1-Introduction to Histology.pptx

Introduction to Histology Anatomy Unit Objectives Understand what is Histology Realize the size of histological tissues Know basic steps in tissue preparation and staining Understand the importance of different stains Identify different techniques of imaging in histology ...

Introduction to Histology Anatomy Unit Objectives Understand what is Histology Realize the size of histological tissues Know basic steps in tissue preparation and staining Understand the importance of different stains Identify different techniques of imaging in histology  The name "Histology" is derived from Greek: "Histos“: tissue "logos" : the study of What is Histology: Is the study of the microscopic Histology? anatomy of cells and tissues How? Examining a thin slice (section) of tissue under a light microscope or electron microscope by using different histological stains  Cells: The smallest independently living highly complex structure Levels of Tissues: Functional unit: group of cells of similar function body and origin organizatio Organs: n Several tissues grouped together e.g liver, lung & kidney Systems: Several organs working for a common function e.g Digestive ,Respiratory & Urinary. Tissues Tissues are made of: cells extracellular matrix Four types tissues: Epithelial tissue Connective tissue Muscular tissue Nervous tissue  Basic Techniques Steps in the preparation of histological sections: 1. Fixation 2. Processing (dehydration and clearing) 3. Embedding 4. Cutting 5. Staining 6. Permanent Mounting 1.Fixation of tissues: Keep volume and shape of tissue Prevents autolysis (preserves) Examples of Fixatives (preservatives): Formaldehyde  2. Processing: i. Dehydration Removes fixative and water from the tissue and replace them with dehydrating fluid (Ethanol). ii. Clearing Alcohol is removed in xylene.  3. Embedding  4. Cutting - process by which tissues - Small (micro) sections are are embedded in (wax) cut by a Microtome. -Section thickness: - When solidified gives 2 to 25 micrometers thick sufficient support during for light microscope sectioning. 60 to 100 nanometers thick for electron microscopy 5-Staining Various stains are used to see and highlight specific structures and molecules  1. Hematoxylin and Eosin (H & E) H & E stains are universally used for routine histological examination of tissue sections.  Hematoxylin Blue colored basic dye Stains acidic components of cells e.g. nuclei of cells & rER of cytoplasm. The ability to react with a basic dye - basophilia Positively charged {colors negatively charged molecules (anionic ) e.g., DNA, rER Eosin Red colored acidic dye. Stains the basic components of cells reddish-pink Most of cytoplasm of cells is stained by eosin. The ability to react with an acidic dye –acidophilia Negatively charged {colors positively charged molecules (cationic ) e.g. amino acids, cytoplasm} H&E Nuclei - blue - with some metachromasia Cytoplasm - various shades of pink- identifying different tissue components  2. Periodic acid-Schiff (PAS) staining :mucus, basal lamina, H&E stain does not preserve Mucous in Goblet cells, which Appear empty PAS stain preserves Mucous in Goblet cells, which and stains it with a magenta colour glycogen (carbohydrates) Glyco gen Carbohydrate content in liver tissue stained magenta with PAS stain  3. Toluidine blue (Metachromatic stain) Blue stain. Stains specific components of tissues a purple color Metachromasia is seen in: hyaline cartilage IJDVL, Omar El Safoury Blue stain of Hyaline Metachromasia of Mast cells, skin cartilage  4. Oil Red O Stain lipids red-orange in frozen sections 5. Sudan black Stains lipids black in frozen sections  7. Silver impregnation 8. Reticulin Stain Reticular fibers black Stain Reticular fibers black Lymph node, Silver Impregnation 9. Giemsa stain Blood smears, for disease Plasmodiun Falciparum (Malaria) Trypanosoma sp. 6- Mounting Covered with a thin glass cover-slip  Total preparation time for light microscopy: 12 hours to 2½ days Immediate Frozen section time: 5 minutes For immediate results (in tumor surgery)  Types of Microscopes Light Microscopy Bright-Field Bright-Field Polarizing Confocal Fluorescence Fluorescence Phase-Contrast Types of Microscopes Electron Microscopy Transmission Scan E.M E.M Bright-field microscopy Most commonly used by both students and pathologists Phase- Contrast Microsco py Does not require staining to Epithelial cell from cheek view the slide Can be used with living, cultures cells Polarizin g Light Microsco py Tissue structures e.g. collagen appear as bright structures against a dark background. Fluorescence Microscopy Uses UV light, under which only fluorescent molecules are visible.  Transmission Electron Microscopy With wavelengths much shorter than those of light, electron beams Allow very-high-resolution images at high magnification, called ultrastructural images. Bronchiole, epithelial cells, X Surface of epithelial cell, X 100,000 5000 Scanning Electron Microscopy Bruce Wetzel/Harry Schaefer, Clarke F. Millette, Univ. of South courtesy National Cancer Institute Carolina, USA  Case Scenario A tissue sample(biopsy) was taken from a patient complaining of a lump in the neck region. The pathologist then fixed the tissue using formaldehyde which step immediately comes after fixation? A. Dehydration B. Clearing C. Staining D. Embedding Thank you