Histology And Its Methods Of Study PDF 2024

University of Tripoli Faculty of Medicine Histology and Genetics Department Histology and its methods of study Subject- HS120 1...

University of Tripoli Faculty of Medicine Histology and Genetics Department Histology and its methods of study Subject- HS120 1 SUN. 30-6-2024 FIRST LECTURE-SPRING 2024 Prepared by assistant lecturer Suleiman Mohamed Shalaaby Histology & its methods of study 7/10/2024 2  Histology & its methods of study Chapter -1 Introduction 7/10/2024 3 Histology & its methods of study ❖ Objective By The End Of This Chapter, The Student Should Know: 1) Definition of histology. 2) The organization of the human body. 3) The components of the human body. 4) The different techniques for preparing tissue sections. 5) The different techniques for staining tissue sections. 6) The basic tools used to study its microscopic structure. 7) The different types of microscopes. 7/10/2024 4 Introduction ❖ Histology This is a word derived from 2 Greek words: (Gr. Histo, tissue or web + logos, study). most tissues are webs of interwoven filaments and fibers, both cellular and noncellular, with membranous linings tissue and organs. ❖ Definition Of Histology is the study of the tissues of the body and how these tissues arranged to constitute organs. 7/10/2024 5 Introduction ❖ Subject Of Histology Involves All aspects of tissue biology, with the focus on how cells’ structure and arrangement optimize functions specific to each organ ❖ The Organization Of The Human Body The human body is highly complicated. The human body subdivided into four structural subunits: Cells, Tissues, Organs, systems. 7/10/2024 6 Body Of Living Organism 7/10/2024 7 Introduction 1) Cells The cell are the smallest structural and functional units in the body. The cell are vary in size from 4 to 200 micrometer. To study the cells, must be look at them under a microscope. ❖ The Basic Parts Of The Cell: A. The cytoplasm: which is bounded by its cell membrane. B. The nucleus: bounded by the nuclear envelope. 7/10/2024 8 Introduction 2) Tissue All organs of the body consist of four basic types in various combination. Each basic tissue consists of special types of cells that have the same general characteristics. These Basic Tissues: 1. Epithelial tissue. 2. Connective tissue. 3. Muscle tissue. 4. Nervous tissue. 7/10/2024 9 Introduction 3) Organs Each organ is made up of different types of tissue that together perform a special function. For Example: the kidneys, liver and lung are different organs. 4) Systems A systems are an organization of different organs that together perform more complex functions in the body. For Example: the urinary system, gastrointestinal and respiratory systems are different systems. 7/10/2024 10 Introduction ❖ The Components Of The Human Body Tissues Have Two Interacting Components A. Extracellular matrix (ECM). B. Cells. ❖ A. Extracellular Matrix : Consists of many kinds of molecules, some of which are highly organized and form complex structures, such as: 1) Collagen fibrils. 2) Basement membranes. 7/10/2024 11 Introduction ❖ The Main Function Of Extracellular Matrix Furnish mechanical support for the cells. Transport nutrients to the cells. Excretion of metabolic wastes and secretory products. Extracellular matrix molecules sometimes regulate the functioning of cell. 7/10/2024 12 Introduction ❖ B. Cells Cells produce the ECM locally and are in turn strongly influenced by matrix molecules. Many matrix components bind to specific cell surface receptors that span the cell membranes and connect to structural components inside the cells forming continuum in which cells and the ECM function together in a well coordinated manner. So that the cell and ECM work and interact with each other with stimulant and inhibitors. 7/10/2024 13 Introduction ❖ During Development Cells and their associated matrix become functionally specialized and give rise to fundamental types of tissues with characteristic structural and functional features. Organs are formed by an orderly combination of these tissues, and their precise arrangement allows, the functioning of each organ and the organism as a whole. The small size of cells and matrix components makes histology dependent on the use of microscopes and molecular methods of study. 7/10/2024 14 Introduction ❖ Advances In Biochemistry, molecular biology, physiology, immunology, and pathology are essential for a better knowledge of tissue biology. ❖ Familiarity With The tools and methods of any branch of science is essential for a proper understanding of the subject. ❖ This Chapter Reviews Common methods used to study cells and tissues, focusing on microscopic approaches. 