INTRODUCTION TO MICROANATOMY By Dr A K Akinloye
INTRODUCTION TO MICROANATOMY By Dr. A. K. Akinloye Department of Veterinary Anatomy University of Agriculture Abeokuta 9/25/2021 1
What is Histology? The term histology, is derived from the Greek histos, meaning tissue (web) and logia, knowledge It is, in the strict sense, the knowledge, or science, of tissues whether they are of plants or animals is the study of the microscopic anatomy of cells and tissues of plants and animals It is performed by examining a thin slice (section) of tissue under a light or electron microscope 9/25/2021 2
What is Veterinary Histology? The science that focuses on the detailed morphology of tissues of domestic animals with the aid of the microscope and correlates specific structures with function 9/25/2021 3
What is Veterinary Microanatomy? Involves the examination and architectural description of the microscopic anatomy of normal cells of the body of domestic animals and all their contents and products 9/25/2021 4
HISTORY l. Robert Hooke: First to observe cells in 1665, by observing slices of cork under a microscope. The cork appeared as little boxes which he called cells 9/25/2021 5
l In 1883 Mathias Schleiden and Theodor Schwann proposed that all plants and animals were composed of cells which were the basic building blocks of life In 1855 Rudolf Virchow stated that new cells arise from the division of pre-existing cells and that chemical reactions needed for life occurred inside the cell l All this work led to the formation of the cell theory: 9/25/2021 6
APPLICATIONS Embryo development study Structure and function Interdisciplinary studies Anat. , Physiol. , Biochem, Chem Organization of tissue 9/25/2021 7
TEACHING AND LEARNING TOOLS Microscopy Light microscopy Phase contrast microcopy Polarizing microscopy, Electron microscopy [Transmission & Scanning] Fluorescent microscopy. 9/25/2021 8
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Electronmicroscope 9/25/2021 11
TEACHING AND LEARNING TOOLS (Continued) Atlases or text-atlases Projectors Micrographs Sections. Others: notebooks, pencils, erasers, lab coats. 9/25/2021 12
LEVELS OF ORGANIZATION Ø Cells Ø Tissues Ø Organ Systems Ø Organism Tissues Cells Organism Organ Systems 9/25/2021 13
What is a Cell? The smallest basic structure of higher organisms capable of independent existence 9/25/2021 14
What is a Tissue? Cells of similar function and origin that form functional units 9/25/2021 15
Histological Classification of Animal Tissues There are four basic types of tissues: ØEpithelial tissue ØMuscle tissue ØConnective tissue ØNervous tissue 9/25/2021 16
Sources of Tissues Histological examination of tissues starts with surgery, biopsy or autopsy (or necropsy, in the case of animal tissues). § Biopsy is an examination of tissue taken from a living body § Autopsy is an examination of post-mortem tissue § Necropsy is an examination of tissue taken from dead animal 9/25/2021 17
Epithelial tissue A sheet of cells of a similar type tightly adhered to each other on the external and internal surfaces of the body Epithelium: the lining of glands, bowel, skin and some organs like the liver, lung, kidney, Endothelium: the lining of blood and lymphatic vessels, Mesothelium: the lining of pleural, and pericardial spaces, 9/25/2021 18
CONNECTIVE TISSUE Provides structural support for the tissues and organs of the body. This mechanical function is important in maintaining the form of the body, organs and tissues. Composed of: § cells § extracellular matrix. 9/25/2021 19
MUSCLE TISSUE Characterized by contraction Responsible for the movements of body and body parts Develops from mesoderm Classified into 3: § Skeletal Muscle § Cardiac Muscle § Smooth Muscle 9/25/2021 20
NERVOUS TISSUE Consists of two cell types: § Nerve cells (Neurons) § Neuroglia. Derived from embryonic neuroectoderm Divided anatomically into: CNS - brain and spinal cord. PNS- nerve fibers, nerve cells, glia and ganglia. 9/25/2021 21
What is an Organ? ØAn organ represents an even greater measure of complexity and is composed of various tissues. 9/25/2021 22
