Methods of studying cells, including the principles and limitations of optical, transmission electron and scanning electron microscopes; magnification and resolution; measurement and calibration using an eyepiece graticule and stage micrometer; cell fractionation and ultracentrifugation to separate organelles.

What this dot point is asking

AQA wants you to compare the three microscope types, distinguish magnification from resolution, calibrate measurements with a graticule and stage micrometer, do magnification and size calculations, and describe how cell fractionation separates organelles.

The answer

Magnification versus resolution

The key equation:

Rearrange to find any value. Always convert both measurements to the same unit first. Useful conversions: $1 \text{ mm} = 1000 \text{ micrometres}$ and $1 \text{ micrometre} = 1000 \text{ nanometres}$.

The three microscopes

Optical (light) microscope

Uses light focused by glass lenses. Maximum magnification about times 1500; resolution about 200 nanometres (limited by the wavelength of light). Specimens can be living and in colour, and the equipment is cheap and portable. Cannot resolve small organelles such as ribosomes.

Transmission electron microscope (TEM)

A beam of electrons passes through a very thin specimen; denser regions absorb more electrons and appear darker. Resolution about 0.1 nanometres, so internal ultrastructure (cristae, ribosomes) is visible. But the specimen must be dead and in a vacuum, preparation is complex and can create artefacts, and the image is 2D and not in colour.

Scanning electron microscope (SEM)

A beam scans the surface of a specimen and reflected electrons build a 3D surface image. Resolution is lower than TEM (about 3 to 10 nanometres) but it shows surface detail. Again the specimen is dead and in a vacuum.

Electron microscopes resolve far more than light microscopes because electrons have a much shorter wavelength than light, and resolution is limited by wavelength.

Measuring with a graticule and stage micrometer

An eyepiece graticule is a scale in the eyepiece, but its divisions have no fixed real size, they change with each objective lens. To find the real size of one division you calibrate it against a stage micrometer, a slide engraved with an accurate scale (often 100 divisions of 10 micrometres each).

Procedure:

1. Line up the graticule scale against the stage micrometer.
2. Count how many graticule divisions match a known length on the stage micrometer.
3. Calculate the real value of one graticule division.
4. Remove the stage micrometer and measure your specimen in graticule divisions, then convert.

For example, if 10 graticule divisions line up with 100 micrometres on the stage micrometer, one graticule division equals $\frac{100}{10} = 10$ micrometres. Recalibrate every time you change the objective lens.

Cell fractionation

Cell fractionation separates organelles so they can be studied in bulk.

Preparation. The tissue is placed in a solution that is:

• Cold to reduce enzyme (and especially digestive enzyme) activity that would damage organelles.
• Isotonic (same water potential as the cells) so organelles do not shrink or burst by osmosis.
• Buffered to keep pH constant and protect protein structure.

Homogenisation. The tissue is blended to break open the cells and release the organelles into the solution, then filtered to remove debris.

Ultracentrifugation. The filtered homogenate is spun, slowest first then increasing the speed in stages. The densest organelles sediment first as a pellet; the supernatant is decanted and spun again at a higher speed to pellet the next densest organelle. The order is:

1. Nuclei (most dense)
2. Mitochondria (and chloroplasts in plants)
3. Lysosomes
4. Endoplasmic reticulum
5. Ribosomes (least dense)

Examples in context

Example 1. Choosing a microscope for a task. To watch the movement of cytoplasm (cyclosis) in a living pondweed cell you must use a light microscope, because electron microscopy requires dead specimens in a vacuum. To resolve the cristae inside a mitochondrion you need a TEM, because the light microscope cannot resolve structures below 200 nanometres.

Example 2. Isolating mitochondria for a respiration study. Researchers studying respiration grind liver tissue in cold, isotonic, buffered sucrose, filter the homogenate, then spin it at a low speed to remove nuclei before spinning the supernatant faster to pellet the mitochondria. Keeping the solution cold and isotonic preserves the organelles so their enzyme activity can be measured.

Try this

Q1. Explain why an electron microscope has a higher resolution than a light microscope. [2 marks]

• Cue. Electrons have a much shorter wavelength than light; resolution is limited by wavelength, so shorter wavelength gives higher resolution.

Q2. A cell image is 50 mm wide and the magnification is times 2500. Calculate the actual width in micrometres. [2 marks]

• Cue. $50 \text{ mm} = 50\,000$ micrometres; $50\,000 / 2500 = 20$ micrometres.

Q3. Explain why the solution used in cell fractionation must be isotonic and buffered. [2 marks]

• Cue. Isotonic so organelles do not gain or lose water by osmosis and burst or shrink; buffered to keep pH constant so proteins and organelle structure are not damaged.