Introduction to Histology PDF

HISTOLOGY OF BASIC TISSUES BY DR. ADEKEYE, ADESHINA DEPT OF ANATOMY ABUAD  Histology & Its Methods of Study Histology is the study of the tissues of the body and how these tissues are arranged to constitute organs. It is a branch of science concerned wit...

HISTOLOGY OF BASIC TISSUES BY DR. ADEKEYE, ADESHINA DEPT OF ANATOMY ABUAD  Histology & Its Methods of Study Histology is the study of the tissues of the body and how these tissues are arranged to constitute organs. It is a branch of science concerned with the microscopic study of cells, tissues, and organs in relation to their function (microanatomy, microscopic anatomy).  Tissues are made of two interacting components: cells and extracellular matrix. The main functions extracellular matrix Furnish mechanical support for the cells, Transport of nutrients to the cells Transport of catabolites and secretory products Note: The small size of cells and matrix components makes histology dependent on the use of microscopes  A microscope is an instrument used to see objects that are too small for the naked eye. The science of investigating small objects using such instrument is called Microscopy. ❖ Physiology/Medicine (2014 Nobel prize winner) US-British scientist John O’keefe split the Nobel Prize in medicine with Norwegian couple May-Britt Moser and Edvard Moser for breakthroughs in brain cell research that could pave the way for a better understanding of diseases like Alzheimer’s.  Yoshinori Ohsumi is a Japanese cell biologist specializing in autophagy, the process that cells use to destroy and recycle cellular components (2016 Nobel prize winner, Physiology/medicine) 2019 NOBEL PRIZE IN PHYSIOLOGY/MEDICINE? ❑ Microscope and its type a. Optical microscope (light): uses light to image the sample b. Electron microscope (TEM or SEM): are based on the interaction of electrons and tissue components. ❖Four fundamental tissues Epithelial Connective Muscular Nervous Diagram of a typical light microscope. ❑ Preparation of Tissues for Study The most common procedure used in the study of tissues is the preparation of histological sections or tissue slices that can be studied with the aid of the light microscope. Under the light microscope, tissues are examined via a light beam that is transmitted through the tissue.  REASONS: Because tissues and organs are usually too thick for light to pass through them they must be sectioned to obtain thin, translucent sections and then attached to glass slides before they can be examined. In most cases, tissues must be sliced into thin sections and attached on glass slides before they can be examined. The basic steps used in tissue preparation for histology ❑ Tissue Processing a. Fixation Small pieces of fresh tissue are placed in fixative solutions to: prevent autolysis and preserve the structure and molecular composition of cell (10% Formalin: formaldehyde dissolved in an aqueous or buffered medium) b. Dehydration The fixed pieces then undergo “dehydration" by being transferred through a series of increasingly more concentrated alcohol solutions, ending in 100% which effectively removes all water from the tissue (ethanol). c. Clearing The alcohol is then removed in a clearing solution immiscible in both alcohol and melted paraffin (hydrocarbon e.g Toluene and xylene) d. Infiltration/Inpregnation When the tissue is then placed in melted paraffin at 58°C it becomes completely infiltrated with this substance.  After infiltration the tissue is placed in a small mold containing melted paraffin, which is then allowed to harden. The resulting paraffin block is trimmed to expose the tissue for sectioning (slicing) The sections are floated on water and then transferred to glass slides to be stained. This is a process of introducing paraffin wax or molten paraffin wax into the tissue to replace the clearing agent. Tissues are infiltrated by immersion in a substance such as a wax, which is fluid when hot and solid when cold e. Embedding & Sectioning Tissues are usually embedded in a solid medium to facilitate sectioning. To obtain thin sections with the microtome, tissues must be infiltrated after fixation with embedding substances that impart a rigid consistency to the tissue. Embedding materials include paraffin and plastic resins. Embedding is a process by which tissues are surrounded by a medium such as agar, gelatin or wax which when solidified will provides sufficient external support during sectioning. NOTE: Paraffin is used routinely for light microscopy; Resins are used for both light and electron microscopy A microtome  A microtome is used for sectioning paraffin-embedded tissues for light microscopy. After mounting a trimmed block with the tissue specimen, rotating the drive wheel moves the tissue-block holder up and down. Each turn of the drive wheel advances the specimen holder a controlled distance, generally between 1 and 10 m, and after each forward move the tissue block passes over the steel knife edge, which cuts the sections at a certain thickness. Paraffin sections are then adhered to glass slides Deparaffinized, and stained for microscopic examination ❑Staining For microscopically study, sections must typically be stained or dyed because most tissues are colorless. Methods of staining tissues help to make the various tissue components