Histology

• Histology, also known as microscopic anatomy, is the study of cells and tissues

  
• Histology


  • Traditionally done with light microscopy
  • Main objective is for veterinary students to learn how the four basic kinds of tissues (epithelial tissues, connective tissue and mesenchymal cells, muscle and nervous tissues) are assembled into the different organs and organ systems of the body
  • Students will be asked to learn a large number of new vocabulary words and to develop the "microscopic eye" for details that help them tell cells and structures apart
  • While the module will emphasize on identification of normal cells and tissues as they appear in a histologic section examined using a light microscope, when appropriate, cytology preparations, transmission and scanning electron microscopy images will also be employed
  • Although the emphasis will be on normal cells and tissues, the opportunity to see cells and tissues affected by disease will benefit students' understanding of normal structure and function
  • This module will serve as a "teaser" to General Pathology which follows this module and emphasizes the pathophysiology of disease through the microscopic study of diseased tissue

  
• Components of general histology

  • Histology study methods
  • Epithelia
  • Connective tissue
  • Supporting connective tissues (cartilage, bone)
  • Blood
  • Nerve tissue
  • Muscle tissue
• Tissues


  Made of two interacting components: cells and extracellular matrix
• Extracellular matrix provides mechanical support for the cells, transports nutrients to the cells, and carries away catabolites and secretory products
• Cells and extracellular matrix form a continuum that functions together and reacts to stimuli and inhibitors together
• Most organs are formed by an orderly combination of several tissues, except the central nervous system, which is formed almost solely by nervous tissue
• Fixation


  Process to avoid tissue digestion by enzymes present within the cells (autolysis) or by bacteria and to preserve the structure and molecular composition
• Chemical fixation


  Tissues are immersed in solutions of stabilizing or cross-linking agents called fixatives
• Formalin


  One of the best fixatives for routine light microscopy, a buffered isotonic solution of 37% formaldehyde
• Glutaraldehyde


  Another widely used fixative, reacts with the amine groups (NH2) of tissue proteins and can cross-link proteins
• Double fixation procedure


  Using a buffered glutaraldehyde solution followed by a second fixation in buffered osmium tetroxide, a standard procedure in preparations for fine structural studies
• Embedding


  Tissues are infiltrated with embedding substances that impart a rigid consistency to the tissue, such as paraffin and plastic resins
• Paraffin embedding


  1. Dehydration

  2. Clearing

  3. Impregnation with paraffin
• Plastic embedding


  Dehydration in ethanol, infiltration with plastic solvents, replacement by plastic solutions that are hardened by means of cross-linking polymerizers
• Microtome


  Instrument used to slice the hard blocks containing the tissues into sections 1 to 10 µm thick

  
• Freeze-etching technique


  Cells are rapidly frozen in liquid nitrogen, fractured, and the exposed surfaces are shadowed and coated with layers of platinum and carbon to form a replica of the surface
• Staining


  Methods of staining tissues that make the various tissue components visible and permit distinctions to be made between them

  
• Basophilic


  Tissue components with a net negative charge that stain more readily with basic dyes
• Acidophilic


  Cationic tissue components, such as proteins with many ionized amino groups, that have affinity for acidic dyes
• Hematoxylin and eosin (H&E)


  The most commonly used dye combination, where hematoxylin stains DNA of the cell nucleus and other acidic structures blue, and eosin stains other cytoplasmic components and collagen pink
• Microscope resolution


  The ability of a lens to separate or distinguish between small objects that are close together, the maximal resolving power of the light microscope is approximately 0.2 µm

  
• Video cameras and image analysis software

  Enhance the power of the light microscopes, allow the capture of digitized images suitable for computerized image analysis and printing, and enable studying living cells for long periods of time
• Bright-field microscopy


  The ordinary microscope, where the specimen is illuminated from below and observed in the light that passes through it

  
• Resolution


  The ability to distinguish two objects as separate when they are close together
• Objects less than 0.2 µm apart will be seen as only one object
• Image quality


  Clarity and richness of detail, depends on the microscope's resolving power

  
• Video cameras


  • Highly sensitive to light
  • Enhance the power of bright-field and other light microscopes
  • Allow capture of digitized images suitable for computerized image analysis and printing
• The frontiers of light microscopy have been redefined by the use of video cameras and digital image enhancement
• Bright-field microscopy


  The ordinary microscope that forms a dark image against a brighter background

  
• Bright-field microscope operation


  1. Condenser collects and focuses light, producing a cone of light that illuminates the object

  2. Objective lenses enlarge and project the illuminated image of the object

  3. Eyepiece or ocular lens further magnifies the image and projects it onto the viewer's retina, photographic film, or a detector like a CCD camera
• Total magnification


  Obtained by multiplying the magnifying power of the objective and ocular lenses
• Electron microscope


  Uses an electron beam to create an image, with electromagnets acting as lenses
• TEM and SEM are based on the interaction of electrons and tissue components
• Electron wavelength


  Much shorter than light, allowing a thousand-fold increase in resolution
• Transmission electron microscopy (TEM)

  • Electron beam is produced by a cathode and passes down through the chamber in a vacuum
  • Electrons can be focused by passing through electric coils which act as electromagnetic lenses

  
• TEM image


  Areas where electrons passed appear bright (electron lucent), areas that absorb or deflect electrons appear dark (electron dense)
• TEM specimen preparation


  1. Fixed to prevent decomposition

  2. Stained with electron dense material

  3. Dehydrated

  4. Embedded in plastic

  5. Cut into thin slices using an ultramicrotome

  
• TEM requires very thin sections (40-90 nm) to allow useful interaction between the specimen and the electrons
• Negative staining


  Specimen is spread out in a thin film with heavy metals that do not penetrate but render the background dark, making the specimen appear bright