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AQA A Level Biology Your notes
2.2 The Microscope in Cell Studies
Contents
2.2.1 Methods of studying cells
2.2.2 Microscopy & Drawing Scientific Diagrams
2.2.3 Iodine Detects Starch Grains
2.2.4 Resolution & Magnification
2.2.5 Magnification Calculations
2.2.6 Cell Fractionation & Ultracentrifugation
2.2.7 Scientific Research into Cell Organelles
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2.2.1 Methods of studying cells
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The Principles & Limitations of Microscopy
Microscopes can be used to analyse cell components and observe organelles
Magnification and resolution are two scientific terms that are very important to understand and
distinguish between when answering questions about microscopy (the use of microscopes):
Magnification tells you how many times bigger the image produced by the microscope is than the
real-life object you are viewing
Resolution is the ability to distinguish between objects that are close together (i.e. the ability to
see two structures that are very close together as two separate structures)
There are two main types of microscopes:
Optical microscopes (sometimes known as light microscopes)
Electron microscopes
Optical (light) microscopes
Optical microscopes use light to form an image
This limits the resolution of optical microscopes
Using light, it is impossible to resolve (distinguish between) two objects that are closer than half
the wavelength of light
The wavelength of visible light is between 500-650 nanometres (nm), so an optical microscope
cannot be used to distinguish between objects closer than half of this value
This means optical microscopes have a maximum resolution of around 0.2 micrometres (µm) or 200
nm
This means optical microscopes can be used to observe eukaryotic cells, their nuclei and possibly
mitochondria and chloroplasts
This also means that optical microscopes cannot be used to observe smaller organelles such as
ribosomes, the endoplasmic reticulum or lysosomes
The maximum useful magnification of optical microscopes is about ×1500
Electron microscopes
Electron microscopes use electrons to form an image
This greatly increases the resolution of electron microscopes compared to optical microscopes,
giving a more detailed image
A beam of electrons has a much smaller wavelength than light, so an electron microscope can
resolve (distinguish between) two objects that are extremely close together
This means electron microscopes have a maximum resolution of around 0.0002 µm or 0.2 nm (i.e.
around 1000 times greater than that of optical microscopes)
This means electron microscopes can be used to observe small organelles such as ribosomes, the
endoplasmic reticulum or lysosomes
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The maximum useful magnification of electron microscopes is about ×1,500,000
There are two types of electron microscopes:
Transmission electron microscopes (TEMs) Your notes
Scanning electron microscopes (SEMs)
Transmission electron microscopes (TEMs)
TEMs use electromagnets to focus a beam of electrons
This beam of electrons is transmitted through the specimen
Denser parts of the specimen absorb more electrons
This makes these denser parts appear darker on the final image produced (produces contrast
between different parts of the object being observed)
Advantages of TEMs:
They give high-resolution images (more detail)
This allows the internal structures within cells (or even within organelles) to be seen
Disadvantages of TEMs:
They can only be used with very thin specimens or thin sections of the object being observed
They cannot be used to observe live specimens (as there is a vacuum inside a TEM, all the water
must be removed from the specimen and so living cells cannot be observed, meaning that
specimens must be dead, unlike optical microscopes that can be used to observe live specimens)
The lengthy treatment required to prepare specimens means that artefacts can be introduced
(artefacts look like real structures but are actually the results of preserving and staining)
They do not produce a colour image (unlike optical microscopes that produce a colour image)
Scanning electron microscopes (SEMs)
SEMs scan a beam of electrons across the specimen
This beam bounces off the surface of the specimen and the electrons are detected, forming an image
This means SEMs can produce three-dimensional images that show the surface of specimens
Advantages of SEMs:
They can be used on thick or 3-D specimens
They allow the external, 3-D structure of specimens to be observed
Disadvantages of SEMs:
They give lower resolution images (less detail) than TEMs
They cannot be used to observe live specimens (unlike optical microscopes that can be used to
observe live specimens)
They do not produce a colour image (unlike optical microscopes that produce a colour image)
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Examiner Tip
Your notes
This is a lot of information to learn! First, make sure you know the basics of how each type of
microscope works. Then learn the advantages and disadvantages of each type of microscope. In
particular, make sure you can compare and contrast TEMs and SEMs in terms of their relative
advantages and disadvantages. In an exam question, you could be given a situation and then asked
which type of electron microscope would be most suitable to use and why. A good revision idea is to
make a table of the advantages and disadvantages of each type of microscope...then learn them!
