light changes its speed when passing through cellular and extracellular structures with different refractive indices. These changes are used by the phase-contrast system to cause the structures to appear lighter or darker in relation to each other. Because it does not require fixation or staining, phase-contrast microscopy allows observation of living cells and tissue cultures, and such microscopes are prominent tools in all cell culture labs. Confocal Microscopy With a regular bright-field microscope the beam of light is relatively large and fills the specimen. Stray light reduces contrast within the image and compromises the resolving power of the objective lens. Confocal microscopy avoids stray light and achieves greater resolution by using (1) a small point of high-intensity light provided by a laser and (2) a plate with a pinhole aperture in front of the image detector. The point light source, the focal point of the lens, and the detector's pinpoint aperture are all optically conjugated or aligned to each other in the focal plane (confocal) and unfocused light does not pass through the pinhole. Polarizing Microscopy Polarizing microscopy allows the recognition of structures made of highly organized molecules. When normal light passes through a polarizing filter (such as a Polaroid), it exits vibrating in only one direction. If a second filter is placed in the microscope above the first one, with its main axis perpendicular to the first filter, no light passes through. If, however, tissue structures containing oriented macromolecules are located between the two polarizing filters, their repetitive structure rotates the axis of the light emerging from the polarizer and they appear as bright structures against a dark background. ELECTRON MICROSCOPY Transmission and scanning electron microscopes are based on the interaction of electrons and tissue components. The wavelength in the electron beam is much shorter than of light, allowing a thousand- fold increase in resolution. Transmission Electron Microscopy The transmission electron microscope (TEM) is an imaging system that permits resolution around 3 mm. This high resolution allows magnifications of up to 400,000 times to be viewed with details. Scanning Electron Microscopy Scanning electron microscopy (SEM) permits pseudo–three- dimensional views of the surfaces of cells, tissues, and organs. Like the TEM this microscope produces and focuses a very narrow beam of electrons, but in this instrument the beam does not pass through the specimen. Instead the surface of the specimen is first dried and coated with a very thin layer of metal atoms through which electrons do not pass readily. When the beam is scanned from point to point across the specimen it interacts with the metal atoms and produces reflected electrons or secondary electrons emitted from the metal. CELL & TISSUE CULTURE Live cells and tissues can be maintained and studied outside the body. In a complex organism, tissues and organs are formed by several kinds of cells. These cells are bathed in fluid derived from blood plasma, which contains many different molecules required for growth. HISTOCHEMISTRY & CYTOCHEMISTRY The terms histochemistry and cytochemistry indicate methods for localizing cellular structures in tissue sections using unique enzymatic activity present in those structures. To preserve these enzymes histochemical procedures are usually applied to unfixed or mildly fixed tissue, often sectioned on a cryostat to avoid adverse effects of heat and paraffin on enzymatic activity. HISTOCHEMISTRY & CYTOCHEMISTRY Enzyme histochemistry usually works in the following way: (1) tissue sections are immersed in a solution that contains the substrate of the enzyme to be localized; (2) the enzyme is allowed to act on its substrate; (3) at this stage or later, the section is put in contact with a marker compound; (4) this compound reacts with a molecule produced by enzymatic action on the substrate; (5) the final reaction product, which must be insoluble and which is visible by light or electron microscopy only if it is colored or electron-dense, precipitates over the site that contains the enzyme. Immunohistochemistry A highly specific interaction between molecules is that between an antigen and its antibody. For this reason, methods using labeled antibodies have become extremely useful in identifying and localizing many specific proteins, not just those with enzymatic activity that can be demonstrated by histochemistry. The body's immune cells are able to discriminate its own molecules (self) from foreign ones. When exposed to foreign molecules—called antigens—the body responds by producing antibodies that react specifically and bind to the antigen, thus helping to eliminate the foreign substance. Antibodies belong to the immunoglobulin family of glycoproteins, produced by lymphocytes. Immunohistochemistry In immunohistochemistry, a tissue section (or cells in culture) that one believes contains the protein of interest is incubated in a solution containing an antibody to this protein. The antibody binds specifically to the protein, whose location in the tissue or cell can then be seen with either the light or electron microscope, depending on the type of compound used to label the antibody. Antibodies are commonly tagged with fluorescent compounds, with peroxidase or alkaline phosphatase for histochemical detection, or with electron-dense gold particles.  