Histology 1-1-10 PDF

1 Block 102-PMS Principles of microscopic and macroscopic structures Histology  2 Contents Histology and Cytology techniques 1. Paraffin technique 2. Types of microscopes 3. Cell & tissue cultur...

1 Block 102-PMS Principles of microscopic and macroscopic structures Histology  2 Contents Histology and Cytology techniques 1. Paraffin technique 2. Types of microscopes 3. Cell & tissue culture: The cell 1. Membranous organelles 2. Non membranous organelles 3. Inclusions 4. Nucleus 5. Cell cycle and Cell death Tissues of the body 1. Epithelial tissues 2. Connective tissues 3. Cartilage 4. Bone  3 Histology and Cytology Definition of Histology: “Histo” comes from Greek which means “tissue” “Ology” comes from Greek which means “branch of knowledge” Definition of Cytology: “Cyto” comes from Greek which means “cell” Histology requires the use of “Microscopes” to view the structures under increasing magnifications. Because most tissues and organs are too thick for light to pass through, thin translucent sections are cut from them and placed on glass slides for microscopic examination of the internal structures. For tissue preparation for light microscope, the paraffin technique is the most commonly used technique. Paraffin technique Steps as follow: 1. Fixation using solution of formaldehyde (formalin). Aims of fixation:  Prevent autolysis by stabilizing lysosomes of the cells preserving tissue structure.  Fixation kills bacteria thus preventing putrefaction.  Hardens the tissue to facilitate cutting. 2. Washing in running tap water. 3. Dehydration: removing water by passing the tissue through increasing concentrations of ethyl alcohol (from 0 to 100%), this occurs gradually to prevent sudden tissue shrinkage. 4. Clearing: the alcohol is replaced with xylene, which is a paraffin solvent. Xylene also makes the tissue transparent. 5. Impregnation and Embedding: using hot oven, the tissue is placed in warm paraffin wax (55-60 C) to fill the spaces between cells. 6. Sectioning: serial thin sections (5-7 µm thickness) are cut using rotatory microtome and then mounted on a slide. 7. Staining: unfortunately, most staining solutions are aqueous, so to stain the sections, the wax has to be dissolved and replaced with water (rehydration). The sections are passed through xylene, and then decreasing strengths of alcohol (100% to and finally Rotatory microtome  4 water). Once stained, the section is then dehydrated once again, and placed in xylene and a coverslip is placed to protect the sample. Hematoxylin and eosin (H&E) Both are the universally used stains for routine histological examination of tissue sections. Hematoxylin Eosin Basic dye that stains acidic components Acidic dye that stains the cell cytoplasm of the cell (nucleus) giving blue color giving reddish pink color (acidophilia) (basophilia) A B A Section of scalp fixed, embedded in paraffin, and mounted on a slide without being stained. B, Nearby section from the same block stained with H&E Clinical applications: Biopsies are tissue samples removed during surgery or routine medical procedures then processed for microscopic analysis in a pathology laboratory. N.B: sometimes intra-operative consultation and immediate pathological reports (especially in tumor surgery) might be needed for rapid decision (takes only minutes)  5 during the surgery, so a much more rapid processing method (freezing technique) is used. In freezing technique, biopsy is rapidly frozen in liquid nitrogen then sectioned using a cryostat. Types of microscopes Light microscopes:  Bright-Field microscope: It is the most common and widely used microscope in histology. Resolution and magnification  6 Resolving power Magnification power  The ability of microscope to show two  The ability of microscope to magnify points very close to each other as two objects. separate points.  Magnification power = Power of ocular x objective lens For light microscope is 0.2 µm. For light microscope: nearly 1500-2000 For electron microscope is 0.2 nm For electron microscope: nearly 500000 White blood cell from the peripheral circulation (1600×). This micrograph illustrates the limit of resolution of the light microscope.  Fluorescence microscope: tissues are stained using a fluorescent dye then examined with the immunofluorescence microscope in the dark.  Phase-Contrast Microscope: this microscopy is a technique most commonly used to examine living, unstained cells growing in laboratory tissue culture plates.  7 A Phase-contrast image of an epithelial cell from a rat kangaroo grown in tissue culture. B, The same cell as seen in an immunofluorescence microscope after the cell was stained with an antibody to the intermediate filament protein vimentin. Electron microscopes: Transmission and scanning electron microscopes are based on the interaction of tissue components with beams of electrons. The wavelength in an electron beam is much shorter than that of light, allowing a 1000-fold increase in resolution.  8 Comparison between light and electron microscopes Light microscope (compound) Electron microscope Small Large and non-portable Relatively inexpensive Expensive Does not need a lot of training Training is required Colored image Black and white image Specimen can be alive and unharmed Specimen must be dead Lower resolving power Greater resolving power Lower magnification Greater magnification Skeletal muscle by light microscopy Skeletal muscle by electron microscopy  9 Cell & tissue culture:  Cell culture refers to the removal of cells from an animal and their subsequent growth in a favorable artificial environment (in vitro).  Cell culture allows the direct observation of cellular behavior under a phase-contrast microscope.  It is essential for many experiments which are technically impossible to be performed in intact animals. Medical application:  Cell biology: cell culture supplies biologists with different cells for different experiments.  Karyotyping (will be discussed later).  Cancer research for novel chemotherapeutic drug screening or interactions between cancer cells and the immune system.  Virology for virological studies and vaccine production which requires the culture of infected cells to obtain virus proteins.  Regenerative medicine through using of cultured stem cells, tissues or organ to replace damaged cells and tissues in patients suffering from organ failure.  Manufacturing of biopharmaceuticals as the production of antibiotics or therapeutic hormone formulations (e g. to treat growth hormone deficiency in children).  10 Enzyme histochemistry (cytochemistry): it is a method for localizing cellular structures using a specific enzymatic activity present in those structures. To preserve the endogenous enzymes, histochemical procedures usually use unfixed or mildly fixed tissue, which is sectioned on a cryostat to avoid adverse effects of heat and organic solvents which are commonly used in paraffin technique. Immunohistochemistry: labeled antibodies are routinely used in immunohistochemistry to identify and localize many specific proteins, not just those with enzymatic activity that can be demonstrated by histochemistry. For studying