Methods of Studying Cells

Magnification and Resolution

Microscopes are used to study the structure of cells and their organelles. They all rely on two key principles:

• Magnification — how many times bigger an image is than the real object
• Resolution — the ability to distinguish between two points that are close together. This is determined by the wavelength used — a shorter wavelength gives higher resolution; a longer wavelength gives lower resolution.

Different types of microscope use different sources — light or electrons — and this affects how much detail they can show.

Magnification Formula

$$ \text{Magnification} = \frac{\text{size of image}}{\text{size of real object}} $$

Rearranged:

• Real size = image size ÷ magnification
• Image size = real size × magnification

Practice — Magnification Calculations

Use the image below to work out each question. The image size of each feature is shown directly on the diagram. Click Show working to reveal the step-by-step solution.

Types of Microscope

There are two main types of microscope used to study cells: optical (light) and electron. Electron microscopes are further split into two types: TEM and SEM.

Optical (Light) Microscope

• Uses light to form the image
• Wavelength is long (longer than that of electrons)
• Maximum resolution ≈ 0.2 μm — anything smaller than this cannot be seen as a separate structure
  • Cannot see ribosomes, ER or lysosomes (all smaller than 0.2 μm)
  • Can see the nucleus and sometimes mitochondria (no internal detail)
• Maximum magnification ≈ × 1500
• Can be used to view living cells in colour

Electron Microscope

• Uses a beam of electrons to form the image
• Wavelength of electrons is much shorter than light → much higher resolution
• Higher resolution and magnification than optical microscopes
• Specimens must be in a vacuum, so cells are dead (no living cells)

Transmission Electron Microscope (TEM)

• Uses electromagnets to focus a beam of electrons that is transmitted through the specimen
• Denser parts of the specimen absorb more electrons → appear darker in the image
• Produces 2D images
• Higher resolution than SEM → shows the internal structure of organelles (e.g. cristae in mitochondria, thylakoids in chloroplasts)
• Limitation: requires thin specimens

Scanning Electron Microscope (SEM)

• A beam of electrons is scanned across the surface of the specimen
• Reflected/scattered electrons are detected to build up an image
• Produces 3D images showing the surface of the specimen
• Can use thicker specimens than TEM
• Lower resolution than TEM

Compare the three microscopes

Test yourself — click a button to reveal that column.

Cell Fractionation

Cell fractionation breaks open cells and separates their organelles so each type can be studied individually. There are two main stages: homogenisation & filtration, then ultracentrifugation.

Before any of these steps begin, the cells are placed in a solution that must be:

• Ice-cold — to reduce the activity of hydrolytic enzymes that would damage the organelles
• Isotonic — the same water potential as the organelles, preventing osmotic damage (organelles shrinking or bursting)
• Buffered — to maintain a constant pH so enzymes and proteins are not denatured

1. Homogenisation and Filtration

The cells are vibrated or ground in a blender. This breaks open the cell-surface membrane and releases the organelles into the solution.

The mixture is then filtered through a gauze to remove large debris such as connective tissue and unbroken cells. The organelles pass through, leaving a filtrate that contains the mixture of organelles ready for separation.

2. Ultracentrifugation

The filtrate is spun in a centrifuge at increasing speeds to separate the organelles by density (heaviest first):

1. The filtrate is poured into a tube and spun at a low speed.
2. The heaviest organelles (e.g. nuclei) move to the bottom of the tube, forming a pellet.
3. The remaining organelles stay suspended in the liquid above — called the supernatant.
4. The supernatant is drained into a new tube and spun at a higher speed, repeating the process.
5. This continues at progressively higher speeds until each type of organelle has been separated.

Order of pelleting (heaviest to lightest): nuclei → chloroplasts → mitochondria → lysosomes → endoplasmic reticulum → ribosomes.

Identifying Starch Grains

A common required-practical activity for studying plant cells under an optical microscope:

• Place a thin section of plant tissue on a slide
• Add a drop of iodine in potassium iodide solution
• Cover with a coverslip and view under the optical microscope
• Starch grains turn blue-black — they show up clearly against the rest of the cell

Artefacts vs Organelles

An artefact is a structure seen in a microscope image that is not actually part of the real cell — e.g. air bubbles, distortion or debris from preparing the specimen.

For a long time, scientists couldn’t tell which small structures were real organelles and which were artefacts. As new techniques developed (especially the electron microscope), images became clearer and more reproducible — letting the scientific community confirm which structures were real (e.g. mitochondria, ER) and rule out artefacts.

Exam Question Practice