Histology st Week 1 (1 sem.): Cytoplasm and Cell Nucleus Glenn Nathaniel S.D. Valloso, MD, DPSP Anatomic and Clinical Pathologist BulSU College of Medicine Department of Anatomy A.Y. 2024-2025 PART 1 - THE CYTOPLASM: INTRODUCTION Cells and extracellular material together comprise all the tissues that make up the organs of multicellular animals. In all tissues, cells themselves are the basic structural and functional units, the smallest living parts of the body. Animal cells are eukaryotic (Gr. eu, good, + karyon, nucleus), with distinct membrane-limited nuclei surrounded by cytoplasm containing many varied membrane-limited organelles. CELL DIFFERENTIATION The first cellular divisions of the zygote produce cells called blastomeres and as part of the inner cell mass blastomeres give rise to all tissue types of the adult. Explanted to tissue culture such cells have been termed embryonic stem cells. During their specialization process, called cell differentiation, the cells synthesize specific proteins, change their shape, and become very efficient in specialized functions. CYTOPLASMIC ORGANELLES The cell is composed of two basic parts: cytoplasm (Gr. kytos, cell, + plasma, thing formed) and nucleus (L. nux, nut). Individual cytoplasmic components are usually not clearly distinguishable in common hematoxylin-and-eosin–stained preparations; the nucleus, however, appears intensely stained dark blue or black. The outermost component of the cell, separating the cytoplasm from its extracellular environment, is the plasma membrane (plasmalemma). The cytosol contains hundreds of enzymes, such as those of the glycolytic pathway, that produce building blocks for larger molecules and break down small molecules to liberate energy. All the machinery converging on the ribosomes for protein synthesis (mRNA, transfer RNA, enzymes, and other factors) is also contained within the cytosol. Plasma Membrane All eukaryotic cells are enveloped by a limiting membrane composed of phospholipids, cholesterol, proteins, and chains of oligosaccharides covalently linked to phospholipid and protein molecules. The plasma, or cell, membrane functions as a selective barrier that regulates the passage of certain materials into and out of the cell and facilitates the transport of specific molecules. ENDOCYTOSIS Endocytosis is a process in which a cell takes in material from the extracellular fluid using dynamic movements and fusion of the cell membrane to form cytoplasmic, membrane-enclosed structures containing the material. Such cytoplasmic structures formed during endocytosis fall into the general category of vesicles or vacuoles. Three major forms of endocytosis. (a): Phagocytosis involves the extension from the cell of large folds called pseudopodia which engulf particles, for example bacteria, and then internalize this material into a cytoplasmic vacuole or phagosome. (b): In pinocytosis the cell membrane invaginates (dimples inward) to form a pit containing a drop of extracellular fluid. The pit pinches off inside the cell when the cell membrane fuses and forms a pinocytotic vesicle containing the fluid. (c): Receptor-mediated endocytosis includes membrane proteins called receptors which bind specific molecules (ligands). When many such receptors are bound by their ligands, they aggregate in one membrane region which then invaginates and pinches off to create vesicle or endosome containing both the receptors and the bound ligands. EXOCYTOSIS In exocytosis a membrane-limited cytoplasmic vesicle fuses with the plasma membrane, resulting in the release of its contents into the extracellular space without compromising the integrity of the plasma membrane. Often exocytosis of stored products from epithelial cells occurs specifically at the apical domains of cells, such as in the exocrine pancreas and the salivary glands. The fusion of membranes during exocytosis is a highly regulated process involving interactions between several specific membrane proteins. Exocytosis is triggered in many cells by transient increase in cytosolic Ca2+. SIGNAL RECEPTION AND TRANSDUCTION Endocrine signaling, the signal molecules (called hormones) are carried in the blood to target cells throughout the body. Paracrine signaling, the chemical mediators are