microscopy + cell fractionation and ultracentrifugation (topic 2)

Question 1

resolution

Answer 1

the ability to distinguish between 2 points - if you can’t do this you see 2 points as 1 point

Question 2

magnification

Answer 2

tells you how many times bigger the microscope image is than the real life object

increasing the magnification increases the size of the image but not the resolution

size of image
——————
actual size

Question 3

two main types of microscopes

Answer 3

light microscope

electron microscope
- transmission electron microscope (TEM)
- scanning electron microscope (SEM)

Question 4

light microscope

Answer 4

• specimens are illuminated with light
• the light is focussed using glass lenses and viewed using the eye. all LMs today are compound microscopes, so they use several lenses to obtain high magnification.
• poor resolution as a result of the relatively long wavelength of light. the resolution of a LM is 200nm, so objects closer than that will only be seen as one point.
• specimens can be living or dead, but often need to be stained with a coloured dye to make them visible.

Question 5

transmission electron microscope (TEM)

Answer 5

• uses an electron beam which has a much shorter wavelength, so a higher resolution
• beam of electrons fired by electron gun and focused using electromagnets
• whole system must be in a vacuum (else air will deflect electrons as they’re so small)
• vacuum means living specimens cannot be observed, cells are first killed and chemically ‘fixed’ in a complicated and harsh treatment
• electron beam passes through thin section of the specimen. parts of it absorb electrons and appear dark, other parts allow electrons to pass through and appear bright.
• specimen must be really thin.
• image produced on a screen which can be photographed to give a photomicrograph
• flat 2-D image
• image in black and white
• can resolve 0.1nm

Question 6

scanning electron microscope (SEM)

Answer 6

• SEM directs a beam of electrons onto the surface of the specimen and the beam is passed back and forth across the specimen. electrons are scattered and detected.
• limitations of TEM apply to SEM except that specimen doesn’t need to be really thin.
• 3D image can be generated. allows detailed study of surfaces.
• resolving power 20nm (lower than TEM)

Question 7

conversions - mm to μm

Answer 7

x 1000

Question 8

how to measure size in microscopes

Answer 8

eyepiece graticule - (a ruler looking thing) but must be calibrated (not a specific measurement)

Question 9

cell fractionation use

Answer 9

used to isolate different organelles of cells, cells are broken up so that organelles are separated out

Question 10

cell fractionation method

Answer 10

1 - before - tissue is cut up and kept in a COLD (slow down enzyme activity so no organelles are broken down), ISOTONIC (prevent organelles shrinking/bursting due to osmotic gain/loss of water) BUFFERED (to maintain constant pH to prevent denaturing) solution

2 - homogenation - cells broken up by a homogeniser (blender). releases organelles from cell membrane. the resultant fluid is known as the homogenate. filter to remove large pieces of debris, then called filtrate.

3 - ultracentrifugation - spin filtrate in tubes at very high speed to separate the organelles. centrifugal force separates organelles into densities.
(dense organelle forms a pellet at the bottom. the remaining fluid is called the supernatant

Question 11

cell fractionation result

Answer 11

densest organelle (nuclei) forced to bottom of the tube, forming a PELLET
fluid at the top called the SUPERNATANT

• supernatant transferred to another tube and spun in the ultracentrifuge at faster speed than before
  next densest organelle (mitochondria) is forced to bottom of the tube