What you need to know (from the AQA specification)
What you need to know (from the AQA specification)
- The principles and limitations of optical microscopes, transmission electron microscopes and scanning electron microscopes.
- Measuring the size of an object viewed with an optical microscope. The difference between magnification and resolution.
- Use of the formula: magnification = size of image / size of real object.
- Principles of cell fractionation and ultracentrifugation as used to separate cell components.
- Students should be able to appreciate that there was a considerable period of time during which the scientific community distinguished between artefacts and cell organelles.
Magnification and Resolution
Microscopes are used to study the structure of cells and their organelles. They all rely on two key principles:
- Magnification: How many times bigger an image is than the real object
- Resolution: The ability to distinguish between two points that are close together. This is determined by the wavelength used. A shorter wavelength gives higher resolution, whereas a longer wavelength gives lower resolution.
Different types of microscope use different sources (light or electrons), which affects how much detail they can show.
What does wavelength mean, and why does it matter for resolution?
What does wavelength mean, and why does it matter for resolution?
Wavelength is the distance between two peaks of a wave (like the distance between two ripples on water).
- Light has a relatively long wavelength (~400–700 nm)
- Electrons behave like waves too, but with a much shorter wavelength (~0.005 nm)
The shorter the wavelength, the closer together two points can be and still be seen as separate points. If two points are too close together for the wavelength used, they blur into a single point.
Because electrons have a wavelength about 100,000 times shorter than light, an electron microscope can resolve objects that much smaller, which is why we can see organelles, ribosomes and even viruses using electron microscopes, but not light microscopes.
Don't confuse magnification with resolution
You can magnify an image as much as you want, but if the wavelength is too long, two close objects will still appear as one blurred point.
Magnification Formula
$$ \text{Magnification} = \frac{\text{size of image}}{\text{size of real object}} $$
Rearranged:
- Real size = image size ÷ magnification
- Image size = real size × magnification
Exam tip: unit conversions
Most magnification questions involve converting between units. Get the image and real size into the same units before dividing.
- 1 cm = 10 mm
- 1 mm = 1000 μm
- 1 μm = 1000 nm
Memorise: 1 mm = 1000 μm (a common mistake is dividing by 100 instead of 1000).
Practice: Magnification Calculations
Use the image below to work out each question. The image size of each feature is shown directly on the diagram. Click Show working to reveal the step-by-step solution.
Worked example: real size of the nucleus
From the diagram:
- Image size of the nucleus = 20 mm
- Magnification = × 2000
Find: the real size of the nucleus in μm.
Show working
Step 1: convert image size to μm: 20 mm × 1000 = 20,000 μm
Step 2: apply the formula: Real size = image size ÷ magnification = 20,000 ÷ 2000 = 10 μm
Practice 1: real size of the chloroplast
From the diagram:
- Image size of the chloroplast = 14 mm
- Magnification = × 2000
Find: the real size of the chloroplast in μm.
Show working
Step 1: convert image size to μm: 14 mm × 1000 = 14,000 μm
Step 2: apply the formula: Real size = image size ÷ magnification = 14,000 ÷ 2000 = 7 μm
Practice 2: find the magnification (mitochondrion)
From the diagram:
- Image size of the mitochondrion = 5 mm
- Real size of the mitochondrion = 2 μm
Find: the magnification of the image.
Show working
Step 1: get both values into the same unit: Convert 5 mm to μm: 5 × 1000 = 5000 μm
Step 2: apply the formula: Magnification = image size ÷ real size = 5000 ÷ 2 = × 2500
Types of Microscope
There are two main types of microscope used to study cells: optical (light) and electron. Electron microscopes are further split into two types: TEM and SEM.
Optical (Light) Microscope
- Uses light to form the image
- Wavelength is long (longer than that of electrons)
- Maximum resolution ≈ 0.2 μm: anything smaller than this cannot be seen as a separate structure
- Cannot see small organelles in detail, e.g. ribosomes, ER or lysosomes
- Can see the nucleus and sometimes mitochondria (no internal detail)
- Maximum magnification ≈ × 1500
- Can be used to view living cells in colour
Electron Microscope
- Uses a beam of electrons to form the image
- Wavelength of electrons is much shorter than light, giving much higher resolution
- Higher resolution and magnification than optical microscopes
- Specimens must be in a vacuum, so cells are dead (no living cells)
Transmission Electron Microscope (TEM)
- Uses electromagnets to focus a beam of electrons that is transmitted through the specimen
- Denser parts of the specimen absorb more electrons, so they appear darker in the image
- Produces 2D images
- Higher resolution than SEM, so it shows the internal structure of organelles (e.g. cristae in mitochondria, thylakoids in chloroplasts)
- Limitation: requires thin specimens
Scanning Electron Microscope (SEM)
- A beam of electrons is scanned across the surface of the specimen
- Reflected/scattered electrons are detected to build up an image
- Produces 3D images showing the surface of the specimen
- Can use thicker specimens than TEM
- Lower resolution than TEM
Compare the three microscopes
Test yourself: click a button to reveal that column.