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2.2.2 Microscopy & Drawing Scientific Diagrams
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Practical Skill: Microscopy & Drawing Scientific Diagrams
Many biological structures are too small to be seen by the naked eye
Optical microscopes are an invaluable tool for scientists as they allow for tissues, cells and organelles
to be seen and studied
For example, the movement of chromosomes during mitosis can be observed using a microscope
When using an optical microscope always start with the low power objective lens:
It is easier to find what you are looking for in the field of view
This helps to prevent damage to the lens or coverslip incase the stage has been raised too high
A graticule must be used to take measurements of cells:
A graticule is a small disc that has an engraved scale. It can be placed into the eyepiece of a
microscope to act as a ruler in the field of view
As a graticule has no fixed units it must be calibrated for the objective lens that is in use. This is
done by using a scale engraved on a microscope slide (a stage micrometer)
By using the two scales together the number of micrometers each graticule unit is worth can be
worked out
After this is known the graticule can be used as a ruler in the field of view
The stage micrometer scale is used to find out how many micrometers each graticule unit represents
Electron microscopes can produce highly detailed images of animal and plant cells
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The key cellular structures within animal and plant cells are visible within the electron micrographs
below
Your notes
Some internal features of animal (top) and plant cells (bottom) can be identified using transmission
electron microscopy
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Drawing Cells
To record the observations seen under the microscope (or from photomicrographs taken) a labelled Your notes
biological drawing is often made
Biological drawings are line pictures which show specific features that have been observed when the
specimen was viewed
There are a number of rules/conventions that are followed when making a biological drawing
The conventions are:
The drawing must have a title
The magnification under which the observations shown by the drawing are made must be
recorded
A sharp HB pencil should be used (and a good eraser!)
Drawings should be on plain white paper
Lines should be clear, single lines (no thick shading)
No shading
The drawing should take up as much of the space on the page as possible
Well-defined structures should be drawn
The drawing should be made with proper proportions
Label lines should not cross or have arrowheads and should connect directly to the part of the
drawing being labelled
Label lines should be kept to one side of the drawing (in parallel to the top of the page) and drawn
with a ruler
Drawings of cells are typically made when visualizing cells at a higher magnification power, whereas
plan drawings are typically made of tissues viewed under lower magnifications (individual cells are
never drawn in a plan diagram)
Plant cell biological drawing
Bacterial cell biological drawing
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Your notes
Examiner Tip
When producing a biological drawing, it is vital that you only ever draw what you see and not what you
think you see.
To accurately reflect the size and proportions of structures you see under the microscope, you should
get used to using the eyepiece graticule.
You should be able to describe and interpret photomicrographs, electron micrographs and drawings
of typical animal cells.
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2.2.3 Iodine Detects Starch Grains
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Practical Skill: Using Iodine to Detect Starch Grains
Starch is the storage polysaccharide of plants
Sugars formed during photosynthesis are stored as starch inside starch grains
These starch grains are commonly found in:
the stroma of chloroplasts
storage organs such as potato tubers
the seeds of cereals and legumes
Starch grains are large enough to be seen with a light microscope but they first require staining in
order to be seen easily
Iodine (I2) in potassium iodide (KI) solution can be used to stain starch grains
This can be done by adding a drop of potassium iodide onto the specimen (mounted on a slide)
before placing a coverslip over the top
This makes the starch grains darker in colour, making them easier to see
Remember, iodine in potassium iodide solution has a light orange-brown colour but turns blue-
black in the presence of starch
Examiner Tip
Remember - only a very small amount of stain is required (usually just a drop or two will be enough)!
Also, when adding a cover slip, remember to slowly lower the cover slip onto the specimen at an angle
to avoid any air bubbles getting trapped (these could create ‘artefacts’ and obstruct your view of the
specimen).