What is an Organ System? At an even higher level of organization: An organ system is composed of several organs (such as the gastrointestinal system, respiratory system, cardiovascular system, endocrine system). 9/25/2021 23
What is an Organism? An organism can be seen to be formed of different levels of organization, with increasing levels of complexity and each of which plays important roles in the physiological homeostasis of the body. 9/25/2021 24
UNITS OF MEASUREMENT Angstrom unit (A 0) = 10 -10. Nanometer (nm) = 10 -9. Micrometer (μm) = 10 -6 replaces Micron (μ) = 10 -6. meter 9/25/2021 25
SECTIONS Extremely thin, transparent shavings Cut from a little piece of tissue Laid flat on a glass slide Stained Covered with mounting fluid and cover-slip. 9/25/2021 26
STEPS IN THE PARAFFIN TECHNIQUE Tissue collection Fixation Dehydration Clearing Embedding Sectioning Staining and mounting 9/25/2021 27
Technical Procedure Fixation ØThe tissues are mechanically and biochemically stabilized in a fixative. The most common fixative is neutral buffered formalin (10% formaldehyde in phosphate buffered saline (PBS)) 9/25/2021 28
Other Examples of Fixatives 4% solution of buffered formaldehyde Acetic acid Picric acid Potassium dichromate Osmic acid Glutaraldehyde Ethanol Bouin’s fluid Os. O 4 (Osmium tetroxide) 9/25/2021 29
Action of fixatives Keep from washing away of components (hardening? ) Good antiseptics that kill bacteria, etc Affects tissue to the extent that reaction to other stains is favoured. 9/25/2021 30
Technical Procedure Processing Ø The most common technique is wax processing either manually or in machine. The samples are immersed in multiple baths of progressively more concentrated ethanol to dehydrate the tissue, followed by a clearing agent such as, xylene or Histoclear, and finally hot molten paraffin wax (impregnation). During this 12 to 16 hour process, paraffin wax will replace the xylene: 9/25/2021 31
Processing methods and routine schedules Machine processing manual processing 9/25/2021 32
Technical Procedure Embedding Sectioning Staining 9/25/2021 33
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Embedding materials Paraffin Wax (commonest) Polyester wax Nitrocellulose Synthetic resins Freezing 9/25/2021 36
MOULD SYSTEMS 1 - Paper boats 2 - Embedding irons or metal containers 3 - Disposable plastic moulds Embedding rings or cassette-bases which become an integral part of the block and serve as the block holder in the microtome. 9/25/2021 37
Embedding Moulds Tissue processing Embedding moulds: (A) paper boat; (B) metal boat mould; (C) Dimmock embedding mould; (D) Peel-a-way disposable mould; (E) base mould used with embedding ring ( F) or cassette bases (G) 9/25/2021 38
Precaution while embedding The wax is clear of clearing agent. No dust particles must be present. Immediately after tissue embedding, the wax must be rapidly cooled to reduce the wax crystal size. 9/25/2021 39
General Embedding Procedure 1 - Open the tissue cassette, check against worksheet entry to ensure the correct number of tissue pieces are present. 2 - Select the mould, there should be sufficient room for the tissue with allowance for at least a 2 mm surrounding margin of wax. 3 - Fill the mould with paraffin wax. 4 Using warm forceps select the tissue, taking care that it does not cool in the air; at the same time. 5 - Chill the mould on the cold plate, orienting the tissue and firming it into the wax with warmed forceps. This ensures that the correct orientation is maintained and the tissue surface to be sectioned is kept flat. 6 - Insert the identifying label or place the labeled embedding ring or cassette base onto the mould. 7 - Cool the block on the cold plate, or carefully submerge it under water when a thin skin has formed over the wax surface. 8 - Remove the block from the mould. 9 - Cross check block, label and worksheet. 9/25/2021 40
Cutting the thin slices Slices of a few microns thick are difficult to cut. 1μ (one micron) is 1/1000 mm. Special machines are widely employed for this purpose. These machines are called MICROTOMES 9/25/2021 41
CUTTING using the microtome 9/25/2021 42
