3.2.1 - Methods of studying cells Flashcards
Topic 2
Magnification
Number of times greater image is than size of the real (actual) object
How do you calculate magnification?
Magnification = size of image / size of real object
Resolution
The minimum distance apart 2 objects can be to be distinguished as separate objects
Principle of optical microscopes
- Light focused using
glass lenses - Light passes through specimen,
different structures absorb different amounts & wavelengths - Generates a 2D image of a cross-section
What are some limitations of an optical microscope?
- Low resolution due to the long wavelength of light
- Can’t see internal structure of
organelles or ribosomes - Specimen must be thin
- Low magnification (x 1500)
What are the benefits of using an optical microscope?
- Can view living organisms
- Simple preparation
- Can show colour
Explain the principles of a 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
What are the benefits of using a transmission electron microscope?
- Very high resolution due to short wavelength of electrons
- Can see internal structures of organelles and ribosomes
- High magnification (x 1,000,000)
What are the disadvantages of using a transmission electron microscope?
- Specimen must be very thin
- Can only view dead / dehydrated specimens as uses a vacuum
- Complex preparation so
artefacts often present - Does not show colour
Explain the principles of a scanning electron microscope (SEM)
- Electrons focused using
electromagnets - Electrons deflected / bounce off specimen surface
- Generates a 3D image
of surface
What are the benefits of using a scanning electron microscope?
- generates a 3D image
- High resolution due to short wavelength of electrons
- Specimen does not need to be thin
- High magnification (x 1,000,000)
What are the disadvantages of using a scanning electron microscope?
- Can’t see internal structures
- Can only view dead / dehydrated specimens as uses a vacuum
- Complex preparation so
artefacts often present - Does not show colour
Suggest how the scientific community distinguished between artefacts (eg. dust, air bubbles occurring during preparation) and cell organelles
● 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
List the steps in calculations involving magnification, real size & image size
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
How do you convert from a nanometre (nm) to a micrometre (μm) ?
divide by 1000
How do you convert from a micrometre (μm) to a millimetre (mm) ?
divide by 1000
How do you convert from a millimetre (mm) to a metre (m) ?
divide by 1000
How do you convert from a metre (m) to a millimetre (mm) ?
X 1000
How do you convert from a millimetre (mm) to a micrometre (μm) ?
X 1000
How do you convert from a micrometre (μm) to a nanometre (nm) ?
X 1000
Describe how the size of an object viewed with an optical microscope can be measured
- Line up (scale of) eyepiece graticule with (scale of) stage micrometre
- Calibrate eyepiece graticule - use stage micrometre to calculate size of divisions on eyepiece graticule
- Take micrometre away and 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
Describe and explain the principles of cell fractionation and
ultracentrifugation as used to separate cell components
- Homogenise tissue /
use a blender
> Disrupts the cell membrane, breaking open cells to release
contents / organelles - 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/change shape
> Buffered to keep pH constant
○ So enzymes don’t denature - changing shape of proteins - Filter homogenate
> To remove large, unwanted debris eg. whole cells, connective tissue - Ultracentrifugation -
separates organelles
in order of density /
mass
> Centrifuge homogenate in a tube at a low speed
> Remove pellet of heaviest organelle and re-spin 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)
What is the order of density of the organelles in a cell?
nuclei → chloroplasts /
mitochondria → lysosomes → ER → ribosomes
When using a light microscope why do you need a thing specimen (often a single layer of cells)
- so light can pass through
