2.

1 Cell structure
2.1.3 Methods of studying cells

Microscopes - principles and limitations

Optical microscope Transmission Scanning

electron microscope (TEM) electron microscope (SEM)

● Light focused using glass ● Electrons focused using ● Electrons focused using
lenses electromagnets electromagnets
● Light passes through thin ● Electrons pass through very ● Electrons scan specimen
specimen thin specimen surface
● Light that passes through or ● Denser = absorb more ● Reflected electrons are
reflects the surface can be electrons = darker detected
seen ● Generates a 2D image of a ● Generates a 3D image of the
● Generates a 2D image of a cross-section surface
cross-section

× Low resolution - 200 nm ✓ V. high resolution - 0.2 nm ✓ High resolution - 2 nm

× Due to long ✓ Due to short ✓ Due to short
wavelength of light wavelength of electrons wavelength of electrons
× Can’t see internal structure ✓ Can see internal structures ✓ Can see external / 3D /
of organelles or organelles of organelles and small surface structures
smaller than 200nm eg. organelles eg. ribosomes × Lower resolution than TEM
ribosomes ✓ High magnification - ✓ High magnification
× Low magnification - x 1,000,000 - x 1,000,000
x 1500

✓ Can view living organisms × Dead / dehydrated × Dead / dehydrated

× Only thin specimens specimens, uses vacuum specimens, uses vacuum
✓ Simple preparation × Only v. thin specimens ✓ Thick / 3D specimens
✓ Can show colour × Complex preparation so × Complex preparation so
artefacts often present artefacts often present
× Does not show colour × Does not show colour

Early scientific research into cell organelles - artefacts in microscopy

You should be able to appreciate that there was a considerable period of time during which the scientific
community distinguished between artefacts (eg. dust, air bubbles) and cell organelles.

● Artefacts often occur during preparation of a sample, particularly for electron microscopes
● To overcome this, scientists prepared specimens in different ways
● If an object could be seen with one technique but not another → more likely to be an artefact than an
organelle
Magnification

● Number of times greater the image is than the size of the real (actual) object
● Magnification = size of image / size of real object

Resolution

● Minimum distance apart 2 objects can be for them to be distinguished as separate objects
● Ie. level of detail
● Limited by wavelength of radiation → electron wavelength shorter than light so higher magnification

Converting units

When doing magnification calculations, all units must be the same.

Unit Number of metres

Centimetre (cm) 1/100 m 0.01 m 10-2 m

Millimetre (mm) 1/1000 m 0.001 m 10-3 m

Micrometre (µm) 1/1000000 m 0.000001 m 10-6 m

Nanometre (nm) 1/1000000000 m 0.000000001 m 10-9 m

Measuring the size of objects viewed with optical microscopes using a stage
micrometre and eyepiece graticule

Eyepiece graticule Stage micrometre

Where In the eyepiece; spans across entire field of view On a microscope slide; placed on the stage

Units No fixed units → must be calibrated to work out Does have fixed units - 1mm long micrometre is
size of divisions at a particular magnification normally divided into 100 equal units of 10µm

Use Remains in field of view when viewing specimen Used to calibrate the eyepiece graticule at a
so is used to measure size of object particular magnification

1. Line up the (scale of the) eyepiece graticule with (the scale of) the stage micrometre
2. Calibrate the eyepiece graticule - use stage micrometre to calculate size of divisions on the eyepiece
graticule
3. Take the micrometre away and use the graticule to measure how many divisions make up the object
4. Calculate the size of the object by multiplying the number of divisions by the size of division
5. Recalibrate eyepiece graticule at different magnifications
Step 2 worked example - using the stage micrometre to
calculate size of divisions on the eyepiece graticule

● When the scales are lined up, you can see (in the box)
that 4 eyepiece graticule divisions = 10 stage
micrometre divisions
● In this stage micrometre, one subdivision is 10µm
● So 4 eyepiece graticule divisions = 10µm x 10 = 100µm
● So 1 eyepiece graticule division = 100µm/4 = 25µm

Principles of cell fractionation and ultracentrifugation as used to separate cell

components

1. Homogenise (break up) tissue using a blender

● Disrupts cell membrane → breaks open cell
● Releases contents / organelles
2. Keep in a cold, isotonic, buffered solution

Cold To reduce enzyme activity → so organelles aren’t 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 → separate organelles in order of density / mass

Nuclei → chloroplasts / mitochondria → lysosomes → endoplasmic reticulum → ribosomes

1. Centrifuge homogenate in a tube at a low speed

2. Remove pellet of heaviest organelle and respin supernatant at a higher speed
3. Repeat at increasing speeds until organelles separated out, each time pellet is made of
lighter organelles