methods of studying cells

• what are the 3 different microscopes

  - optical microscope

  - transmission electron microscope

  - scanning electron microscope
• what are the 4 principles of light/optical microscopes

  1. use visible light to view specimens

  
  

  2. use glass lenses to focus light + magnify images

  
  

  3. specimen is mounted onto the glass slide in water, oil or wax

  
  

  4. light passes through the specimen + viewed by the eye
• what are the 2 different types of electron microscopes

  - transmission electron microscopes (TEM)

  - scanning electron microscopes (SEM)
• 3 principles of electron microscopes

  1. use beams of electrons to view specimens

  
  

  2. use electromagnetic lens to focus electron beam

  = electrons cant pass through glass

  
  

  3. specimen must be in a vacuum to allow electrons to travel without being deflected by air molecules
• transmission electron microscope


  - electrons pass through specimen + hit fluorescent screen/detector

  = our eye cant detect/"see" electrons

  
  

  - denser areas of the specimen appear darker on the image

  = fewer electrons pass through as more are absorbed
• scanning electron microscope


  - electrons bounce off the surface of the specimen

  
  

  - electrons scatter + are collected by a detector, forming a 3 dimensional image on the screen
• 5 advantages of optical/light microscopes

  1. image in colour

  = can see true colour of specimens

  = eg. RBCs

  
  

  2. can view living specimens

  
  

  3. easier to use than electron microscope

  
  

  4. simple, quicker preparation of specimens for viewing

  
  

  5. cheaper
• 3 disadvantages of light/optical microscope

  1. lower resolution (less visible detail) compared with TEM

  
  

  2. lower effective magnification

  
  

  3. cant see ultrastructure of cells
• 4 advantages of electron microscopes

  1. higher resolution of images

  = can see more detail than optical microscope

  
  

  2. higher effective magnification

  
  

  3. can see ultrastructure of cells

  = ie. at the nanometre scale

  = eg. ribosomes, ER

  
  

  4. SEM provides 3D images of the surface of specimens
• 5 disadvantages of electron microscopes

  1. more complex + time consuming preparation of specimens

  
  

  2. cannot view living specimens

  = dead only due to vacuum

  
  

  3. only black + white images produced

  = cant see true colour of specimens

  = false colour can be added using computers

  
  

  4. more difficult to use

  
  

  5. expensive
• what is an eye piece gradual (EPG)

  a glass disc inside the eye piece lens

  = used to measure objects

  
  

  BUT - EPG must be calibrated agains a known scale

  = stage micrometer
• how to work out magnification

  Image size/actual size

  
• how to turn mm into μm

  x1000
• how to turn μm into nm

  x 1000
• how to turn nm into μm


  divide by 1000
• how to turn μm into mm


  divide by 1000
• how to turn cm into mm

  x 10
• what is cell fractionation

  used to extract organelles from cells for study
• what are 3 important conditions/characteristics of cell fractionation

  samples are kept:

  
  

  - very cool

  = reduces rate of hydrolytic enzyme activity

  = metabolism is slowed + self digestion is prevented

  
  

  - buffered solution

  = maintain a constant PH

  = prevents denaturing of protein

  
  

  - isotonic solution

  = organelles do not change volume

  = prevents organelles from bursting due to osmosis
• what is an isotonic solution

  same concentration inside + outside of the organelle
• what happens before centrifugation (stages 1 + 2)

  - sample is chilled over ice + cut into small pieces in cold, buffered, isotonic solution

  
  

  - sample is equalised thoroughly before centrifugation

  = cell organelles remain intact

  = blend sample
• stage 3 of cell fractionation

  equalised suspension is filtered to remove cellular debris

  = kept cool throughout
• stage 4 of cell fractionation

  the filtrate is centrifuged at low speed to remove partially opened cells + small pieces of debris
• stage 5 of cell fractionation

  the supernatant containing the organelles is carefully decanted off

  = supernatant is used for the next round of centrifuging

  = spins faster + for longer each time
• what is the supernatant

  a liquid or medium which remains above a pellet after centrifugation and is composed of lighter or smaller materials
• what is the pellet

  Under centrifugal force, denser particles migrate toward the bottom of the tube to eventually form a pellet

  = the lighter particles will remain in the supernatant
• why is it possible to separate cell organelles using centrifugation

  different organelles have different densities
• Why is a sample homogenised before centrifugation?

  to break open cell walls/membranes to release organelles into the solution