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2.2.4 Resolution & Magnification
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Resolution & Magnification
Magnification
Magnification is how many times bigger the image of a specimen observed is in compared to the
actual (real-life) size of the specimen
A light microscope has two types of lens:
An eyepiece lens, which often has a magnification of x10
A series of (usually 3) objective lenses, each with a different magnification
To calculate the total magnification the magnification of the eyepiece lens and the objective lens are
multiplied together:
eyepiece lens magnification x objective lens magnification = total magnification
Resolution
Resolution is the ability to distinguish between two separate points
If two separate points cannot be resolved, they will be observed as one point
The resolution of a light microscope is limited by the wavelength of light
As light passes through the specimen, it will be diffracted
The longer the wavelength of light, the more it is diffracted and the more that this diffraction will
overlap as the points get closer together
Electron microscopes have a much higher resolution and magnification than a light microscope as
electrons have a much smaller wavelength than visible light
This means that they can be much closer before the diffracted beams overlap
The concept of resolution is why the phospholipid bilayer structure of the cell membrane cannot be
observed under a light microscope
The width of the phospholipid bilayer is about 10nm
The maximum resolution of a light microscope is 200nm (half the smallest wavelength of visible
light, 400nm)
Any points that are separated by a distance less than 200nm (such as the 10nm phospholipid
bilayer) cannot be resolved by a light microscope and therefore will not be distinguishable as
“separate”
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Your notes
The resolving power of an electron microscope is much greater than that of the light microscope, as
structures much smaller than the wavelength of light will interfere with a beam of electrons
Comparison of the electron microscope & light microscope
Light microscopes are used for specimens above 200 nm
Light microscopes shine light through the specimen, this light is then passed through an objective
lens (which can be changed) and an eyepiece lens (x10) which magnify the specimen to give an
image that can be seen by the naked eye
The specimens can be living (and therefore can be moving), or dead
Light microscopes are useful for looking at whole cells, small plant and animal organisms, tissues
within organs such as in leaves or skin
Electron microscopes, both scanning and transmission, are used for specimens above 0.5 nm
Electron microscopes fire a beam of electrons at the specimen either a broad static beam
(transmission) or a small beam that moves across the specimen (scanning)
The electrons are picked up by an electromagnetic lens which then shows the image
Due to the higher frequency of electron waves (a much shorter wavelength) compared to visible
light, the magnification and resolution of an electron microscope is much better than a light
microscope
Electron microscopes are useful for looking at organelles, viruses and DNA as well as looking at
whole cells in more detail
Electron microscopy requires the specimen to be dead however this can provide a snapshot in
time of what is occurring in a cell eg. DNA can be seen replicating and chromosome position within
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the stages of mitosis are visible
Light v Electron Microscope Table Your notes
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2.2.5 Magnification Calculations
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Magnification Calculations
Magnification is how many times bigger the image of a specimen observed is in comparison to the
actual (real-life) size of the specimen
The magnification (M) of an object can be calculated if both the size of the image (I), and the actual size
of the specimen (A), is known
An equation triangle for calculating magnification
Worked example
An image of an animal cell is 30 mm in size and it has been magnified by a factor of X 3000.
What is the actual size of the cell?
