Microscopes

Microscopes

1. Light microscopes

   • How it works
     • Light passes from bulb under stage -> condenser lens -> specimen
     • Beam of light focused through objective lens -> then eyepiece lens
       • Eyepiece lens magnifies image again - x10
     • Different no. of objective lenses
       • x4 , x10 , x40 , x100 (oil immersion lens)
     • Total mag. = objective mag. multiplied by the eyepiece mag.
   • Advantages and disadvantages
     • Maximum mag. = x1500
     • Maximum res. = 200 nm
       • Not high
     • Wide range of specimens can be viewed
       • Living organisms
       • Thin sections of larger organisms
       • Smear preps of blood or cheek cells
   • Preparation of specimens
     • Staining
       • Coloured stains are chemicals that bind to chemicals in specimens
       • Allows it to be seen
     • Sectioning
       • Specimen embedded in wax
       • Thin sections cut without distorting structure
       • Useful for soft tissue

2. Cell size and magnification

   • Graticule
     • Transparent with small ruler
     • Super imposed onto specimen
     • Dimensions of specimen can be measured
     • Eyepiece lens has to be calibrated for each objective lens
   • Actual size = image size / magnification

3. Electron microscopes

   • How it works
     • Generate beam of electrons
       • Wavelength = 0.004 nm
     • Resolution = 0.2 nm
       • 500,000 times greater than human eye
     • Magnets focus beam of electrons
     • Image projected onto screen or photographic paper
       • Image = black and white
       • Electron micrographs
   • Transmission Electron TEM
     • Electron beam passes through very thin sample
     • Electrons pass through denser parts less easily - contrast given
     • 2D image
     • Mag = x500,000
   • Scanning Electron SEM
     • Electron beam directed onto sample
       • Not through specimen
     • Electrons 'bounced off' sample
     • 3D view
     • Mag = x100,000
   • Advantages and Limitations
     • Samples have to be in vacuum
     • Produce detailed images of organelles
     • SEM = 3D images
     • Expensive
     • Preparing and using needs high degree of skill and training