rapidly metabolized so that they act only on local cells very close to the source. Synaptic signaling, a special kind of paracrine interaction, neurotransmitters act only on adjacent cells through special contact areas called synapse SIGNAL RECEPTION AND TRANSDUCTION Autocrine signaling, signals bind receptors on the same cell type that produced the messenger molecule. Juxtacrine signaling, important in early embryonic tissue interactions, signaling molecules remain part of a cell's surface and bind surface receptors of the target cell when the two cells make direct physical contact. Mitochondria Mitochondria (Gr. mitos, thread, + chondros, granule) are membrane- enclosed organelles with enzyme arrays specialized for aerobic respiration and production of adenosine triphosphate (ATP), which contains energy stored in high-energy phosphate bonds and is used in most energy-requiring cellular activities. Glycolysis converts glucose anaerobically to pyruvate in the cytoplasm, releasing some energy. Mitochondria are usually elongated structures 0.5–1 micrometer in diameter and lengths up to ten times greater. Ribosomes Ribosomes are small electron-dense particles, about 20 x 30 nm in size. Ribosomes found in the cytosol are composed of four segments of rRNA and approximately 80 different proteins. Those of the mitochondria (and chloroplasts), like prokaryotic ribosomes, are somewhat smaller with fewer constituents. All ribosomes are composed of two different-sized subunits. Ribosomes In eukaryotic cells, the RNA molecules of both subunits are synthesized within the nucleus. Their numerous proteins are synthesized in the cytoplasm but then enter the nucleus and associate with rRNAs. The assembled large and small subunits then leave the nucleus and enter the cytoplasm to participate in protein synthesis. The large and small ribosomal subunits come together by binding an mRNA strand and typically numerous ribosomes are present on an mRNA as polyribosomes (or polysomes). Endoplasmic Reticulum The cytoplasm of eukaryotic cells contains an anastomosing network of intercommunicating channels and sacs formed by a continuous membrane which encloses a space called a cisterna. In sections cisternae appear separated, but high-resolution microscopy of whole cells reveals that they are continuous. This membrane system is called the endoplasmic reticulum (ER). In many places the cytosolic side of the membrane is covered by polyribosomes synthesizing protein molecules which are injected into the cisternae. This permits the distinction between the two types of endoplasmic reticulum: rough and smooth. ROUGH ENDOPLASMIC RETICULUM Rough endoplasmic reticulum (RER) is prominent in cells specialized for protein secretion, such as pancreatic acinar cells (digestive enzymes), fibroblasts (collagen), and plasma cells (immunoglobulins). The RER consists of saclike as well as parallel stacks of flattened cisternae, limited by membranes that are continuous with the outer membrane of the nuclear envelope. SMOOTH ENDOPLASMIC RETICULUM Regions of ER that lack bound polyribosomes make up the smooth endoplasmic reticulum (SER), which in most cells is less abundant that RER but is continuous with it. SER cisternae are often more tubular and more likely to appear as a profusion of interconnected channels of various shapes and sizes than as stacks of flattened cisternae. SMOOTH ENDOPLASMIC RETICULUM SER contains enzymes associated with a wide variety of specialized functions. A major role of SER is the synthesis of the various phospholipid molecules that constitute all cellular membranes. The phospholipids are transferred to other membranes from the SER (1) by direct communication with the RER allowing lateral diffusion, (2) by vesicles that detach, move to and fuse with other membranous organelles, or (3) by being carried individually by phospholipid transfer proteins. Functions of rough and smooth ER As seen with the TEM the cisternae of rough ER are flattened, with polyribosomes on their outer surfaces and concentrated material in their lumens. Such cisternae appear separated in sections made for electron microscopy, but they actually form a continuous channel or compartment in the cytoplasm. Smooth ER is continuous with rough ER but is involved with a much more diverse range of functions. Three major activities associated with smooth ER are (1) lipid biosynthesis, (2) detoxification of potentially harmful compounds, and (3) sequestration of Ca++ ions. Specific cell types with well-developed smooth ER are usually specialized for one of these functions. Golgi Apparatus The highly dynamic Golgi apparatus, or Golgi complex, completes posttranslational modifications and then packages and addresses proteins synthesized in the RER. In polarized secretory cells with apical and basal ends, such as mucus- secreting goblet cells, the Golgi