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Spec mapping: AQA 7402 Section 3.2.1.2 — methods of studying cells (refer to the official AQA specification document for exact wording). Required Practical 1 — microscopy is anchored in this lesson.
To study organelles, biochemists must do two things: isolate them from cells (cell fractionation) and visualise them (microscopy). These two methodologies underpin nearly every other lesson in this unit. Without cell fractionation, the metabolic functions of organelles could not be assigned with confidence. Without microscopy, the structural claims you make about cells would have no observational basis. This lesson covers the techniques, the underlying physics, the key calculations and the AQA-style mark-scheme literacy you need to handle questions on magnification, resolution, scale bars and equipment selection.
Cell fractionation is the process of separating the different organelles of a cell so that they can be studied individually. It involves two main stages: homogenisation and differential centrifugation.
The tissue is placed in a cold, isotonic, buffered solution and broken up using a blender or homogeniser (e.g., a Potter–Elvehjem homogeniser) to produce a homogenate — a suspension of disrupted cells and their contents.
The solution must be:
| Condition | Reason |
|---|---|
| Cold (ice-cold, around 4 °C) | Reduces the activity of enzymes (especially hydrolytic enzymes from lysosomes) that could digest the organelles |
| Isotonic (same water potential as the cell contents) | Prevents organelles from swelling and bursting (in a hypotonic solution) or shrinking (in a hypertonic solution) by osmosis |
| Buffered (maintained at a constant pH, typically around pH 7.4) | Prevents changes in pH that could denature enzymes or alter the structure of organelles |
The homogenate is then filtered through gauze or cheesecloth to remove large debris such as connective tissue fibres and intact cells.
The filtered homogenate is placed in a centrifuge tube and spun at progressively increasing speeds.
Key Definition: Differential centrifugation is a technique that separates organelles based on their size and density. The heaviest and densest organelles sediment first at low centrifugal forces; lighter organelles require higher forces.
Exam Tip: Remember the order of sedimentation: Nuclei → Mitochondria → Lysosomes/ER → Ribosomes. A useful mnemonic: Naughty Mice Love Running.
For greater resolution, a density gradient (e.g., of sucrose solution, with the densest solution at the bottom) can be used. The sample is layered on top and centrifuged at very high speeds. Organelles migrate through the gradient until they reach the point where their density equals that of the surrounding solution (the isopycnic point), forming distinct bands. This allows separation of organelles with similar sizes but different densities.
Two key concepts underpin all microscopy:
Key Definition: Resolution (resolving power) is the ability to distinguish between two closely spaced objects as separate entities. It depends on the wavelength of the radiation used — shorter wavelengths provide better resolution.
The formula triangle to remember is:
I = A × M
Where:
Rearranged:
Exam Tip: Always ensure that units are consistent before calculating. Convert all measurements to the same unit (µm is usually most convenient). Remember: 1 mm = 1000 µm; 1 µm = 1000 nm.
A mitochondrion has an actual length of 2 µm. On an electron micrograph it appears as 50 mm long.
Magnification = image size ÷ actual size = 50 mm ÷ 0.002 mm = ×25 000
(Note: 2 µm = 0.002 mm, so the calculation uses consistent units.)
Electron micrographs often include a scale bar rather than stating magnification directly. To use a scale bar:
| Feature | Light Microscope | TEM | SEM |
|---|---|---|---|
| Radiation | Visible light | Electrons | Electrons |
| Maximum resolution | ~200 nm | ~0.1 nm | ~3–10 nm |
| Maximum magnification | ~×1500 | ~×500 000 | ~×200 000 |
| Image type | 2D (colour) | 2D (black and white) | 3D (black and white) |
| Specimen | Live or dead; thin sections or whole mounts | Dead; ultrathin sections in vacuum | Dead; surface-coated in vacuum |
| Lenses | Glass | Electromagnets | Electromagnets |
| Staining | Coloured dyes | Heavy metal salts | Metal coating (e.g., gold) |
| Cost | Relatively low | Very high | Very high |
Exam Tip: A frequent exam question asks you to explain why a TEM has greater resolution than a light microscope. The answer must refer to the shorter wavelength of the electron beam compared with visible light, which allows two closely spaced points to be distinguished.
An artefact is a structure visible in a micrograph that is not a genuine part of the specimen but has been introduced during preparation. Examples include:
Scientists minimise artefacts by using careful preparation techniques and comparing images from multiple specimens, ideally using more than one preparation method (cryo-EM avoids chemical fixation by freezing samples at high pressure, preserving them in a near-native state, and is increasingly important in structural biology).
The classical TEM preparation is a multi-step process, each step a potential source of artefact:
Each step takes hours; total preparation often spans days. The resulting micrograph is a static 2D snapshot of a chemically-modified specimen — a long way from the living cell.
A revolution in EM over the last decade has been cryo-electron microscopy (cryo-EM), recognised by the 2017 Nobel Prize in Chemistry (Dubochet, Frank, Henderson). The technique:
Cryo-EM avoids the chemical fixation, dehydration and staining steps that introduce artefacts; it preserves macromolecular complexes in physiological buffer; and it is particularly powerful for large flexible complexes (ribosomes, ion channels, viruses) that resist crystallisation. The technique is changing structural biology and explaining how the COVID-19 spike protein was characterised so rapidly. While not in the AQA spec, awareness of cryo-EM positions a candidate for university interview questions on modern microscopy.
Biological stains exploit specific chemical interactions to render different cellular components visible. Common stains and their targets:
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