7/10/2024 15  The different techniques for preparing tissue sections. ❖There Are Different Techniques Used For Microscopic Examination Including 1) Tissue culture. 2) Micro technique(paraffin & freezing) technique. 3) Blood films and tissue smear. 7/10/2024 16 1. Tissue Culture ❖ This is A special technique by which living cells of the body are isolated and allowed to live, divide and grow outside the body by incubating them in a special growing media. ❖ The cultured cells are examined using phase contrast microscopes. e.g. (Blood cells or tissue cells) ❖ The Tissue Culture Is Used In: Studying chromosomal pattern. Cultivation of bacteria in order to prepare different vaccines. 7/10/2024 17 1-Tissue Culture ❖In vitro Study :(outside the body) The tissue piece is treated with trypsin to separate its components. The cells are placed in a sterile dish with the appropriate cultured medium. 7/10/2024 18 2. Micro technique Biopsies Stain Study 7/10/2024 19 2.Micro techniques ❖ Methods For Preparing Sections For Examination By The Light Microscope ❑ There Are Two Main Techniques: A. The paraffin technique is the (Most common method). B. The freezing technique is the (Most rapid method). The Student Must Understand And Learn The Paraffin Technique In Details. 7/10/2024 20  1. Paraffin technique The most commonly used technique. It is important for the student to understand this technique and its applications. ❖ The Main Objective Of Microtechnique Is: Prepare a piece of fresh soft animal tissue to be sectioned by the knife of the microtome into a thin section. Stain the sections to be examined with a microscope. 7/10/2024 21 The basic steps used in tissue preparation for light microscopy 7/10/2024 22 1.Paraffin technique ❖Steps 1) Tissue sample. 2) Fixation of tissues. 3) Dehydration. 4) Clearing. 5) Impregnation. 6) Embedding. 7) Sectioning. 8) Mounting. 7/10/2024 23 1. Paraffin technique 1.Tissue Sample A small sample (1 cm³) of tissue is obtained by biopsy under anesthesia or autopsy taken immediately after death using a sharp instrument. 7/10/2024 24 1. Paraffin technique 2. Fixation Put the specimen immediately in a fixative solution. Most common and routine one is formal saline which is 10% formaldehyde in normal saline. 7/10/2024 25 1. Paraffin technique ❖Aim Of Fixation : 1) Coagulate the protein and harden the tissue thus making it easier to cut thin sections. 2) Prevent autolysis by the lysosomal enzymes, thus preserving the structure organization of cells, and tissues. 3) Prevent putrefaction by killing the bacteria. 4) Make staining easier by increasing the affinity of the tissues for staining. 7/10/2024 26  1. Paraffin Technique 3. Dehydration Pass the pieces of tissue in ascending grades of alcohol (50%-70% 90%) then absolute alcohol (100%). Dehydration is done gradually to minimize shrinkage and distortion of tissues 7/10/2024 27 1. Paraffin Technique ❖ Aim Of dehydration Remove water from the tissues which is immiscible with paraffin wax solvents by alcohol which is miscible with clearing agents. ❑ Example. Xylol and then paraffin. 7/10/2024 28 1. Paraffin Technique 4. Clearing Put the piece of tissue in (clearing agent). e.g. xylol which is paraffin wax solvent. The tissue block will appear clear and transparent. ❖ Aim Of Clearing 1) To replace absolute alcohol by xylol which is miscible with paraffin wax. 1) Make the tissue translucent. 7/10/2024 29 1.The Paraffin Technique 5) Impregnation: OR (= infiltration with soft paraffin). The cleared fixed tissue is put in warm melted soft paraffin(1&2) in an oven at 52°- 60°c for 30 mint until the wax infiltrates the tissue and replaces all the xylol. 7/10/2024 30 1. Paraffin Technique 6) Embedding In Hard Paraffin The infiltrated tissue is transferred by melted hard paraffin from oven Then the specimen is transferred into a cast. Then filled with melted hard paraffin by embedding device. 31 7/10/2024 1. Paraffin Technique 6) Embedding In Hard Paraffin Now, the block is formed of hard paraffin with the tissue in the Centre. The block is now ready for sectioning. 7/10/2024 32 1. Paraffin Technique 7) Section: The block is cut into thin sections by means of microtome. Sections are cut at a thickness of 4-8 μm. 7/10/2024 33 1. Paraffin Technique 8) Mounting The thin paraffin sections are put on clean glass slides smeared with albumin glycerin and then warm them on a hot plate. Leave the sections for several hours in the incubator to dry. The sections are now fixed on the slides and are ready to be stained. 