Bio

• Tissue preparation


  The process of preparing biological tissues (from biopsy or autopsy) to be observed with light and electron microscopes

  
• Tissue preparation steps


  1. Fixation

  2. Dehydration

  3. Clearing

  4. Infiltration and embedding

  5. Sectioning

  6. Staining
• Fixation


  The process of preserving tissues permanently in as life-like a state as possible, using chemical fixatives like formaldehyde and glutaraldehyde

  
• Dehydration


  The process of removing water from fixed tissues by using a series of alcohols at increasingly higher concentrations, followed by replacement with an intermediate solvent like xylene
• Clearing


  The process of removing the dehydrant (e.g. xylene) with a substance that is miscible with the embedding medium (e.g. paraffin)
• Infiltration and embedding


  The process of replacing the intermediate solvent with the liquid form of the embedding compound (e.g. paraffin wax), which is then allowed to solidify to provide rigidity for sectioning
• Sectioning


  The process of cutting the embedded tissue into thin sections using a microtome, which is a knife with a mechanism for advancing the paraffin block

  
• Microtome


  • A knife with a mechanism for advancing a paraffin block standard distances across it to cut thin sections
  • It is important to have a properly fixed and embedded block to avoid artifacts like tearing, ripping, holes, folding etc.
• Section thickness


  Light microscopy: 1-20 micrometers

  Electron microscopy: 60-100 nanometers
• Section planes


  Cross-section (perpendicular to long axis)

  Longitudinal section (parallel to long axis)

  Oblique section (any other angle)
• Staining


  The process of applying stains to tissue sections to provide contrast and visualize structures, based on charge interactions
• Conventional staining


  Stains that bind to tissue elements based on charge interactions, e.g. hematoxylin binds to negatively charged nucleic acids
• Cover slipping


  The process of covering the stained tissue section on the slide with a thin piece of plastic or glass to protect it, provide better optical quality, and preserve the section

  
• Nanometer


  A unit of length equal to one billionth of a meter, used to measure the thickness of structures observed in electron microscopy
• Electron microscopy


  A type of microscopy that uses a beam of electrons to produce highly magnified images of objects or structures, with a thickness range of 60-100 nanometers
• Light microscopy


  A type of microscopy that uses visible light to produce images of small objects or structures, with a thickness range of 1-20 micrometers
• Micrometer


  A unit of length equal to one millionth of a meter, used to measure the thickness of structures observed in light microscopy