AQA A-level Biology: Microscopy, Magnification and Cell Fractionation (7402)

This guide is aligned to the AQA AS and A-level Biology specification (A-level 7402), for first teaching 2015 with exams from June 2016, Version 1.5. It covers section 3.2.1.3, Methods of studying cells. Nothing in this section is marked as A-level only. Use it with the revision notes and the practice questions. The organelles you are looking at are covered in the cell structure revision notes. Course hub: AQA A-level Biology. Printable checklist. If you are unsure where you stand, try the free diagnostics.

What the specification asks for

Specification wording (3.2.1.3)	What you must be able to do
Principles and limitations of optical, transmission electron and scanning electron microscopes	Explain how each forms an image, and what each cannot do
Measuring the size of an object viewed with an optical microscope	Calibrate an eyepiece graticule against a stage micrometer and use it
Difference between magnification and resolution	Define both and explain why more magnification need not give more detail
magnification = size of image / size of real object (mathematical skill 1.8)	Use and rearrange the formula, converting units
Principles of cell fractionation and ultracentrifugation	Describe how organelles are separated and why each step is needed
Artefacts and cell organelles	Appreciate why scientists took a long time to decide which structures were real

The specification links this section to apparatus and techniques d, e and f (light microscope use including a graticule, scientific drawing, and qualitative reagents). It suggests that you could use iodine in potassium iodide solution to identify starch grains in plant cells.

Magnification and resolution

Magnification tells you how much bigger the image appears than the specimen really is. Resolution is the smallest separation at which two neighbouring points are still seen as two, not one blob.

These are different. A blurred photograph enlarged on a screen has high magnification and poor resolution: you see a bigger blur, not more detail. Once you pass the resolution limit of an instrument, extra magnification adds nothing.

Resolution depends on the wavelength of the radiation used. Shorter wavelengths can separate points that are closer together. Visible light has a relatively long wavelength, so optical microscopes have a low resolution limit. Electrons behave as radiation with a far shorter wavelength, so electron microscopes resolve much finer detail.

The three microscopes

Optical (light) microscope

Principle: light passes through a thin specimen and is focused by glass lenses. You view the image through the eyepiece.

Strengths: it is cheap and portable, specimens can be living, colour is visible, and preparation is quick (a temporary mount with a stain such as iodine in potassium iodide solution, which turns starch grains blue-black).

Limitations: the resolution limit is about 0.2 µm, and the useful magnification is around 1500 times. You cannot see ribosomes, membranes or the internal detail of mitochondria. The specimen must be thin enough for light to pass through.

Transmission electron microscope (TEM)

Principle: a beam of electrons passes through an extremely thin slice of specimen. Electromagnets focus the beam. Denser regions, stained with heavy metals, absorb or scatter more electrons, and the image forms on a screen or detector.

Strengths: very high resolution, so internal ultrastructure such as membranes, ribosomes and the cristae of mitochondria can be seen.

Limitations:

• The specimen sits in a vacuum, because air would scatter the electrons, so living material cannot be examined.
• The specimen must be cut into extremely thin sections, so a 2D slice is seen and a 3D structure must be inferred from many sections.
• The image is black and white.
• Preparation is long and complex, which can introduce artefacts.

Scanning electron microscope (SEM)

Principle: a narrow beam of electrons is scanned across the surface of the specimen. Electrons knocked off or reflected from the surface are detected and used to build the image.

Strengths: the specimen does not need to be thin, and the image shows the 3D surface. Resolution is far better than an optical microscope.

Limitations: the resolution is lower than that of a TEM. The specimen is in a vacuum, so it is not living, and the image is black and white. You see surfaces only, not internal structure.

Side by side

Feature	Optical	TEM	SEM
Radiation	Light	Electrons through specimen	Electrons scanned over surface
Typical resolution limit	about 200 nm	about 0.5 nm	about 3 to 10 nm
Specimen	Thin, can be living	Extremely thin, dead	Any thickness, dead
Image	Colour, 2D	Black and white, 2D	Black and white, 3D surface
Shows	Cells, large organelles	Ultrastructure	Surface features

The resolution figures are typical values for comparison, not exact values for every instrument. Learn the order: TEM best, then SEM, then optical.

Measuring size with an optical microscope

The eyepiece contains a graticule, a ruler with arbitrary divisions. Its divisions have no fixed size, because their real length depends on the objective lens. You calibrate it with a stage micrometer, a slide with a ruled scale of known length. In this guide each stage micrometer division is 10 µm.

1. Put the stage micrometer slide under the objective and bring its ruled scale into focus.
2. Turn the eyepiece until the graticule sits beside the micrometer scale, and make one pair of lines coincide at the left-hand end.
3. Find a second point where lines on both scales coincide.
4. Count the divisions on each scale between the two points.
5. Calculate the real length of one eyepiece division.
6. Replace the micrometer with the specimen and measure in eyepiece divisions.
7. Recalibrate whenever you change objective lens.

Worked example: calibration

At the ×10 objective, 40 eyepiece divisions line up with 28 stage divisions.

