Methods of studying cells

• 3 types of microscopes


  - light/optical microscope

  - transmission electron microscope (TEM)

  - scanning electron microscope (SEM)
• Magnification


  - How many times larger the image is compared to the object
• Magnification equation

  magnification = image size/actual size

  
• Resolution


  - The minimum distance between two objects in which they can still be viewed as separate

  - determined by wavelength of light (for optical microscopes) or electrons (for electron microcopes)
• Optical microscope


  - Beam of light used to create image

  - glass lens used for focusing

  - 2D coloured image produced
• Evaluate optical microscopes


  - Poorer resolution as long wavelength of light - small organelles not visible

  - lower magnification

  - can view living samples

  - simple staining method

  - vaccum not required
• Transmission electron microscope (TEM)


  - Beam of electrons passes through the sample used to create an image

  - focused using electromagnets

  - 2D, black & white image produced

  - can see internal ultrastructure of cell

  - structures absorb electrons and appear dark
• Evaluate TEMs


  - Highest resolving power

  - high magnification

  - extremely thin specimens required

  - complex staining method

  - specimen must be dead

  - vaccum required
• Scanning Electron Microscope (SEM)


  - Beam of electrons pass across sample used to create image

  - focused using electromagnets

  - 3D, black and white image produced

  - electrons scattered across specimen producing image
• Evaluate SEMs


  - High resolving power

  - high magnification

  - thick specimens usable

  - complex staining method specimen

  - must be dead

  - vaccum required
• Why calibrate eyepiece graticule?


  - Calibration of the eyepiece is required each time the objective lens is changed

  - calibrate to work out the distance between each division at that magnification
• Purpose of cell fractionation


  - Break open cells & remove cell debris

  - so organelles can be studied
• Homogenisation


  - Process by which cells are broken open so organelles are free to be separated

  - done using homogeniser (blender)
• Homogenisation conditions


  - Cold - reduces enzyme activity preventing organelle digestion

  - Isotonic - prevents movement of water by osmosis - no bursting / shrivelling of organelles - Buffered - resists pH changes preventing organelle + enzyme damage
• Ultra-centrifugation


  - Homogenate solution filtered to remove cell debris

  - solution placed in a centrifuge which spins at a low speed initially

  - then increasingly faster speeds to separate organelles according to their density
• Differential centrifugation


  - Supernatant first out (spun at lowest speed) is most dense = nuclei

  - spun at higher speeds

  - chloroplasts -> mitochondria -> lysosomes -> RER/SER -> ribosomes (least dense)