microscopy

• Microscopy


  The technical field of using microscopes to view samples and objects that cannot be seen with the unaided eye

  
• Resolving power


  Capacity for resolving two closely located points
• The resolving power of the human eye is 0.2 mm (200 µm)
• A typical eukaryote cell is commonly between 10 and 50µm in size (1 µm=10-6 mm)
• Cell membranes are about 7nm thick (1 nm = 10 -6 µm)
• Microscopes


  Devices that magnify and visualize very small structures like cells and cell compartments

  
• Types of microscopes


  • Light Microscope
  • Electron Microscope

  
• Light microscopes


  • Use visible light and glass lenses to magnify samples up to 1000 times, with a resolving power of 0.2 µm
  • Common for general observation of tissues and cells

  
• Types of light microscopes

  • Phase contrast microscope
  • Polarizing Microscope
  • Confocal Microscope
  • Fluorescence Microscope

  
• Electron microscopes


  • Based on the very short wavelength of electrons and get a resolution power of 1 nm
  • Used for studying the ultrastructure of cells and tissues, that is, the subcellular level organelles, membranes and molecular complexes

  
• Types of electron microscopes

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

  
• Transmission Electron Microscope (TEM)


  Used to study ultrastructure of tissues in ultrathin sections

  
• Scanning Electron Microscope (SEM)


  Intended for the observation of surfaces

  
• Light microscope


  Essential for histological studies as it allows the visualization of cells and morphological features of tissues

  
• Light microscope


  • Relies on glass lenses and visible light to magnify tissue samples
  • Contains two main lenses: objectives and eyepieces
  • Objective gathers the light that passes through the tissue, whereas the eyepiece projects the tissue image on the eye
  • Total magnification is the result of multiplying the objective magnification by the eyepiece magnification

  
• Magnification and resolution power must not be confused, because not matter how we can magnify an image, including digital methods, the resolution cannot be increased
• More advanced light microscopes can get 1000 to 1500 magnifications (100x objective and 10x or 15x eyepiece)

  
• Phase-contrast microscopy


  Based on the principle that light changes its speed when passing through cellular and extracellular structures with different refractive indices, allowing the examination of cells without fixation or staining

  
• Fluorescence microscopy


  Tissue sections are irradiated with ultraviolet (UV) light and the emission is in the visible portion of the spectrum, allowing fluorescent substances to appear bright on a dark background

  
• Fluorescent compounds


  Compounds with affinity for specific cell macromolecules that can be used as fluorescent stains, e.g. Acridine orange, DAPI
• The limit of resolution of the light microscope is 0.2 µm (greatest magnification is x 1,400)

  
• Electron microscopes


  Use electron beams instead of visible light, allowing several million times magnification and a resolving power as small as 1 nm

  
• Electron microscopes


  Do not have glass lenses, they use magnets that work as magnetic lenses by concentrating the electron beam emitted by an electron gun
• Transmission Electron Microscope (TEM)

  Permits resolution around 3 nm and allows isolated particles magnified as much as 400,000 times to be viewed in detail

  
• Scanning Electron Microscope (SEM)


  Visualizes sample surfaces by scanning the specimen with a narrow beam of electrons and capturing the reflected electrons to produce a black-and-white image
• Electron microscope images are always black, white, and shades of gray

  
• Differences between light and electron microscopes

  • Light microscopes use visible light and glass lenses, electron microscopes use electron beams and magnetic lenses
  • Light microscopes have a resolution limit of 0.2 µm, electron microscopes can reach 1 nm resolution
  • Light microscopes magnify up to 1000x, electron microscopes can magnify several million times