Methods of studying cells
AQA
A-Level
An optical microscope passes visible light through a specimen using glass lenses. It is limited by the relatively long wavelength of light, giving a maximum resolution of about 0.2 micrometres. It cannot resolve small organelles but can view living, coloured specimens. Electron microscopes use electron beams with much shorter wavelengths, providing significantly higher resolution. A transmission electron microscope (TEM) transmits electrons through an ultra-thin section to reveal 2D internal ultrastructure. A scanning electron microscope (SEM) sweeps a beam across a surface to produce a 3D image. Electron microscopy requires a vacuum, meaning specimens must be dead, and involves complex preparation that can create artefacts.
16
Objectives
10
Exam Tips
18
Pitfalls
19
Key Terms
20
Mark Points
Subtopics in this area
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.
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.
Methods of studying cells Revision Guide
Learning Objectives
What you need to know and understand
- Explain why the resolution of an optical microscope cannot be improved beyond about 0.2 micrometres however much the image is magnified.
- Choose the appropriate microscope for a stated purpose and justify the choice from resolution and the type of image produced.
- Describe two limitations of electron microscopy that arise from specimen preparation rather than from the instrument.
- Suggest why a cell structure first reported in the 1950s could not have been seen with a light microscope.
- Calibrate an eyepiece graticule against a stage micrometer and state the value of one division for a given objective lens.
- Calculate the magnification of a printed image from a measured image length and a stated real size, showing the conversion to a common unit.
- Rearrange the magnification equation to find the real size of a structure from an image and a stated magnification.
- Convert confidently between millimetres, micrometres and nanometres inside a microscopy calculation.
- Distinguish magnification from resolution by naming the quantity each measures and the units each carries.
- Explain why two structures 0.1 micrometres apart cannot be separated by an optical microscope at any magnification.
- Suggest why a discovery about cell structure had to wait for the electron microscope, referring to resolution rather than magnification.
- Correct an answer that uses the word magnification where resolution is required.
- Define an artefact and give two preparation steps that can create one.
- Explain how scientists established that a newly seen structure was a genuine organelle rather than an artefact.
- Suggest, for a structure seen in only one electron micrograph, why other scientists would not accept it immediately.
- Evaluate a claim about cell structure by judging whether it has been repeated with different preparation methods.
Marking Points
Key points examiners look for in your answers
- Optical microscopes are limited by the relatively long wavelength of light, restricting resolution to approximately 0.2 micrometres.
- Electron microscopes achieve much higher resolution because electrons have a significantly shorter wavelength than visible light.
- TEM produces a high-resolution two-dimensional image of internal ultrastructure from ultra-thin sections.
- SEM produces a three-dimensional image of the specimen's surface by detecting scattered electrons.
- Limitations of electron microscopy include the requirement for a vacuum (specimens must be dead), complex staining, and the risk of artefacts.
- one mark for measuring the image accurately and converting it to the same unit as the real object, for example 46 mm recorded as 46 000 micrometres
- one mark for correct use of magnification equals size of image divided by size of real object, shown as the division even if the arithmetic is wrong
- one mark for a final magnification consistent with the measurement, quoted without units, for example about 20 000 times
- one mark for calibrating an eyepiece graticule against a stage micrometer, and recalibrating after changing the objective lens
- one mark for rearranging the formula correctly when the real size rather than the magnification is the unknown
- One mark for defining magnification as the number of times larger the image is than the object (size of image divided by size of real object).
- One mark for defining resolution as the minimum distance at which two points can still be distinguished as separate.
- One mark for stating resolution is limited by the wavelength of radiation, meaning electron microscopes resolve more detail than optical microscopes.
- One mark for explaining that increasing magnification beyond the resolving power gives a larger but no clearer image (empty magnification).
- One mark for quoting resolution with a unit of distance and magnification without any unit.
- one mark for defining an artefact as a structure produced during preparation that was not present in the living cell
- one mark for a named cause: fixing, staining, dehydration, sectioning, or air bubbles and debris on the slide
- one mark for the idea that a structure is accepted as real when it is seen repeatedly using different preparation techniques or different microscopes
- one mark for the delay being explained by the need for independent repetition and scrutiny by other scientists before a claim is accepted
- one mark for recognising that electron microscopy requires more preparation than light microscopy, so more artefacts can be introduced
Examiner Tips
Expert advice for maximising your marks
- 💡Clearly distinguish between instrument limitations (e.g., wavelength affecting resolution) and specimen preparation limitations (e.g., vacuum requirement, artefacts).
- 💡Always link the shorter wavelength of electrons directly to the higher resolution achieved by electron microscopes.
- 💡Always show the conversion and the division; AQA awards a mark for correct use of the equation or the correct image size in micrometres even when the final answer is wrong.
- 💡Convert everything to micrometres before dividing, then check the conversion rather than the size of the answer: a printed image has been enlarged far beyond what the microscope itself did, so an optical-microscope image of a bacterium can legitimately work out at about 20 000 times.
- 💡Measure to the ends the question specifies, for instance excluding a flagellum if the stem says so.
- 💡If a question asks about what can be seen or told apart, the answer is resolution; if it asks about how much bigger, it is magnification.
- 💡In an explanation question, name the shorter wavelength of electrons as the cause for higher resolution in electron microscopes.
- 💡Anchor your answer in preparation: name the step (fixing, staining, sectioning) that could have created the structure.
- 💡When asked how scientists became confident, use the language of repeatability, different techniques and independent workers.
- 💡This idea returns in practical questions, so use it when asked whether an unexpected structure on a slide is real.
Common Mistakes
Pitfalls to avoid in your exam answers
- Using magnification and resolution interchangeably. Correction: Magnification is how much larger the image is, while resolution is the ability to distinguish two close points; higher magnification does not improve resolution.
- Claiming a TEM gives a 3D surface image or an SEM shows internal structure. Correction: TEM shows 2D internal ultrastructure, whereas SEM shows 3D surface topography.
- Stating living processes can be observed using an electron microscope. Correction: Electron microscopes require a vacuum, so all specimens must be dead.
- Explaining better resolution by stating electrons are smaller. Correction: Higher resolution is due to electrons having a much shorter wavelength than light, not their physical size.
- dividing the real size by the image size, which inverts the magnification
- leaving the image in millimetres and the object in micrometres, so the answer is out by a factor of one thousand
- attaching a unit to a magnification, which is a ratio and has none
- treating eyepiece graticule divisions as fixed micrometres without calibrating against a stage micrometer
- measuring the image carelessly; the mark scheme allows only a narrow band of ruler measurements, so a millimetre either way can cost the answer mark
- Writing magnification where the question requires resolution, such as when discussing the ability to see detail.
- Defining resolution merely as clarity or amount of detail, without the specific idea of distinguishing two separate points.
- Giving magnification a unit such as micrometres; magnification is a ratio and has no units.
- Stating an electron microscope resolves better because it magnifies more, rather than because electrons have a shorter wavelength.
- defining an artefact as a mistake made by the scientist rather than a structure created by the preparation process
- saying the problem was solved by using a higher magnification instead of by repeating with different techniques
- assuming only electron microscopy creates artefacts, when squashes and stained slides produce them too
- claiming one very clear image is enough to prove a structure is genuine
- describing artefacts as structures the scientist imagined rather than ones genuinely visible in the specimen