1. INTRODUCTION TO MICROANATOMYBy
Dr. A.K. Akinloye
Department of Veterinary Anatomy
University of Agriculture
Abeokuta
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2. 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
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3. 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
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4. 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
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5. HISTORY
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
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6. All this work led to the formation of the cell theory:
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
All this work led to the formation of the cell theory:
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7. Anat.,Physiol.,Biochem, Chem
APPLICATIONS
Embryo development study
Structure and function
Interdisciplinary studies
Anat.,Physiol.,Biochem, Chem
Organization of tissue
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8. TEACHING AND LEARNING TOOLS
Microscopy
Light microscopy
Phase contrast microcopy
Polarizing microscopy,
Electron microscopy [Transmission & Scanning]
Fluorescent microscopy.
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11. Electronmicroscope
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12. TEACHING AND LEARNING TOOLS (Continued)
Atlases or text-atlases
Projectors
Micrographs
Sections.
Others: notebooks, pencils, erasers, lab coats.
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13. LEVELS OF ORGANIZATION
Cells
Tissues
Organs
Organ Systems
Organism
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14. What is a Cell?
The smallest basic structure of higher organisms capable of independent existence
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15. What is a Tissue?
Cells of similar function and origin that form functional units
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16. Histological Classification of Animal Tissues
There are four basic types of tissues:
Epithelial tissue
Muscle tissue
Connective tissue
Nervous tissue
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17. 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
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18. 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,
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19. 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.
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20. 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
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21. 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.
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22. What is an Organ?
An organ represents an even greater measure of complexity and is composed of various tissues.
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23. 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).
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24. 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.
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25. UNITS OF MEASUREMENT Angstrom unit (A0) = 10-10 meter.
Nanometer (nm) = 10-9 meter.
Micrometer (μm) = 10-6meter
replaces
Micron (μ) = 10-6 meter.
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26. 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.
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27. STEPS IN THE PARAFFIN TECHNIQUE
Tissue collection
Fixation
Dehydration
Clearing
Embedding
Sectioning
Staining and mounting
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28. Technical Procedure Fixation
The tissues are mechanically and biochemically stabilized in a fixative. The most common fixative is neutral buffered formalin (10% formaldehyde in phosphate buffered saline (PBS))
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29. Other Examples of Fixatives
4% solution of buffered formaldehyde
Acetic acid
Picric acid
Potassium dichromate
Osmic acid
Glutaraldehyde
Ethanol
Bouin’s fluid
OsO4 (Osmium tetroxide)
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30. Keep from washing away of components (hardening?)
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.
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31. Technical Procedure Processing
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:
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32. Processing methods and routine schedules
Machine processing
manual processing
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33. Technical Procedure Embedding Sectioning Staining
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36. Paraffin Wax (commonest) Polyester wax Nitrocellulose Synthetic resins
Embedding materials
Paraffin Wax (commonest)
Polyester wax
Nitrocellulose
Synthetic resins
Freezing
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37. 2- Embedding irons or metal containers 3- Disposable plastic moulds
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.
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38. 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)
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39. 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.
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40. 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.
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41. Cutting the thin slices
Slices of a few microns thick are difficult to cut. 1μ (one micron) is 1/1000mm.
Special machines are widely employed for this purpose. These machines are called MICROTOMES
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42. CUTTING
using the microtome
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43. 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
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46. 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)
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47. 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.
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48. 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-to-red colour to the tissue constituents i.e. acidophilic constituents. It is thus, an acidic dye.
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49. 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
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50. Examples of Stains Routine (Nucl & cyto) - H &E DNA - Fuelgen
Connective Tissue Fibre -
Masson’s Trichrome
Silver impregnation
Fat Sudan IV
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51. More Stains Other compounds used to colour tissue sections include:
safranin
oil red o
congo red
fast green FCF
silver salts
numerous natural and artificial dyes
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52. Stains (ctd) Carbohydrate Periodic Acid-Shiff (PAS)
Mucopolysaccharide Toluidine blue (metachromatic)
Acian blue
Hale’s Colloidal Iron
Elastic fibers- Mallory Azan
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53. Staining machine
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54. STAINED TISSUES
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55. 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.
                                                                                                                                                                                                                                                                                                                         
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56. Reaction of substances to Dyes
Reaction of proteins
Nucleic acids and Nucleoproteins
Inorganic materials
Lipids
Carbohydrates
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57. There are four (4) categories: Simple sugars e.g. glucose
Carbohydrates
There are four (4) categories:
Simple sugars e.g. glucose
Sugar Polymers e.g. glycogen
Glycoproteins (carbohydrate + protein)
Mucopolysaccharides
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58. Other ways of Preparing Section
The frozen section techniques
Bone tissue section
Blood smear
Paraffin infiltration by the freeze-drying method
Celloidin method
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59. 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
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60. 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
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61. INTERPRETATION OF TISSUE SECTION
Artifacts
Shrinkage
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62. Causes of Artifacts Shrinkage Folds and wrinkles
Nicks in microtone knife
Rough handling of fresh tissue
Postmortem degeneration
Dirty stains leading to precipitates
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65. THREE DIMENSIONS Understanding organ architecture: Longitudinal
Transverse
Oblique
Tangential
Combinations of the above
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68. The End
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