2.3 Methods of Studying Cells

Question 1

What is meant by magnification?

Answer 1

• Number of times greater image is than size of real object
• Magnification = image size / actual size

Question 2

What is meant by resolution?

Answer 2

Minimum distance apart two objects can be to be distinguished as separate objects

Question 3

Optical microscope

Answer 3

Light focussed using glass lens
Light passes through specimen, different structures absorb different amounts/wavelengths
2D image of cross-section
Low resolution as wavelength of light is long
Can’t see internal structures of organelles
Thin specimen
Low magnification
Can view living organisms
Simple preparation
Colour

Question 4

TEM

Answer 4

Electrons focussed using electromagnets
Electrons pass through specimen, denser parts absorb more and appear darker
2D image of cross-section
Very high resolution as wavelength of electrons is short
Can see internal structures of organelles
Very thin specimen
High magnification
Can only view dead specimens as system in a vacuum
Complex preparation so artefacts often present
Not colour

Question 5

SEM

Answer 5

Electrons focussed using electromagnets
Electrons deflected off surface of specimen
3D image of surface
High resolution as wavelength of electrons is short
Can’t see internal structures of organelles
Specimen doesn’t need to be thin
High magnification
Can only view dead specimens as system in a vacuum
Complex preparation so artefacts often present
Not colour

Question 6

How can the size of an object be measured?

Answer 6

• Line up eyepiece graticule with stage micrometer
• Calibrate using eyepiece graticule, use stage micrometer to calculate size of divisions
• Use graticule to measure how many divisions make up the object
• Calculate size of object by multiplying number of divisions by size of division
• Recalibrate eyepiece graticule at different magnifications

Question 7

How can a sample of organelles be isolated by cell fractionation?

Answer 7

• Homogenise using a blender to break cell membrane, releasing organelles
• Place in an ice cold, isotonic, buffered solution
• Ice cold to reduce enzyme activity so organelles not damaged, isotonic so water doesn’t move into cells by osmosis causing them to burst, buffered to maintain constant pH so enzymes don’t denature
• Filter homogenate to remove large debris
• Centrifuge filtrate at low speed
• Densest organelles flung to bottom, forming pellet
• Remove pellet and recentrifuge supernatant at higher speed