Microscopes

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Magnification and Resolution

Magnification --> how much bigger the image appears than the actual object/thing itself

Resolution --> the ability to distinguish two separate points on an image 

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Light Microscopes

  • 2 lenses: objective lens which is near the specimen and magnifies the image and the eyepiece lens where the specimen is viewed and magnified again 
  • uses light (illumination - light underneath)
  • certain wavelengths are filtered to produce an image
  • natural colour of sample seen 
  • maximum magnification = x 2000
  • maximum resolution = 200nm (limiting factor)
  • ^ lower than electron microscopes 
  • specimens can be living or dead
  • inexpensive to buy and operate, small and portable, simple sample preparation, no vacuum required
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Electron Microscopy (General)

  • beam of electrons
  • wavelength less than 1nm used to illuminate the specimen 
  • more detail, magnification high and still a clear resolution  
  • very expensive to buy and operate 
  • can only be used inside a carefully controlled environment in a dedicated space (large and needs to be installed)
  • specimens can be damaged by the electron beam
  • vacuum required 
  • black and white images (can be coloured digitally)
  • complex preparation process- problem with artefacts (structures produced due to preparation processes, however can be eliminated as techniques improve) 
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Transmission Electron Microscope (TEM)

  • uses electromagnets to focus a beam of electrons which is then transmitted through a specimen to produce an image 
  • denser parts of the specimen absorb more electrons and therefore appear darker
  • 2D image of a thin sample
  • maximum magnification = x 500,000
  • best resolution, power = 0.5nm 
  • produces black and white images 
  • specimens are dead 
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Scanning Electron Microscope (SEM)

  • beam of electrons sent across the surface of a specimen and reflected electrons are collected which are gathered in a cathode ray tube to form an image
  • produces 3D images which gives valuable information about the appearance of different organisms
  • maximum magnification = x 500,000
  • maximum resolution power = 3-10 nm (not as good as TEM)
  • dead specimens
  • produce black and white images 
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Laser Scanning Confocal Microscopy

  • fluorescent miscroscopes- higher light intensity used to illuminate a specimen treated with a fluorescent chemical (absorption and re-radiation of light)
  • moves a single spot of focused light across a specimen (point illumination) this causes flourescence from components labelled with 'dye'
  • emitted light from a specimen is filtered through a pinhole arpeture (confocal because positions of 2 pinholes- light waves and laser take same pathway)
  • only light radiated from very close to the focal plane (sharpest focus) is detected
  • Laser instead of light- higher intensities and improves illumination 
  • very high resolution images can be obtained
  • 2D but 3D images can be produced- images, different focal planes
  • used with eyes- non-invasive 
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