1 - Traditional histological technique: tissues are hardened by replacing water with paraffin. The tissue is then cut in the microtome at thicknesses varying from 2 to 25 micrometers thick. From there the tissue can be mounted on a microscope slide, stained and examined using a light microscope 9/25/2021 43
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Alternative techniques CRYOSECTION - tissue frozen and cut with cryostat - staining similar to those of wax sections PLASTIC EMBEDDING - for electron microscopy. - embedment in epoxy resin. - ultrathin sections (less than 0. 1 micrometers) cut using diamond or glass knives. - sections stained with electron dense stains (uranium and lead) 9/25/2021 46
CRYOSECTION Water-rich tissues are hardened by freezing and cut frozen; sections are stained and examined with a light microscope. This technique is much faster than traditional histology (5 minutes vs. 16 hours) and are used in operations to achieve a quick diagnosis. Cryosections can also be used in immunohistochemistry as freezing tissue does not alter or mask its chemical composition as much as preserving it with a fixative. 9/25/2021 47
Stains and staining Routine stain = H&E Basic component: Haematoxylin imparts a blue-to-purple colour to the tissue constituents i. e. basophilic constituents. It is thus, a basic dye. Acidic component: Eosin imparts a pink-tored colour to the tissue constituents i. e. acidophilic constituents. It is thus, an acidic dye. 9/25/2021 48
Staining Routinely done to give contrast as, without staining, it is difficult to see differences in cell morphology Haematoxylin and Eosin (H&E) most common stains. Haematoxylin colours nuclei blue, eosin colours the cytoplasm pink To see the tissue under a microscope, the sections are stained with one or more pigments 9/25/2021 49
Examples of Stains Routine (Nucl & cyto) - H &E DNA - Fuelgen Connective Tissue Fibre Masson’s Trichrome Silver impregnation Fat 9/25/2021 - Sudan IV 50
More Stains Other compounds used to colour tissue sections include: Ø safranin Øoil red o Øcongo red Øfast green FCF Øsilver salts Ønumerous natural and artificial dyes 9/25/2021 51
Stains (ctd) Carbohydrate Periodic Acid-Shiff (PAS) Mucopolysaccharide Toluidine blue (metachromatic) Acian blue Hale’s Colloidal Iron Elastic fibers- Mallory Azan 9/25/2021 52
Staining machine 9/25/2021 53
STAINED TISSUES 9/25/2021 54
Hematoxylin and Eosin (H & E) H & E is a charge-based, general purpose stain. Hematoxylin stains acidic molecules shades of blue. Eosin stains basic materials shades of red, pink and orange. H & E stains are universally used for routine histological examination of tissue sections. 9/25/2021 55
Reaction of substances to Dyes Reaction of proteins Nucleic acids and Nucleoproteins Inorganic materials Lipids Carbohydrates 9/25/2021 56
Carbohydrates There are four (4) categories: Simple sugars e. g. glucose Sugar Polymers e. g. glycogen Glycoproteins (carbohydrate + protein) Mucopolysaccharides 9/25/2021 57
Other ways of Preparing Section The frozen section techniques Bone tissue section Blood smear Paraffin infiltration by the freeze-drying method Celloidin method 9/25/2021 58
Histochemistry The science of chemical reactions on components of tissue. A commonly performed histochemical technique is the Perls Prussian blue reaction, used to demonstrate iron deposits in diseases like Hemochromatosis 9/25/2021 59
Immunohistochemistry Recently, antibodies are used to specifically visualize proteins, carbohydrates and lipids: this is called Immunohistochemistry Other advanced techniques include in situ hybridization to identify specific DNA or RNA molecules, and confocal microscopy 9/25/2021 60
INTERPRETATION OF TISSUE SECTION Artifacts Shrinkage 9/25/2021 61
Causes of Artifacts – – – 9/25/2021 Shrinkage Folds and wrinkles Nicks in microtone knife Rough handling of fresh tissue Postmortem degeneration Dirty stains leading to precipitates 62
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THREE DIMENSIONS Understanding organ architecture: Longitudinal Transverse Oblique Tangential Combinations of the above 9/25/2021 65
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The End 9/25/2021 68
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