| Feature | Optical | TEM | SEM |
|---|---|---|---|
| Source | Light | Beam of electrons (transmitted through) | Beam of electrons (scanned across surface) |
| Resolution | Low (~0.2 μm) | Very high (~0.2 nm), about 1000× better than optical | High (~10 nm), lower than TEM but still much better than optical |
| Max magnification | Low (× 1500) | Very high (× 500,000+), about 300× more than optical | High (× 100,000+), lower than TEM but still far higher than optical |
| Image | 2D, in colour | 2D, black & white | 3D, black & white |
| Specimen | Living or dead | Dead (must be in vacuum), thin | Dead (must be in vacuum), can be thicker |
| Best for seeing | Whole cells, nucleus (no internal detail) | Internal structure of organelles (cristae, thylakoids) | Surface of specimens (e.g. pollen, whole cells) |
Cell Fractionation
Cell fractionation breaks open cells and separates their organelles so each type can be studied individually. There are two main stages: homogenisation & filtration, then ultracentrifugation.
Before any of these steps begin, the cells are placed in a solution that must be:
- Ice-cold: to reduce the activity of hydrolytic enzymes that would damage the organelles
- Isotonic: the same water potential as the organelles, preventing osmotic damage (organelles shrinking or bursting)
- Buffered: to maintain a constant pH so enzymes and proteins are not denatured
1. Homogenisation and Filtration
The cells are vibrated or ground in a blender. This breaks open the cell-surface membrane and releases the organelles into the solution.
The mixture is then filtered through a gauze to remove large debris such as connective tissue and unbroken cells. The organelles pass through, leaving a filtrate that contains the mixture of organelles ready for separation.
2. Ultracentrifugation
The filtrate is spun in a centrifuge at increasing speeds to separate the organelles by density (heaviest first):
- The filtrate is poured into a tube and spun at a low speed.
- The heaviest organelles (e.g. nuclei) move to the bottom of the tube, forming a pellet.
- The remaining organelles stay suspended in the liquid above. This is called the supernatant.
- The supernatant is drained into a new tube and spun at a higher speed, repeating the process.
- This continues at progressively higher speeds until each type of organelle has been separated.
Order of pelleting (heaviest to lightest): nuclei → chloroplasts → mitochondria → lysosomes → endoplasmic reticulum → ribosomes.
Exam tip: explaining the conditions
A common 6-mark question asks why the solution must be cold, isotonic and buffered. Be specific:
- Cold: reduces enzyme activity (don’t just say “preserves the cells”)
- Isotonic: prevents osmotic damage to organelles (don’t confuse with whole-cell osmosis)
- Buffered: maintains pH so proteins / enzymes aren’t denatured
Identifying Starch Grains
Practical context
Practical context
Students could use iodine in potassium iodide solution to identify starch grains in plant cells.
A practical activity for studying plant cells under an optical microscope:
- Place a thin section of plant tissue on a slide
- Add a drop of iodine in potassium iodide solution
- Cover with a coverslip and view under the optical microscope
- Starch grains turn blue-black: they show up clearly against the rest of the cell
Artefacts vs Organelles
An artefact is a structure seen in a microscope image that is not actually part of the real cell, e.g. air bubbles, distortion or debris from preparing the specimen.
For a long time, scientists couldn’t tell which small structures were real organelles and which were artefacts. As new techniques developed (especially the electron microscope), images became clearer and more reproducible, letting the scientific community confirm which structures were real (e.g. mitochondria, ER) and rule out artefacts.
How this topic is tested
This analysis is based on past paper data from 2017 to 2025. It is intended for interest only and is not predictive of what will appear in future papers.
- Tested in 8 of 9 years (2017–2025): 20 question parts worth 54 marks.
- 16th most-examined topic overall by marks, 4th in Unit 2.
Marks by year
Most-tested spec points
- Cell Fractionation & Ultracentrifugation: tested in 6 parts (19 marks)
- Principles & Limitations of Microscopes: tested in 5 parts (15 marks)
- Magnification & Resolution: tested in 4 parts (10 marks)
Also links to: Organelle Structure & Function, Prokaryotic Cell Structure, Absorption in the Ileum.