7/10/2024 34 1.The Paraffin Technique ❖ Advantage Of The Paraffin Technique Paraffin technique takes A short time (few days). It gives thin serial sections which is important for research work (4.8 μm). Paraffin sections are easily stained. ❖ Disadvantage Fat cannot be demonstrated by paraffin method as the used fixatives and alcohol dissolve it. Paraffin is not ideal for histochemistry activity because heat destroys enzymes. 7/10/2024 35 1. Paraffin Technique Note: Electron microscopic preparation The same as LM with some differences: 1. EM very much small pieces (1 mm³). 2. EM usually double fixatives are used; glutaraldehyde 2.5% in 0.1M phosphate buffer and osmium tetroxide 1.0% in the same buffer. 3. EM ascending grades of alcohol or acetone are used. 4. EM propylene oxide is used as clearing agent. 5. EM especial hard embedding material called epoxy resin is used to facilitate cutting into ultra thin sections. 7/10/2024 36 1. Paraffin Technique Note: Electron microscopic preparation The same as LM with some differences: 6. EM ultra microtome with glass or diamond knives is used to cut resin blocks ultra thin sections (50-100 nm thick). 7. EM staining, in the conventional color sense, does not exist. however, when sections are exposed to salts of heavy metals, such as uranyl acetate and lead nitrate, light (electron- lucent) and dark (electron-dense) images can be detected. 8. EM the stained ultra thin sections on copper grids are examined by the transmission electron microscope. 7/10/2024 37 2- freezing technique ❖In this method: The fresh tissues are frozen, hardened and cut with A freezing microtome in the cryostat apparatus. 7/10/2024 38 2- Freezing technique ❖ Advantages Of The Freezing Technique Quick and simple method. Used during operations for rapid diagnosis of tumors Used in histochemistry to demonstrate the enzyme activity. No heat is used ❖ Disadvantages Of The Freezing Technique It gives thick and sometimes fragmented sections. very difficult to cut and stained. 7/10/2024 39 Medical application ❖ Biopsies or tissue samples are removed during surgery or routine medical procedures In the operating room as the follows: ✓ Place and fix the biopsies in vials of formalin for processing and microscopic analysis in a pathology laboratory. ❖ If results of such analyses are required before the medical procedure is completed, for example to know whether a growth is malignant before the patient is closed: ✓ A much more rapid processing method is used and the biopsy is rapidly frozen in liquid nitrogen, preserving cell structures and making the tissue hard and ready for sectioning. Medical application ❖ A microtome called a cryostat in a cabinet at subfreezing temperature is used to section the block with tissue, and the frozen sections are placed on slides for rapid staining and microscopic examination by a pathologist. ❖ Freezing of tissues is also effective in histochemical studies of very sensitive enzymes or small molecules because freezing, unlike fixation, does not inactivate most enzymes. ❖ Finally, because clearing solvents often dissolve cell lipids in fixed tissues, frozen sections are also useful when structures containing lipids are to be studied histologically.  3- blood films and tissue smear ❖ Blood film pertains to examining blood, while a tissue smear pertains to examining tissue. 7/10/2024 42  1- Blood Films ❖ This is a test where a sample of blood is examined under a microscope. ❖ A drop of blood from Peripheral blood is placed on a glass slide and then spread into a thin layer that allows individual cells to be examined. ❖ Peripheral blood is the blood circulating throughout the body. 7/10/2024 43 1- Blood Films The Cellular Components That Could Be Isolated From Human Peripheral Blood Include: 1) Erythrocytes (red blood cells). 2) Leukocytes (white blood cells). 3) Thrombocytes (platelets). ❖ This test is used to diagnose various blood disorders such as: 1) Anemia. 2) Infections. 3) Hematological malignancies like leukemia. 7/10/2024 44 3- blood films and tissue smear Preparation Of A Thin And Thick Blood Film On The Same Slide 3- Blood Films And Tissue Smear Staining Of Peripheral Blood Smear 2- Tissue Smear 2) This is a test where a sample of tissue is examined under a microscope. Small piece of tissue (which may be from the skin, or a mucosal surface like the mouth or cervix) is taken and spread on a glass slide. This test is used to diagnose diseases affecting tissues such as: 1. Cancer. 2. Infections. 3. Inflammatory conditions. 