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 5 principles and limitations of optical, transmission election and scanning electron microscopes

Answer 2

Optical:
1. Generates a 2D image of cross-section
2. Light passes through specimen, different structures absorb different amount & wavelengths
3. Low resolution due to long wavelength of light
4. Can’t see internal structure of organelles or ribosomes
5. Specimen = thin
TEM:
1. Genrâtes a 2D image of a cross-section
2. Electrons pass through specimen, denser parts absorb more and appear darker
3. Very high resolution due to short wavelength of electrons
4. Can see internal structures of organelles and ribosomes
5. Specimen = very thin
SEM:
1. Generates a 3D image of surface
2. Electrons deflected / bounce off specimen surface
3. High resolution due to short wavelength of electrons
4. Can’t see internal structures
5. Specimen does not need to be thin

Question 3

Equation for magnification + variations

Answer 3

Magnification = image / real
Image = mag x real
Real = image / mag

Question 4

Describe how to convert between different units

Answer 4

Cm -> mm = x10
Mm -> um = x1000
Um -> nm = x1000

Nm -> um = /1000
Um -> mm = /1000
Mm -> cm = /10

Question 5

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

Answer 5

1. Line up scale of eyepiece graticule with scale of stage micrometre
2. Calibrate eyepiece graticule
3. Take micrometre away and use graticule to measure how many divisions make up the object
4. Calculate size of object by multiplying number of divisions by egu
5. Recalibrate for different magnifications

Question 6

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

Answer 6

1. Homogenate tissue / use a blender
   = disrupts the cell membrane, breaking open cells to release contents / organelles
2. Place in a cold, buffered isotonic solution
   Cold = 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
   = to remove large, unwanted debris e.g. whole cells, connective tissue
4. Ultracentrifugation - separated organelles in order of density / mass
   = centrifuge homogenate in a tube at a low speed
   = remove pellet of heaviest organelles and response 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