Length of 28 stage divisions = 28 × 10 µm = 280 µm. One eyepiece division = 280 ÷ 40 = 7 µm. A cell that spans 5 eyepiece divisions is 5 × 7 = 35 µm long.

At the ×40 objective, 100 eyepiece divisions line up with 22 stage divisions. Length = 22 × 10 = 220 µm, so one eyepiece division = 220 ÷ 100 = 2.2 µm. A nucleus spanning 5 divisions is 5 × 2.2 = 11 µm across.

The eyepiece division is smaller at higher power, which is why one calibration cannot be reused.

The magnification formula

In symbols, with M for magnification, I for image size and A for actual size: M = I ÷ A.

Rearranged:

• I = M × A
• A = I ÷ M

Always put image and object in the same unit before dividing. Convert millimetres to micrometres by multiplying by 1000, and micrometres to nanometres by multiplying by 1000. Magnification has no unit.

A useful triangle: write I over (A × M), where I is image size, A is actual size and M is magnification. Cover the one you want.

Worked example 1: TEM photograph

A mitochondrion is measured on a TEM print as 62 mm long. Its real length is 2.5 µm.

Image size = 62 mm = 62 × 1000 = 62 000 µm. Magnification = 62 000 ÷ 2.5 = ×24 800.

Worked example 2: using a scale bar

A micrograph has a scale bar 32 mm long labelled 2 µm. A cell measures 112 mm across.

Magnification = 32 000 µm ÷ 2 µm = ×16 000. Real cell width = 112 mm ÷ 32 mm × 2 µm = 7.0 µm.

Worked example 3: a drawing

A student draws a plant cell 84 mm wide. Using the graticule, the real cell is 70 µm wide.

Magnification = 84 000 ÷ 70 = ×1200.

Order of magnitude

Mathematical skill 1.8 also covers order-of-magnitude comparisons. A cell is 20 µm across and a mitochondrion in it is 2 µm long. The ratio is 20 ÷ 2 = 10, so the cell is one order of magnitude larger. A ratio of 100 is two orders, 1000 is three.

Cell fractionation and ultracentrifugation

Cell fractionation separates organelles so that each can be studied. Organelles can then be tested for their function, or examined with a microscope.

Step 1: homogenisation

The tissue is broken up in a blender or homogeniser to burst the cells and release their contents. The solution is:

• Cold, to reduce enzyme activity, so enzymes released from damaged lysosomes do not digest the organelles.
• Isotonic, with the same water potential as the cells, so organelles do not gain water and burst or lose water and shrink by osmosis.
• Buffered, to hold the pH constant, so enzymes and other proteins do not denature.

Step 2: filtration

The homogenate is filtered to remove unbroken cells and large debris such as cell wall fragments. The filtrate contains the organelles.

Step 3: ultracentrifugation

The filtrate is placed in a tube in a centrifuge and spun at a low speed first. The largest, densest organelles are thrown to the bottom as a pellet. The liquid above, the supernatant, is poured into a new tube and spun faster. This is repeated at increasing speeds, and each time a lighter organelle forms the next pellet.

The usual order of separation is:

1. Nuclei
2. Chloroplasts (plant tissue only)
3. Mitochondria
4. Lysosomes
5. Endoplasmic reticulum (as small membrane pieces)
6. Ribosomes

The exact speeds depend on the tissue and the centrifuge, so a question will give them if it needs them.

Worked reasoning: why that order?

Pellet formation depends on mass and density. Nuclei are the largest organelles, so they sediment at the lowest speed. Ribosomes are the smallest, so they need the highest. If a question says a pellet obtained at low speed contains DNA and no respiratory activity, that pellet is nuclei.

Artefacts and organelles

An artefact is a structure seen in a microscope image that is not part of the living cell. It is produced by preparation or observation: fixing, dehydrating, staining, sectioning, a vacuum, or the electron beam. Examples are folds or scratches in a section, air bubbles, and shrunken membranes.

The specification asks you to appreciate that scientists spent a considerable period distinguishing between artefacts and real organelles. When a new microscope or stain shows an unfamiliar structure, nobody can say at once whether it is real. Scientists gain confidence if the structure:

• appears with several different preparation methods, since an artefact usually depends on one;
• is seen with different types of microscope;
• is seen by different research groups who repeat the work;
• has a function that can be shown, for example by testing a fractionated sample.

Common errors

• Writing that electron microscopes have a higher “magnification” as the reason for detail. The reason is the higher resolution, due to the shorter wavelength.
• Saying a TEM shows 3D surface detail. That is the SEM.
• Not converting mm to µm before dividing.
• Reusing a graticule calibration after changing objective.
• Forgetting that “isotonic” protects organelles from osmosis, and “buffered” protects proteins from pH change.
• Describing the pellet as the “heavy liquid”. The pellet is the solid at the bottom; the supernatant is the liquid.

Official syllabus

AQA AS and A-level Biology specification (7401 and 7402), for first teaching 2015, exams June 2016 onwards, Version 1.5, published by AQA. Section 3.2.1.3 Methods of studying cells.