conspicuous and also permit distinctions to be made between them. Most of these dyes behave like acidic or basic compounds. ❖ Cationic ("basic") Dyes: tissue components stained with these dyes are basophilic. ❖ A basic dye carries a net positive charge on its colored portion and is described by the general formula [dye†Cl¯] Examples of basic dyes are Toluidine blue Methyl green Green Alcian blue Methylene blue. Hematoxylin behaves like a basic dye Examples of basophilic tissue components nuclei and nucleoli, Cytoplasmic RNA ❖ Anionic ("acid") Dyes: tissue components stained with these dyes are acidophilic. ❖ An acidic dye, such as eosin, carries a net negative charge on its colored portion and is described by the general formula [Na†dye¯]. Example of acidic dyes are Orange G- orange Eosin- pink-red Acid fuchsin- Red Picric acid: yellow Examples of acidophilic tissue components mitochondria secretory granules Most cytoplasm  Simple combination of hematoxylin and eosin (H&E) is used most commonly. Hematoxylin stains DNA of the cell nucleus blue. Eosin stains other cytoplasmic components and collagen pink With H&E, basophilic cell nuclei are stained purple With PAS, staining is most intense at the cell surface, where projecting while cytoplasm stains pink microvilli have a prominent layer of glycoproteins Hematoxylin & Eosin (H&E) and Periodic acid-Schiff (PAS) staining a kidney renal corpuscle. ❑ PROBLEMS IN THE INTERPRETATION OF TISSUE SECTIONS ✓ Distortions and Artifacts caused by tissue processing : one cause of distortion is the shrinkage produced by the fixative (ethanol) and by heat needed for paraffin embedding. A consequence of shrinkage is the appearance of artificial spaces between cells and other tissue components. Another source of artificial spaces is the loss of molecules that were not properly kept in the tissues by the fixative or that were removed by the dehydrating and clearing fluids.  Glycogen and lipids are often lost during tissue preparation. All these artificial spaces and other distortions caused by the section preparation procedure are called ARTIFACTS ✓ Another point to remember in studying histological sections is the impossibility of differentially staining all tissue components on a slide stained by a single procedure. ❑ Other Techniques include Specialized microscopic techniques Autoradiography Cell and tissue culture Histochemistry and cytochemistry Immunocytochemistry and hybridization techniques Cell and organelle separation by differential centrifugation ❖ LIGHT MICROSCOPY These phenomenon are based on the interaction of light and tissue components. With the light microscope, stained preparations are usually examined by means of light that passes through the specimens. The microscope is composed of 2 parts ✓ Mechanical parts ✓ Optical parts  The optical components consist of three systems of lenses: Condenser Objective Eyepiece. The Condenser: collects and focuses light in order to produce 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 further magnifies this image and projects it onto the viewer’s retina. The total magnification is obtained by multiplying power of the objective and eyepiece (x…..) ❖Autoradiography Autoradiography is a method of localizing newly synthesized macromolecules (DNA, RNA, protein, glycoproteins, and polysaccharides) in cells or tissue sections. Radioactively labeled metabolites (nucleotides, amino acids) incorporated into the macromolecules emit weak radiation that is restricted to the cellular regions where the molecules are located. ❖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. Cell and tissue culture has been very helpful in isolating the effect of a single molecule on one type of cell or tissue. It also allows the direct observation of the behaviour of living cells under a microscope. Several experiments that cannot be performed in the living animal can be reproduced In Vitro (outside) In vivo: studies that are within the living organisms. ❑ Medical Application Cell culture has been widely used for the study of the metabolism of normal and cancerous cells Also for the development of new drugs Useful in the study of parasites that grow only within the cells such as viruses and some protozoa ❖ Histochemistry & Cytochemistry The terms histochemistry and cytochemistry are used to 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, Sectioned are often on a cryostat to avoid adverse effects of heat and paraffin on enzymatic activity. Enzyme histochemistry usually works in the following way: (a) tissue sections are immersed in a solution that contains the substrate of the enzyme to be localized (b) the enzyme is allowed to act on its substrate  (c) at this stage or later, the section is put in contact with a marker compound (d) this compound reacts with a molecule produced by enzymatic action on the substrate (e) 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. Examples of enzymes that can be detected histochemically include the following Phosphatases Dehydrogenases Peroxidase ❖ Immunohistochemistry It involves a highly specific interaction between molecules (between an antigen and its antibody) Useful in identifying and localizing many specific proteins. 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. ❖ 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.