HISTOOOO (TRANS: Introduction)

• Microscopy


  • Light Microscopy
  • Electron Microscopy
• Light Microscopy


  • Bright-field Microscopy
  • Phase-contrast Microscopy
  • Dark-field Microscopy
  • Fluorescence Microscopy
  • Confocal Microscopy
  • Ultraviolet Microscope
  • Polarizing Microscopy

  
• Bright-field Microscope


  • Most used microscope of students and researchers
  • Has light source, condenser lens, stage, objective lens, ocular lens
• Resolving power


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

  
• Resolution


  Ability of the microscope to distinguish details. To detect 2 objects as different objects.

  
• Magnification


  Ability of microscope to see small objects seem larger

  
• Human Eye > Bright-field microscope> SEM> TEM (Theoretical) > TEM (Tissue Section) > Atomic force Microscopy in terms of distance between resolvable points

  
• Micrometer


  1 x 10 -1000 in M
• Nanometer


  1 x 10 – 9000 in M
• Phase Contrast Microscope


  • Enables examination of unstained cells and tissues, especially useful for living cells
  • Interference microscope allows quantification of tissue mass
  • Differential interference microscope useful for assessing surface properties of cells and other biologic objects

  
• Dark-field Microscope


  • Object will appear bright in a dark background
  • Useful in examining autoradiographs, urine for crystals, and demonstrating specific bacteria
  • Beneficial in Microbiology to appreciate shape and movement of bacteria

  
• Fluorescence Microscope


  • Used to display autofluorescent molecules (Fluorochromes)
  • Widespread application in detection of antigens or antibodies in immunocytochemical staining procedures
  • Polyclonal antibodies are tolerant to antigen epitope change but prone to cross-reactivity
  • Monoclonal antibodies are more specific with minimal cross-reactivity

  
• Direct Fluorescence Assay


  Using one antibody to detect antigen
• Indirect Fluorescence Assay


  Using two antibodies to detect antigen
• Confocal Scanning Microscope


  • Allows visualization of a biologic specimen in three dimensions (3D configuration)
• Ultraviolet Microscope


  • Uses quartz lenses with an ultraviolet light source
  • Image depends on the absorption of UV light by molecules in the specimen
  • Useful in detecting nucleic acids and certain proteins
• Polarizing Microscope


  • Uses the fact that highly ordered molecules or arrays of molecules can rotate the angle of the plane of polarized light
  • Has additional filters compared to Light Microscope
• Electron Microscopes


  • Transmission Electron Microscope (TEM)
  • Scanning Electron Microscope (SEM)
• Transmission Electron Microscope (TEM)


  • Uses the interaction of a beam of electrons with a specimen to produce an image
  • Enables observation of structures found in the inner portion of the organism, its organelles
• Scanning Electron Microscope (SEM)


  • The electron beam does not pass through the specimen but is scanned across its surface
  • Allows appreciation of the specimen's appendages

  
• Steps in tissue preparation

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


  To preserve cell and tissue structure in a life like manner
• Dehydration


  Remove intracellular and extracellular water to prepare tissue for subsequent steps
• Clearing


  Removal of alcohol from the tissue
• Infiltration


  Filling the spaces between tissue to make the tissue firm to facilitate cutting
• Embedding


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


  Trim the edges to create a perfect block to fit in the microtome and expose the tissue
• Microtome cuts the tissue block into thinner sections, Rotary Microtome is the most common
• Clearing agent


  Alcohol is removed in the tissue by immersing in a clearing agent
• Clearing solutions


  Makes tissues clear
• Infiltration


  Also known as Impregnation, filling the spaces between tissue to make the tissue firm to facilitate cutting
• Paraffin wax


  Most commonly used for impregnation
• Infiltration


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


  Also known as casting, the paraffin-infiltrated tissue is placed in a mold with melted paraffin and allowed to harden
• The medium used in infiltration of tissue is the same medium used for embedding
• Tissue block


  Contains the paraffin infiltrated tissue and the paraffin mold
• Trimming


  Trim the edges to create a perfect block to fit in the microtome, expose the tissue
• Microtome


  Cuts the tissue block into thinner sections
• Rotary Microtome


  Most common, invented by Minot
• Section-cutting


  Cut the tissue block into thin films, tissue ribbon, straighten in water bath, remove excess paraffin in oven