apparatus occupies a characteristic position between the nucleus and the apical plasma membrane. Secretory Vesicles or Granules Originating in the Golgi apparatus, secretory vesicles are found in those cells that store a product until its release by exocytosis is signaled by a metabolic, hormonal, or neural message (regulated secretion). These vesicles are surrounded by a membrane and contain a concentrated form of the secretory product. The contents of some secretory vesicles may be up to 200 times more concentrated than those in the cisternae of the RER. Secretory vesicles with dense contents of digestive enzymes are referred to as zymogen granules. Lysosomes Lysosomes are sites of intracellular digestion and turnover of cellular components. Lysosomes (Gr. lysis, solution, + soma, body) are membrane-limited vesicles that contain about 40 different hydrolytic enzymes and are particularly abundant in cells with great phagocytic activity (eg, macrophages, neutrophils). Although the nature and activity of lysosomal enzymes vary depending on the cell type, the most common are acid hydrolyases such as proteases, nucleases, phosphatase, phospholipases, sulfatases, and -glucuronidase. Proteasomes Proteasomes are abundant cytoplasmic protein complexes not associated with membrane, each approximately the size of the small ribosomal subunit. They function to degrade denatured or otherwise nonfunctional polypeptides. Proteasomes also remove proteins no longer needed by the cell and provide an important mechanism for restricting activity of a specific protein to a certain window of time. Whereas lysosomes digest bulk material introduced into the cell, or whole organelles and vesicles, proteasomes deal primarily with proteins as individual molecules. The proteasome is a cylindrical structure made of four stacked rings, each composed of seven proteins including proteases. Peroxisomes or Microbodies Peroxisomes (peroxide+soma) are spherical membrane-limited organelles approximately 0.5 micrometer in diameter. They utilize oxygen but do not produce ATP and do not participate directly in cellular metabolism. Peroxisomes oxidize specific organic substrates by removing hydrogen atoms that are transferred to molecular oxygen (O2). This produces hydrogen peroxide (H2O2), a substance potentially damaging to the cell which is immediately broken down by catalase, another enzyme in all peroxisomes. THE CYTOSKELETON The cytoplasmic cytoskeleton is a complex network of (1) microtubules, (2) microfilaments (actin filaments), and (3) intermediate filaments. These protein structures determine the shape of cells, play an important role in the movements of organelles and cytoplasmic vesicles, and also allow the movement of entire cells. Microtubules Within the cytoplasmic matrix of eukaryotic, cells are fine tubular structures known as microtubules. Microtubules are also found in cytoplasmic processes called cilia and flagella. They have an outer diameter of 24 nm, with a dense wall 5 nm thick and a hollow lumen. Microtubules are variable in length, but they can become many micrometers long. Occasionally, two or more microtubules are linked by protein arms or bridges, which are particularly important in cilia and flagella. Microfilaments (Actin Filaments) Contractile activity in cells results primarily from an interaction between actin and its associated protein, myosin. Actin is present as thin (5–7 nm diameter) polarized microfilaments composed of globular subunits organized into a double-stranded helix. There are several types of actin and this protein is present in all cells. Actin is usually found in cells as polymerized filaments of F-actin mingled with free globular G-actin subunits. Intermediate Filaments In addition to microtubules and the thin actin filaments, eukaryotic cells contain a class of filaments intermediate in size between the other two cytoskeletal components and with a more variable diameter averaging 10–12 nm. In comparison with microtubules and actin filaments, intermediate filaments are much more stable and vary in their protein subunit structure in different cell types. A dozen or more heterogeneous protein classes that form such intermediate filaments have been identified and localized immunocytochemically. INCLUSIONS Unlike organelles, cytoplasmic inclusions are composed mainly of accumulated metabolites or other substances and are often transitory components of the cytoplasm. Non-motile and with little or no metabolic activity, inclusions are not considered organelles. Fat droplets Glycogen granules Lipofuscin granules PART 2 - THE CELL NUCLES: INTRODUCTION The nucleus contains a blueprint for all cell structures and activities encoded in the DNA of the chromosomes. It also contains the molecular machinery to replicate its DNA and to synthesize an RNA. Macromolecular transfer between