Maths and practical skills
- Units and standard form (MS 0.1, 0.2): e.g. 2023 P1 Q6.4
- Applying knowledge to practical contexts (PS 1.2): e.g. 2018 P3 Q4.4
- Scientific drawings (AT e): e.g. 2017 P1 Q9.4
- Uncertainty and error (MS 1.11): e.g. 2019 P1 Q1.3
- Magnification and order of magnitude (MS 1.8): e.g. 2019 P1 Q1.3
- Using equations (MS 2.2): e.g. 2023 P1 Q6.4
- Area, volume and surface area of shapes (MS 4.1): e.g. 2018 P3 Q4.2
- Using practical equipment and techniques (PS 4.1): e.g. 2017 P1 Q9.4
Tips from examiner reports
What students commonly get wrong
- The fractionation solutionWatch out: when explaining the solution used in cell fractionation, be specific. Isotonic prevents osmotic damage to the organelles (not the cells). Buffered keeps the pH constant so enzymes and proteins aren't denatured: "prevents damage" isn't enough. 2024 P1 Q10.1
- Centrifuge speedsWatch out: to remove or collect large organelles like nuclei, spin at a low speed first, then increase the speed for smaller organelles. Don't jump straight to high speeds. 2022 P1 Q7.2
- What "higher resolution" meansWatch out: higher resolution means the microscope can distinguish points that are closer together. Electron microscopes have higher resolution than optical ones. 2025 P1 Q4.2
- TEM vs SEMWatch out: TEM gives 2D images showing internal structure. SEM gives 3D images of the surface. Don't mix them up. 2025 P1 Q4.2 · 2017 P1 Q10.1
Exam Question Practice
A few exam-style questions on this topic to try. Have a go at each one before opening the answer.
An image from an optical microscope shows a single bacterial cell. In the image, the cell is 54 mm long.
The actual length of this bacterial cell is 1.8 µm.
Calculate the magnification of this image.
Show your working.
Magnification × ________
(2 marks)
Hint
Use the magnification formula. Be very careful with unit conversions between mm and µm.
Mark Scheme
× 30 000 (2 marks)
Working:
- Convert to the same units: 54 mm = 54 000 µm
- Magnification = image size ÷ actual size
- 54 000 ÷ 1.8 = 30 000
If the answer is wrong, 1 mark for any of:
- 54 000 µm (the image size in µm)
- ÷ 1.8 (dividing the image size by the actual size)
Also accepted / rejected
Tips
- Magnification = image size ÷ real size
- Convert units correctly: 54 mm = 54 000 µm, not 5400 µm
- Don’t divide the real size by the image size
Contrast how an optical microscope and a transmission electron microscope work and contrast the limitations of their use when studying cells.
(6 marks)
Hint
For each feature (what they use to form an image, resolution, specimens, colour, preparation, focusing), how do the two microscopes differ?
Mark Scheme
Max 6 marks
- TEM uses electrons and optical uses light (1 mark)
- TEM gives a greater resolution (1 mark)
- (So with TEM) smaller organelles/named cell structures can be seen
OR more detail in organelles/named cell structures can be seen (1 mark) - TEM can only view dead/dehydrated specimens and optical can view live specimens (1 mark)
- TEM does not show colour and optical can (1 mark)
- TEM needs thinner specimens (1 mark)
- TEM needs a more complex/time-consuming preparation (1 mark)
- TEM focuses using magnets and optical uses (glass) lenses (1 mark)
Also accepted / rejected
3. “Clearer” is not the same as “more detail”.
4. Accept “only optical can view live specimens”.
5. Accept “only optical can show colour”.
7. Accept “TEM needs a more difficult preparation”.
Ignore references to artefacts.
Tips
- ‘Contrast’ means every point must state a clear difference between the two microscopes
- Optical microscopes do show the larger organelles; don’t say no organelles are visible
- SEMs and 3D images aren’t relevant
Table 1 gives the resolution of three types of microscope.
Table 1
| Type of microscope | Resolution / µm |
|---|---|
| Optical (light) microscope | 0.2 |
| Scanning electron microscope (SEM) | 0.01 |
| Transmission electron microscope (TEM) | 0.0002 |
Table 2 gives the sizes of algal cells and mitochondria.
Table 2
| Structure | Diameter / µm |
|---|---|
| Algal cell | 8 to 12 |
| Mitochondrion | 0.5 to 1.0 |
For each investigation below, choose the most suitable microscope and give two reasons for your choice.
Use Table 1, Table 2 and your knowledge of microscopes.
Investigation 1 – looking at the living algal cells in a sample of pond water. A blue dye is added that only enters the dead cells.
Investigation 2 – seeing the three-dimensional surface shape of mitochondria.
(4 marks)
Hint
Compare each microscope’s resolution with the size of what you need to see. Which microscope can view living cells and colour, and which one shows the 3-D surface of a structure?
