INTRODUCTION TO MICROANATOMY

1. INTRODUCTION TO MICROANATOMY By Dr. A.K. Akinloye Department of Veterinary Anatomy University of Agriculture Abeokuta

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

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

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

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

6. 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:

7. APPLICATIONS • Embryo development study • Structure and function • Interdisciplinary studies Anat.,Physiol.,Biochem, Chem • Organization of tissue

8. TEACHING AND LEARNING TOOLS Microscopy • Light microscopy • Phase contrast microcopy • Polarizing microscopy, • Electron microscopy [Transmission & Scanning] • Fluorescent microscopy.

11. Electronmicroscope

12. TEACHING AND LEARNING TOOLS(Continued) • Atlases or text-atlases • Projectors • Micrographs • Sections. • Others: notebooks, pencils, erasers, lab coats.

13. LEVELS OF ORGANIZATION • Cells • Tissues • Organs • Organ Systems • Organism

14. What is a Cell? • The smallest basic structure of higher organisms capable of independent existence

15. What is a Tissue? • Cells of similar function and origin that form functional units

16. Histological Classification of Animal Tissues • There are four basic types of tissues: • Epithelial tissue • Muscle tissue • Connective tissue • Nervous tissue

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

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,

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.

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

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.

22. What is an Organ? • An organ represents an even greater measure of complexity and is composed of various tissues.

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).

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.

25. UNITS OF MEASUREMENT • Angstrom unit (A0) = 10-10meter. • Nanometer (nm) = 10-9meter. • Micrometer (μm) = 10-6meter replaces • Micron (μ) = 10-6meter.

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.

27. STEPS IN THE PARAFFIN TECHNIQUE • Tissue collection • Fixation • Dehydration • Clearing • Embedding • Sectioning • Staining and mounting

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))

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)

30. 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.

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:

32. Processing methods and routine schedules • Machineprocessing • manual processing

33. Technical Procedure • Embedding • Sectioning • Staining

36. Embedding materials • Paraffin Wax (commonest) • Polyester wax • Nitrocellulose • Synthetic resins • Freezing

37. 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.

38. Embedding Moulds Tissue processingEmbedding 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)

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.

40. General Embedding Procedure1- 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.

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

42. CUTTING • using the microtome

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

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)

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.

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.

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

50. Examples of Stains • Routine (Nucl & cyto) - H &E • DNA - Fuelgen • Connective Tissue Fibre - Masson’s Trichrome Silver impregnation • Fat - Sudan IV