HISTOOOO (PPT: Introduction)

• Methods used in histology

  • Light Microscopy
  • Electron Microscopy

  
• Microscope

  An instrument that magnifies an image and allows visualization of greater detail than is possible with the unaided eye

  
• Types of microscopes


  • Simple
  • Compound
• Resolving power


  The ability of a microscope lens or optical system to produce separate images of closely positioned objects

  
• Parts of a bright-field microscope

  • Light source
  • Condenser lens
  • Stage
  • Objective lens
  • Ocular lens
• Phase contrast microscope


  • Enables examination of unstained cells and tissues, especially useful for living cells
• Modifications of phase contrast microscope

  • Interference microscope
  • Differential interference microscope (using Nomarski optics)
• Interference microscope


  Allows quantification of tissue mass
• Differential interference microscope


  Useful for assessing surface properties of cells and other biologic objects
• Differential interference contrast image

  • Reveals nucleus, cytoplasmic inclusions, and bacteria on cell surface
• Phase contrast image


  • Features pronounced halos around cellular periphery and nucleus
• Dark-field microscope


  Useful in examining autoradiographs and demonstrating specific bacteria
• Fluorescence microscope


  Used to display autofluorescent molecules and detect antigens or antibodies in immunocytochemical staining procedures

  
• Application of fluorescence microscope

  • Detection of rabies antigen in brain tissues
• Confocal scanning microscope


  Allows visualization of a biologic specimen in three dimensions
• Ultraviolet microscope


  Uses quartz lenses and ultraviolet light source, useful in detecting nucleic acids and certain proteins
• Polarizing microscope


  Uses polarized light to examine highly ordered molecules or arrays of molecules
• Types of electron microscopes

  • Transmission electron microscope
  • Scanning electron microscope
• Transmission electron microscope


  Uses the interaction of a beam of electrons with a specimen to produce an image
• Scanning electron microscope


  The electron beam does not pass through the specimen but is scanned across its surface
• Electron micrographs


  • Escherichia coli cells internalized by a human mast cell
  • E. coli interacting with the surface of human mast cell
• Histopathologic techniques


  • Fixation
  • Dehydration
  • Clearing
  • Infiltration
  • Embedding
  • Trimming
  • Section-Cutting
  • Staining
  • Mounting
  • Labeling
• Fixation


  Small pieces of tissues are immersed in a fixative solution to preserve cell and tissue structure
• Dehydration


  Tissue is transferred through a series of increasingly concentrated alcohol solutions to remove all water
• Clearing


  Alcohol is removed from the tissue by immersing in a clearing agent
• Infiltration


  Tissue is placed in melted paraffin until it becomes completely infiltrated
• Embedding


  The paraffin-infiltrated tissue is placed in a mold with melted paraffin and allowed to harden
• Trimming


  The resulting paraffin block is trimmed to expose the tissue for sectioning
• Section-Cutting


  Tissue block is sliced into thin films using a microtome and placed on glass slides
• Staining


  Methods of staining make various cell components conspicuous and permit distinctions between them
• Mounting


  Stained tissue slides are mounted with a cover slip using a mounting media
• Labeling


  Tissue slides are labeled on the frosted areas with assigned tissue numbers or codes
• Autoradiography


  A method of localizing newly synthesized macromolecules in cells or tissue sections using a photographic emulsion and radioactive material
• Autoradiographs


  • Showing location of newly synthesized glycoproteins containing fucose in mouse salivary gland
• Cell and tissue culture


  Allows the direct observation of the behavior of living cells under a phase contrast microscope
• Medical applications of cell culture

  Study of metabolism of normal and cancerous cells, development of new drugs, study of microorganisms that grow only within cells
• Enzyme digestion


  Can be used to confirm the identity of stained material such as glycogen, DNA, or RNA
• Cell and Tissue Culture

  Cells can be grown from newly explanted tissues or as long established lines and can be examined in the living state by phase-contrast light microscopy
• Cell and Tissue Culture


  • It allows the direct observation of the behavior of living cells under a phase contrast microscope
• Medical Application of Cell Culture

  It has been widely used for the study of the metabolism of normal and cancerous cells and for the development of new drugs