2.1.3 Methods of studying cells

Question 1

Describe the difference between magnification and resolution

Answer 1

● Magnification = number of times greater image is than size of the real (actual) object
○ Magnification = size of image / size of real object
● Resolution = minimum distance apart 2 objects can be to be distinguished as separate objects

Question 2

Compare the principles and limitations of optical microscopes,
transmission electron microscopes and scanning electron microscopes

Answer 2

Optical microscope

• Light focused using
  glass lenses
• Light passes through specimen,
  different structures absorb
  different amounts & wavelengths
• Generates a 2D image of a
  cross-section
• Low resolution due to long
  wavelength of light
• Can’t see internal structure of
  organelles or ribosomes
• Specimen = thin
• Low magnification (x 1500)
• Can view living organisms
• Simple preparation
• Can show colour

Transmission
electron microscope (TEM)

• Electrons focused using
  electromagnets
• Electrons pass through specimen,
  denser parts absorb more and
  appear darker
• Generates a 2D image of a
  cross-section
• Very high resolution due to short
  wavelength of electrons
• Can see internal structures of
  organelles and ribosomes

-Specimen = very thin

• High magnification (x 1,000,000)
• Can only view dead
  specimens as uses a vacuum
• Complex preparation so
  artefacts often present
• Does not show colour

Scanning
electron microscope (SEM)

• Electrons focused using
  electromagnets
• Electrons deflected / bounce
  off specimen surface
• Generates a 3D image
  of surface
• High resolution due to short
  wavelength of electrons
• Can’t see internal structures
• Specimen does not need to be thin
• High magnification (x 1,000,000)
• Can only view dead
  specimens as uses a vacuum
• Complex preparation so
  artefacts often present
• Does not show colour

Question 3

Suggest how the scientific community distinguished between artefacts (eg.
dust, air bubbles occurring during preparation) and cell organelles

Answer 3

● Scientists prepared specimens in different ways
● If an object was seen with one technique but not another, it was more likely to be an artefact than an
organelle

Question 4

List the steps in calculations involving magnification, real size & image size

Answer 4

1 Note formula / rearrange if necessary (I = AM)

2 Convert units if necessary - image and actual size
must be in same unit

3 Calculate answer and check units required or if
standard form etc. is required

Question 5

Describe how to convert between different units

Answer 5

Centimetre (cm) 1/100 m + 10
-2 m
Millimetre (mm) 1/1000 m + 10
-3 m
Micrometre (μm) 1/1000000 m +10
-6 m
Nanometre (nm) 1/1000000000 m + 10
-9 m

Question 6

convert

Answer 6

m
x1000 / 1000
mm
x1000 /1000
μm
x1000 /1000
nm
x1000 /1000

Question 7

Describe how the size of an object viewed with an optical microscope can be
measured

Answer 7

1 ) line eyepiece graticule with stage micrometre.
2 ) Calibrate graticule using micrometre.
3) Measure object with graticule.
4) Calculate object size.
5) Recalibrate at different magnifications.

Question 8

Eg. using the stage micrometre to calculate the size of divisions on the eyepiece graticule (step 2):

Answer 8

● 4 eyepiece graticule divisions
= 10 stage micrometre divisions
● In this stage micrometre, 1 subdivision
= 10 μm
● So 4 eyepiece graticule divisions
= 10 μm x 10 = 100 μm
● So 1 eyepiece graticule division
= 100 μm/4 = 25 μm

Question 9

Describe and explain the principles of cell fractionation and
ultracentrifugation as used to separate cell components

Answer 9

1. Homogenise tissue /
   use a blender

● Disrupts the cell membrane, breaking open cells to release
contents / organelles

2. Place in a cold,
   isotonic, buffered
   solution

● Cold to reduce enzyme activity
○ So organelles not broken down / damaged
● Isotonic so water doesn’t move in or out of organelles by osmosis
○ So they don’t burst
● Buffered to keep pH constant
○ So enzymes don’t denature

3. Filter homogenate

● Remove large, unwanted debris eg. whole cells, connective tissue

4. Ultracentrifugation -
   separates organelles
   in order of density /
   mass

● Centrifuge homogenate in a tube at a low speed
● Remove pellet of heaviest organelle and respin supernatant
at a higher speed
● Repeat at increasing speeds until separated out, each time the
pellet is made of lighter organelles (nuclei → chloroplasts /
mitochondria → lysosomes → ER → ribosomes)