7/10/2024 47  University of Tripoli Faculty of Medicine Histology and Genetics Department Histology and its methods of study Subject- HS120 MON 8-6-2024 FIRST LECTURE-SPRING 2024 PREPARED BY ASSISTANT LECTURER 48 SULEIMAN MOHAMED SHALAABY 2- Staining ❖ Objective: 1. Purpose of staining 2. Selective staining 3. Examples of dyes. 4. A. Common stains used for light microscopy. 5. B. Special methods of staining using the light microscopy. 6. Microscopy. 7/10/2024 49 2- Staining 1. Purpose of staining o Makes colorless tissues visible under a microscope. o Allows differentiation between tissue components. 2. Selective staining o Most dyes form electrostatic (salt) linkages with ionizable chemicals radicals macromolecules in the tissue. o Anionic components (net negative charge) stain with basic dyes (basophilic). o Cationic components (e.g., proteins) stain with acidic dyes (acidophilic). 7/10/2024 50 2. Stanning 3. Examples of dyes: A. Basic dyes: Hematoxylin, toluidine blue, Alcian blue, methylene blue. Is a basic dye which satin acidic structure purplish blue, e.g. nuclei, ribosomes and rough endo reticulum (RER). These structures have strong affinity for this dye due to their high content of DNA & RNA. So these Structures are basophilic. 7/10/2024 51 2- Staining 3. Examples of Dyes B. Acid Dyes: Eosin, Orange G, acid fuchsin. Is an acidic dye which stains basic structures Red or pink, e.g. the cytoplasm protein & collagen fibers is usually the basic component of the cell, So these Structures are acidophilic. 7/10/2024 52 A. Common stains used for light microscopy 1. Hematoxylin & Eosin (H&E) 7/10/2024 53 A. Common stains used for light microscopy 2. Periodic Acid Schiff Reaction (PAS) PAS is one of the histochemical techniques used to demonstrate carbohydrate, glycogen, and some glycoproteins in the cells and tissues into deep red color, e.g. Brush Borders of the Small Intestinal Cells, Basement Membrane, Collagenous Fibers. 7/10/2024 54 A. Common stains used for light microscopy 7/10/2024 55 A. Common stains used for light microscopy 3. Trichrome Stains: is differentiate extracellular components (e.g., Mallory, Masson). Mallory Trichrome: it Stains for Connective Tissue such as , collagenous fibers into blue color, muscle fibers into yellow color, Cytoplasm, nuclei and elastic fibers into red color. 7/10/2024 56 A. Common stains used for light microscopy 4. Van Gieson Stain: this method is used for blood vessels sections to stain, Collagenous fibers into red color, Muscle, epithelia and other tissues into yellow and nuclei into blue. 7/10/2024 57 A. Common stains used for light microscopy 5. Orcein Stain: it used for the demonstration of elastic fibers into brown color. 7/10/2024 58 A. Common stains used for light microscopy 6. Silver Impregnation: this method is used to stain, Reticular fibers into black color, Golgi complex into dark brown and nervous tissues into dark brown. 7/10/2024 59 A. Common stains used for light microscopy 7. Sudan III: it used for the demonstration of fat into orange color. 7/10/2024 60 A. Common stains used for light microscopy 8. Osmium Tetraoxide: Osmic Acid is commonly used in electron microscopy as fixative and as a dye for the demonstration of lipid contents of the cells with light microscope, e.g. Myelin sheath into black color 7/10/2024 61 B. Special Methods of Staining Using the Light Microscopy 1. Vital Staining : it is a means the staining of living cells within the body of a living animal; (in vivo) e.g. Injection of trypan blue into experimental animals, the phagocytic cells (macrophages) engulf the stain and are stained with it. 7/10/2024 62 B. Special Methods of Staining Using the Light Microscopy 2. Supravital Staining : It is the staining of living cells outside the body. e.g. Janus green B for (mitochondria) and brilliant cresyl blue for reticulocytes (immature R BCs). 7/10/2024 63 B. Special Methods of Staining Using the Light Microscopy 3. Metachromatic staining : is the staining of a tissue by a color that differs from the color of the original stain. e.g. mast cells granules react with toluidine blue stain; the reaction will give rise to a new color which is violet. This reaction is called metachromasia. 7/10/2024 64 B. Special Methods of Staining Using the Light Microscopy 4. Neutral stains: ✓Exampl: Leishman's stain:To stain leucocytes and to differentiate between blood cells that contain acidic, basic or neutral granules. 7/10/2024 65 B. Special Methods of Staining Using the Light Microscopy 5. Histochemical methods used for Identification of : a- Organic substance: e.g. Enzymes as: alkaline and acid phosphatase, lipid, and glycogen. B- Inorganic substance: e.g. Iron, phosphate, and calcium. 