To find the actual size of the cell:
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Your notes
The size of cells is typically measured using the micrometre (μm) scale, with cellular structures
measured in either micrometers (μm) or nanometers (nm)
When doing calculations all measurements must be in the same units. It is best to use the smallest unit
of measurement shown in the question
To convert units, multiply or divide depending if the units are increasing or decreasing
Magnification does not have units
Converting units of measurement
There are 1000 nanometers (nm) in a micrometre (µm)
There are 1000 micrometres (µm) in a millimetre (mm)
There are 1000 millimetres (mm) in a metre (m)
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Worked example
Your notes
Step 1: Check that units in magnification questions are the same
Remember that 1mm = 1000µm
2000 / 1000 = 2, so the actual thickness of the leaf is 2 mm and the drawing thickness is 50 mm
Step 2: Calculate Magnification
Magnification = image size / actual size = 50 / 2 = 25
So the magnification is x 25
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2.2.6 Cell Fractionation & Ultracentrifugation
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Cell Fractionation & Ultracentrifugation
Biologists and other scientific researchers may sometimes want to study one specific organelle
This may be to:
Look at the appearance of the specific organelle under an electron microscope
Conduct research on the specific organelle (for example, to learn more about its function)
To do this, a pure sample is needed (containing only the specific organelle being studied)
The process of separating cell organelles from each other is known as cell fractionation
This process involves breaking up a suitable sample of tissue and then centrifuging the mixture at
different speeds
Cell fractionation can be split into three stages:
Homogenisation
Filtration
Ultracentrifugation
Homogenisation
Homogenisation is the biological term used to describe the breaking up of cells
The sample of tissue (containing the cells to be broken up) must first be placed in a cold, isotonic
buffer solution
The solution must be:
Ice-cold to reduce the activity of enzymes that break down organelles
Isotonic (it must have the same water potential as the cells being broken up) to prevent water from
moving into the organelles via osmosis, which would cause them to expand and eventually
damage them
Buffered (have a buffer solution added) to prevent organelle proteins, including enzymes, from
becoming denatured
The tissue-containing solution is then homogenised using a homogeniser
This is a blender-like machine that grinds the cells up (the cells can also be vibrated until they break
up)
This breaks the plasma membrane of the cells and releases the organelles into a solution called the
homogenate
Filtration
The homogenate (containing the homogenised cells) is then filtered through a gauze
This is to separate out any large cell debris or tissue debris that were not broken up
The organelles are all much smaller than the debris and are not filtered out (they pass through the
gauze)
This leaves a solution (known as the filtrate) that contains a mixture of organelles
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Ultracentrifugation
The filtrate is placed into a tube and the tube is placed in a centrifuge Your notes
A centrifuge is a machine that separates materials by spinning
The filtrate is first spun at a low speed
This causes the largest, heaviest organelles (such as the nuclei) to settle at the bottom of the tube,
where they form a thick sediment known as a pellet
The rest of the organelles stay suspended in the solution above the pellet
This solution is known as the supernatant
The supernatant is drained off and placed into another tube, which is spun at a higher speed
Once again, this causes the heavier organelles (such as the mitochondria) to settle at the bottom
of the tube, forming a new pellet and leaving a new supernatant
The new supernatant is drained off and placed into another tube, which is spun at an even higher speed
This process is repeated at increasing speeds until all the different types of organelle present are
separated out (or just until the desired organelle is separated out)
Each new pellet formed contains a lighter organelle than the previous pellet
The order of mass of these organelles (from heaviest to lightest) is usually:
Nuclei
Chloroplasts (if carrying out cell fractionation of plant tissue)
Mitochondria
Lysosomes
Endoplasmic reticulum
Ribosomes
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Your notes
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Your notes
The three stages of cell fractionation (homogenisation, filtration and ultracentrifugation) for separating
organelles
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Examiner Tip
Your notes
There are lots of new biological terms here that you need to know the definitions of, including cell
fractionation, homogenisation, homogenate, isotonic, ultracentrifugation and supernatant. Make sure
you learn the meaning of each of these as you will be expected to be able to answer questions that
require knowledge of these terms and to use them in your answers!
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2.2.7 Scientific Research into Cell Organelles
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Appreciating the Process of Distinguishing Organelles
When looking at a prepared sample (e.g. a cell or a group of cells) under a microscope, you can
sometimes see things that aren’t actually part of the specimen
These are known as artefacts
Artefacts can be a variety of things, such as:
dust
air bubbles
fingerprints
These artefacts often occur during the preparation of a sample
During preparation, a sample is often squashed or stained, which can generate artefacts
The occurrence of artefacts can be decreased by more careful preparation of samples
Artefacts are common in electron micrographs (especially in samples prepared for viewing using a
transmission electron microscope) due to the lengthy treatment required to prepare samples
This was particularly problematic for early research by scientists using the first electron microscopes
To distinguish between artefacts and organelles, they had to repeatedly prepare a specimen in
different ways, using different techniques
If they saw a particular object in a specimen prepared using one preparation technique, but not
another, the object was more likely to be an artefact than an organelle
This was a problem for the scientific community that persisted for a considerable period of time until
preparation techniques and knowledge of organelles improved
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