the nuclear and cytoplasmic compartments is regulated. Because functional ribosomes do not occur in the nucleus, no proteins are produced there. The molecules needed for the activities of the nucleus are imported from the cytoplasm. COMPONENTS OF THE NUCLEUS The nucleus frequently appears as a rounded or oval structure, usually in the center of the cell. Its main components are the nuclear envelope, chromatin consisting of DNA an and a specialized region of chromatin called the nucleolus. The size and morphologic features of nuclei in a specific normal tissue tend to be uniform. Nuclear Envelope The nucleus is surrounded by two parallel unit membranes separated by a narrow (30–50 nm) perinuclear space. Together, the paired membrane intervening space make up the nuclear envelope. Polyribosomes are attached to the outer nuclear membrane, indicating continuity of the nuclear envelope with the endoplasmic reticulum. with the inner nuclear membrane is a meshwork of fibrous proteins called the nuclear lamina, which helps to stabilize the nuclear envelope. Major components of this lamina are filament proteins called lamins which bind to membrane proteins and associate with chromatin in nondividing cells. The pattern of association is regular from cell to cell within a tissue, support that chromosomes have a definite localization within the nucleus. Chromatin In nondividing nuclei, chromatin is the chromosomal material in a largely uncoiled state. Two types of chromatin can be distinguished with both the light and electron microscopes, which reflect chromosomal condensation. Heterochromatin (Gr. heteros, other, + chroma, color), which is electron dense, appears as coarse granules in the electron microscope an in the light microscope. Euchromatin is the less coiled portion of the chromosomes, visible as finely dispersed granular material in the electron microscope and as lightly stained basophilic area microscope. The regions of heterochromatin and euchromatin account for the patchy light- and-dark appearance of nuclei in tissue sections as seen by both light and electron microscopy. Chromatin is composed mainly of coiled strands of DNA bound to basic proteins called histones and to various nonhistone proteins CELL DIVISION Cell division, or mitosis (Gr. mitos, a thread), can be observed with the light microscope. During this process, the parent cell divides, and each of the daughter cells receives a chromosomal se the parent cell. Essentially, a longitudinal duplication of the chromosomes takes place, and these chromosomes are distributed to the daughter cells. MEIOSIS Meiosis is a specialized process involving two closely associated cell divisions that occurs only in the cells that will form sperm and egg cells in the gonads. Mitosis and meiosis Mitosis and meiosis share many aspects of chromatin condensation and separation, but differ in various key ways. As chromosomal condensation begins in meiosis, the two homologous maternal and chromosomes physically align in synapsis and regions are exchanged during crossing over or recombination. This is followed by two meiotic divisions with no intervening S phase. Mitosis produce which are the same genetically. Meiosis with its two successive cell divisions produces four haploid cells. Mitosis and meiosis In summary, meiosis and mitosis share many aspects of chromatin condensation and separation, but differ in key ways: Mitosis is a cell division that produces two diploid cells. Meiosis consists of two connected cell divisions and produces four haploid cells. During meiotic crossing over, new combinations of gene alleles are produced and every haploid cell is genetically unique. Lacking synapsis and the opportunity for DNA recombination cells that are the same genetically. Histology Laboratory Activity – Histology and Its Method of Study 1. Discuss the importance of histology in understanding human biology. How does histology complement other medical fields in diagnosing and treating diseases? Provide examples. 2. Compare and contrast hematoxylin and eosin (H&E) staining with special stains like periodic acid-Schiff (PAS) or Masson’s trichrome. In what contexts would you use these stains, and what cellular components do they highlight? Histology Laboratory Activity – The Cytoplasm and Cell Nucleus 1. Draw a typical eukaryotic cell and label the important structures and organelles. Describe the function of at least five of these structures. 2. Draw and label the structure of the cell membrane. Include the phospholipid bilayer, embedded proteins, and other key components. Explain how these components contribute to membrane function. 3. Illustrate the stages of the cell cycle and briefly explain what happens in each stage. 4. Draw and describe the different phases of meiosis, explaining what happens during each phase. Thank you for listening doctors!!!