Part 1: Magnification and resolution

To study cells well, biologists need two different things: bigger images and clearer images. Those are not the same.

Magnification tells you how many times larger the image is than the real object.

Resolution tells you how close two points can be while still being seen as separate. Higher resolution means more detail.

This is why more magnification is not automatically better. If resolution does not improve, the image just becomes larger and blurrier.

Use:

You can rearrange it to find real size:

Always convert both values into the same units first.

Worked example:

• image size = 20 mm
• real size = 5 μm
• 20 mm = 20,000 μm
• magnification = 20,000 ÷ 5 = ×4000

Part 2: Optical microscopes and measuring size

An optical microscope uses visible light and glass lenses. In a compound microscope, light passes through the specimen, then the objective lens and eyepiece lens magnify the image.

The biggest strength of the optical microscope is that it can be used with living specimens. It is also cheaper, simpler to prepare, and can show natural colour.

Its key limitation is resolution. Visible light has a relatively long wavelength, so the best resolution is about 0.2 μm (200 nm). That means structures closer than this cannot be seen as separate.

Measuring size with an optical microscope

To measure a cell under a light microscope, you use an eyepiece graticule and a stage micrometer.

1. Put the eyepiece graticule in the microscope eyepiece.
2. Place the stage micrometer on the stage. This has a known scale.
3. Line up the two scales using the chosen objective lens.
4. Work out how much one graticule division is worth.
5. Replace the stage micrometer with the specimen and measure the specimen in graticule units.
6. Convert the graticule measurement into a real size.

Important: calibration must be repeated for each objective lens, because magnification changes the value of one graticule unit.

Worked example:

• 10 stage micrometer divisions line up with 40 eyepiece graticule divisions
• 1 stage micrometer division = 10 μm
• so 10 stage micrometer divisions = 100 μm
• 40 graticule divisions = 100 μm
• 1 graticule division = 2.5 μm

If a cell measures 12 graticule divisions, its real diameter is:
12 × 2.5 μm = 30 μm

Part 3: TEM, SEM and artefacts

Electron microscopes were developed because light microscopes cannot resolve the ultrastructure of cells well enough. Electrons have a much shorter wavelength than visible light, so electron microscopes have much higher resolving power.

Both types of electron microscope:

• use a beam of electrons
• use electromagnets rather than glass lenses to focus the beam
• must operate in a near-vacuum because air molecules would scatter the electrons
• cannot be used to observe living specimens directly

A transmission electron microscope (TEM) passes electrons through a very thin specimen. Denser parts absorb more electrons and appear darker. TEM images are:

• very high resolution, around 0.1 nm in theory
• two-dimensional
• good for internal cell ultrastructure

A scanning electron microscope (SEM) scans electrons across the specimen surface. The scattered electrons are detected and used to build an image. SEM images are:

• lower resolution than TEM, around 20 nm
• three-dimensional
• good for surface detail

Main limitations:

• specimen preparation is complex
• images are produced in greyscale
• preparation can introduce artefacts
• the vacuum means living specimens cannot be studied directly

For SEM, preparation is better thought of as fixation, dehydration and metal coating. For TEM, very thin sectioning is also essential.

An artefact is a structure that appears in the image because of preparation, not because it was really present in the cell. Early electron microscopists had to spend time working out which structures were real organelles and which were artefacts. That is one reason why cell biology advanced gradually rather than instantly.

A quick comparison:

Part 4: Cell fractionation and ultracentrifugation

Microscopy shows you where structures are. Cell fractionation lets you separate them so they can be studied on their own.

Stage 1: Homogenisation

Tissue is broken up in a homogeniser and then filtered. The solution used should be:

• ice-cold to slow enzyme activity and reduce damage to organelles
• isotonic so organelles do not gain or lose water by osmosis
• buffered to keep pH stable

The filtrate is called the homogenate.

Stage 2: Ultracentrifugation

The homogenate is spun at increasing speeds. The heaviest structures form a pellet first. The liquid above is the supernatant, which is poured off and spun again at a higher speed.

Typical order of separation:

1. nuclei pellet at low speed
2. in plant tissue, chloroplasts usually pellet before mitochondria because they are more massive
3. mitochondria pellet at the next higher speed
4. lysosomes pellet later
5. very small fragments such as pieces of ER and then ribosomes require the highest speeds

If you are using animal tissue, there is no chloroplast fraction.

This is the principle of differential centrifugation: bigger and heavier components sediment first, lighter ones later.

Part 5: Choosing the right method

A simple way to choose between the methods is to ask what kind of answer you want.

• If you want to look at living cells or measure a whole cell, use an optical microscope.
• If you want the finest detail of internal ultrastructure, use a TEM.
• If you want a 3D surface view, use an SEM.
• If you want to test what an organelle does biochemically, use cell fractionation and ultracentrifugation first.