6. Immunocytochemistry used for: Identification and localization of specific protein e.g. Hormones. It is a highly specific interaction between antigen and its antibody. 7/10/2024 66 MICROSCOPY ❖ They are several type of microscopes used for the study of a histological specimen. The most important types are: 1. The light microscope (LM). 2. The Electron Microscope (EM). 3. Fluorescence microscope. 4. Phase Contrast microscope. 5. The confocal microscope. 7/10/2024 67 1.The light microscope (LM) ❖The light microscope is composed of: 1. The frame with its mechanical parts 2. Optical or Magnification System 3. illumination System 1.The frame: supports the different parts of the optical system; It consists of: 1) Base. 2) Arm. 3) Stage with a central hole for the light to pass through and a body tube carrying the optical system. 7/10/2024 68 1.The light microscope (LM) 2- The Magnifying System : ❑ Is composed of: A- Eyepiece (OCULAR) : with 8, 10, or 12 x of Magnification. B- The Objectives: Four Lenses with Different Magnifications: 3.5, 10, 40, 100 (Oil Immersion Lens). 7/10/2024 69 1.The light microscope (LM) ❖The objective lenses: collect the light transmitted through the section from the condenser, magnify the image and direct it to the ocular lens. ❖The eyepiece (ocular lens) : Magnifies the image & directs it to the viewer's eye or to a screen or a camera. The property of making things appear larger is called magnify. The property of disclosing the fine details is called Resolution. 7/10/2024 70 1.The light microscope (LM) ❑Resolution (R): is the smallest distance between two particles that can be distinguished from each other. Resolution is more important than magnification, since it separates clearly between two points located close together. ❑ Resolution power of: oHuman naked eye: 0.1-0.2 mm. o Lm is 0.1-0.2 um. o EM: 0.1-0.2 nm. 3.The illumination system consists of: light source, condenser and iris diaphragm to regulate the amount of light. 7/10/2024 71 2. The Electron Microscope (EM) There are two types of electron microscopes: A. Transmission. B. Scanning. A. Transmission Electron Microscope (TEM): ❑ In this microscope, the ordinary light source is replaced by electrons, which are emitted by tungsten gun or filament. ❑ Electromagnetic lenses are used and the image is viewed directly on a fluorescent screen. 7/10/2024 72 A. Transmission Electron Microscope (TEM) 7/10/2024 73 B. Scanning Electron Microscope (SEM) ❖B. Scanning Electron Microscope (SEM): This microscope has less resolving power than TEM. It provides a three-dimensional image of the surface of fixed and dehydrated tissues. The sample is coated with gold, which emits secondary electrons after being hit with a primary electron beam. 7/10/2024 74  3. Fluorescence microscope ❖ Certain biological substances absorb invisible ultraviolet light of short wavelength and emit it as visible light of longer wavelength, and are known to exhibit fluorescence. ❖ This fluorescence microscope is provided with a special lamp that has the ability to emit ultraviolet rays which pass through the specimen. ❖ This microscope can be used to examine tissues stained with immune-fluorescence technique and to visualize the antigen antibody reaction. Photomicrograph of kidney cells in culture, stained with acridine orange. Under a fluorescence microscope, DNA (within the nuclei) emits yellow light, and the RNA-rich cytoplasm appears reddish or orange. (Courtesy of A Geraldes and JMV Costa.) 7/10/2024 75 4. Phase Contrast microscope ❖This microscope is based on the fact when light passes through media of different refraction indexes. It changes its direction and speed. These differents of special Lens System are transformed. This microscope is used in Tissue Culture. 7/10/2024 76 5. The confocal microscope 6. Dark field microscope 5. The confocal microscope: This is the most recent type of microscopes. ❖It uses the Laser Light illumination and a highly computerized system to produce a 3Dimensional image. ❖ It is used to visually dissect a sample to study the structure of biological matter. 6.Dark field microscope ❖It is used to examine unstained living specimens that appear bright against a dark fied. 7/10/2024 77 References 1) Basic Histology Text And Atlas Fourteen Edition Jonquiere’s. 2) Medical Histology And Cytogenetics. By Dr. Naama Abdulgader, Md Ph.D. 3) Histology Laboratory Manual For Medical Students.By Dr. Naama Abdulgader Md Ph.D. 4) Histology for the first year medical students part one. by Mohamed Essam Anwar Professor Of Histology Ain Shams. 5) Introduction to functional and clinical histology text and atlas.by Aymen M.Ghallab. part -1. 6) Some Illustrations From The Internet. 7/10/2024 78