Mark Scheme
Max 4 marks
Investigation 1: optical (light) microscope (no mark for the name; max 2 from points 1 to 3)
- It can be used to view living cells (1 mark)
- It shows colour, so the blue dead cells can be told apart (1 mark)
- Its resolution (0.2 µm) is enough to see the algal cells (1 mark)
Investigation 2: scanning electron microscope (SEM) (no mark for the name)
- Its resolution (0.01 µm) is high enough to see mitochondria (1 mark)
- It shows surfaces in three dimensions (1 mark)
Also accepted / rejected
Also accepted
- The opposite, e.g. “electron microscopes can’t show colour” (point 2)
- If points 4 and 5 aren’t given, 1 mark for choosing a TEM to see internal structures, or because it has the highest resolution
Not accepted
- Points 1 to 3 given for any microscope other than an optical microscope
Tips
- Don’t mix up SEM and TEM. An SEM gives 3-D images of surfaces; a TEM gives 2-D images of internal structures
- Use resolution correctly. A smaller value means a higher resolution, so an SEM (0.01 µm) has a higher resolution than an optical microscope (0.2 µm)
- Points about ease of use or specimen preparation gain no marks; give reasons linked to living cells, colour, resolution and 3-D structure
What earns marks
- Choosing an optical microscope for investigation 1, because it can view living cells and show the colour of the stain
A scientist wants a pure sample of nuclei from liver tissue.
Describe and explain how the scientist could use cell fractionation and ultracentrifugation to obtain this sample.
(6 marks)
Hint
Think about why each property of the solution matters (cold, isotonic, buffered). For centrifugation, which organelles settle first and why?
Mark Scheme
Max 6 marks
- Homogenise the tissue to break open the cells and release the organelles (1 mark)
- Filter to remove whole cells and debris (1 mark)
- Use a cold solution to reduce enzyme activity (1 mark)
- Use an isotonic solution (same water potential) so the organelles don’t burst or shrink by osmosis (1 mark)
- Use a buffered solution so proteins don’t denature (1 mark)
- Centrifuge at a low speed, so the nuclei form the pellet (1 mark)
Also accepted / rejected
Also accepted
- “Blend”, “grind” or “chop” for homogenise (point 1)
- Up to 1000 × gravity as a low speed (point 6)
Not accepted
- Saying the solution stops cells bursting (point 4: it’s the organelles)
Tips
- Homogenise to break open the cells, then filter; don’t describe this as “fractionation”, and animal cells have no cell walls
- Explain each feature of the solution: cold to reduce enzyme activity; isotonic to stop organelles (not cells) bursting or shrinking; buffered to stop enzymes/proteins denaturing
- Nuclei are the densest organelles, so centrifuge at a low speed and they form the pellet
- Don’t describe increasing speeds or very high speeds to get the nuclei
What earns marks
- Homogenising and filtering are well known
Scientists investigated enzymes found in the cytoplasm of yeast cells.
The scientists broke open the cells to produce a suspension of cell contents.
Describe how the scientists would remove large organelles from this suspension of cell contents.
(2 marks)
Hint
Which piece of equipment separates organelles by density? Where would the heaviest organelles end up?
Mark Scheme
- Centrifuge the suspension at a low speed (or at increasing speeds) (1 mark)
- The large, dense organelles form the first pellet, which is removed (for example, the nuclei) (1 mark)
Also accepted / rejected
Also accepted
- Small organelles stay in the supernatant (point 2)
- Another named large organelle in the pellet, e.g. mitochondria (point 2)
Tips
- Centrifuge at a low (or gradually increasing) speed; only describing the high speeds of ultracentrifugation misses the mark
- The large, dense organelles form the pellet at the bottom of the tube; smaller ones stay in the supernatant
- Filtering only removes debris, so it isn’t enough on its own
Scientists investigated chloroplasts from spinach leaves. They broke open leaf cells and filtered the mixture to give a suspension of chloroplasts.
They divided the suspension between two tubes. A detergent that dissolves lipids was added to tube P only. Both tubes were then centrifuged at low speed.
Figure 1 shows the tubes after centrifuging.
Explain the appearance of tubes P and Q.
(3 marks)
Hint
Where is chlorophyll found in a chloroplast? Where do large, dense organelles end up when centrifuged? What would the detergent do to the chloroplast membranes?
Mark Scheme
Tube Q
- The chlorophyll is inside the chloroplasts (in the thylakoid membranes) (1 mark)
- Chloroplasts are large and dense, so they form the pellet (1 mark)
Tube P
- The detergent dissolves the chloroplast membranes (phospholipids), so the chlorophyll is released into the liquid (1 mark)
Also accepted / rejected
Not accepted
- “The detergent dissolves lipids” without linking it to the membranes (point 3)
Tips
- Tube Q: the chlorophyll is inside intact chloroplasts, which are large and dense, so they form the green pellet
- Tube P: the detergent dissolved the membranes (phospholipids) of the chloroplasts and thylakoids, so the chlorophyll is released and stays in the liquid; “dissolves lipids” just repeats the stem
- Link each tube to what you can see: where the green colour ends up
Also accepted