Microscopy

Question 1

What can microscopes be used to do?

Answer 1

• analyse cell components + observe organelles

Question 2

What is the difference between magnification + resolution?

Answer 2

• magnification inc. size of an image
  - shows how much bigger image through microscope is than real life object (object appears larger but just as blurred)
• resolution = ability to distinguish between objects close together

Question 3

What are 2 main types of microscope?

Answer 3

• optical (light) microscope
• electron microscope

Question 4

What are the advantages + disadvantages of optical (light) microscopes?

Answer 4

Disadvantages:
- uses light to form an image
- limits resolution as impossible to resolve objects closer than half the wavelength of light
- can’t observe smaller organelles (e.g. ribosomes, lysosomes) as max. useful magnification = x1500
- only 2D slice can be observed

Advantages:
- specimens can be living or dead
- you can see colour

Question 5

How do electron microscopes work?

Answer 5

• uses electrons to form image
  - inc. resolution = more detailed image as a beam of electrons has a smaller wavelength than light
  - max. resolution of 0.2nm so can observe smaller organelles
  - max. useful magnification = x1,500,000

Question 6

What are the 2 types of electron microscopes?

Answer 6

• transmission EM
• scanning EM

Question 7

How do transmission electron microscopes (TEMs) work?

Answer 7

• uses electromagnets to focus a beam of electrons
• beam is transmitted through specimen
• denser parts of specimen absorb more electrons so appear darker, thinner parts allow electrons to pass through so appear lighter

Question 8

What are the advantages + disadvantages of TEMs?

Answer 8

Disadvantages:
- specimens must be thin
- specimens must be dead as must be in a vacuum so electrons can pass through
- no colour
- artefacts can be introduced due to long treatment required to prepare specimens

Advantages:
- high resolution images (more detail)
- allows internal structures within cells to be seen
- can magnify up to x500,000

Question 9

How do scanning electron microscopes (SEMs) work?

Answer 9

• scans a beam of electrons across specimen
• beam bounces off surface of specimen + are scattered + detected

Question 10

What are the advantages + disadvantages of SEMs?

Answer 10

Disadvantages:
- lower resolution images (less detail)
- can’t observe live specimens
- no colour

Advantages:
- can be used on thick/3D specimens
- allows external, 3D structure to be observed

Question 11

What is the equation for total magnification?

Answer 11

total magnification = eyepiece lens magnification x objective lens magnification

Question 12

What is the equation for magnification?

Answer 12

Magnification = image size / actual size

Question 13

What is the conversion from cm - mm - μm - nm?

Answer 13

• cm - mm = x10
• mm - μm = x1000
• μm - nm = x1000

Question 14

What is cell fractionation?

Answer 14

• process of separating cell organelles from each other

Question 15

What does cell fractionation involve?

Answer 15

• breaking up a sample of tissue + then centrifuging the mixture at diff. speeds

Question 16

What are the 3 stages of cell fractionation?

Answer 16

• homogenisation
• filtration
• ultracentrifugation

Question 17

Describe the 1st stage (homogenisation) of cell fractionation.

Answer 17

• place the sample of tissue in a cold, isotonic buffer solution
  
  • cold to reduce activity of enzymes breaking down organelles
  • isotonic to prevent water moving into organelles via osmosis
  • buffered to prevent organelles proteins + enzymes from becoming denatured
• place tissue-containing solution in a homogeniser to break the plasma membrane of the cells + release the organelles into a solution (homogenate)

Question 18

Describe the 2nd stage (filtration) of cell fractionation.

Answer 18

• homogenate is filtered through a gauze
  
  • to remove unwanted large pieces of debris leaving a solution (filtrate)

Question 19

Describe the 3rd stage (ultracentrifugation) of cell fractionation.

Answer 19

• filtrate is spun at a low speed in a centrifuge to form a pellet (contains largest, heaviest organelles)
• supernatant (solution w rest of organelles) is drained off into another tube + spun at a higher speed forming a pellet w a lighter organelle
• process repeats
• centrifuge force separates organelles into densities