Methods of studying cells (AQA A Level Biology)

The Principles & Limitations of Microscopy

• Microscopes can be used to analyse cell components and observe organelles

• Magnification and resolution are two scientific terms that are very important to understand and distinguish between when answering questions about microscopy (the use of microscopes):

  • Magnification tells you how many times bigger the image produced by the microscope is than the real-life object you are viewing

  • Resolution is the ability to distinguish between objects that are close together (i.e. the ability to see two structures that are very close together as two separate structures)

• There are two main types of microscopes:

  • Optical microscopes (sometimes known as light microscopes)

  • Electron microscopes

Optical (light) microscopes

• Optical microscopes use light to form an image

• This limits the resolution of optical microscopes

  • Using light, it is impossible to resolve (distinguish between) two objects that are closer than half the wavelength of light

  • The wavelength of visible light is between 500-650 nanometres (nm), so an optical microscope cannot be used to distinguish between objects closer than half of this value

• This means optical microscopes have a maximum resolution of around 0.2 micrometres (µm) or 200 nm

  • This means optical microscopes can be used to observe eukaryotic cells, their nuclei and possibly mitochondria and chloroplasts

  • This also means that optical microscopes cannot be used to observe smaller organelles such as ribosomes, the endoplasmic reticulum or lysosomes

• The maximum useful magnification of optical microscopes is about ×1500

Electron microscopes

• Electron microscopes use electrons to form an image

• This greatly increases the resolution of electron microscopes compared to optical microscopes, giving a more detailed image

  • A beam of electrons has a much smaller wavelength than light, so an electron microscope can resolve (distinguish between) two objects that are extremely close together

• This means electron microscopes have a maximum resolution of around 0.0002 µm or 0.2 nm (i.e. around 1000 times greater than that of optical microscopes)

  • This means electron microscopes can be used to observe small organelles such as ribosomes, the endoplasmic reticulum or lysosomes

• The maximum useful magnification of electron microscopes is about ×1,500,000

• There are two types of electron microscopes:

  • Transmission electron microscopes (TEMs)

  • Scanning electron microscopes (SEMs)

Transmission electron microscopes (TEMs)

• TEMs use electromagnets to focus a beam of electrons

• This beam of electrons is transmitted through the specimen

• Denser parts of the specimen absorb more electrons

  • This makes these denser parts appear darker on the final image produced (produces contrast between different parts of the object being observed)

• Advantages of TEMs:

  • They give high-resolution images (more detail)

  • This allows the internal structures within cells (or even within organelles) to be seen

• Disadvantages of TEMs:

  • They can only be used with very thin specimens or thin sections of the object being observed

  • They cannot be used to observe live specimens (as there is a vacuum inside a TEM, all the water must be removed from the specimen and so living cells cannot be observed, meaning that specimens must be dead, unlike optical microscopes that can be used to observe live specimens)

  • The lengthy treatment required to prepare specimens means that artefacts can be introduced (artefacts look like real structures but are actually the results of preserving and staining)

  • They do not produce a colour image (unlike optical microscopes that produce a colour image)

Scanning electron microscopes (SEMs)

• SEMs scan a beam of electrons across the specimen

• This beam bounces off the surface of the specimen and the electrons are detected, forming an image

  • This means SEMs can produce three-dimensional images that show the surface of specimens

• Advantages of SEMs:

  • They can be used on thick or 3-D specimens

  • They allow the external, 3-D structure of specimens to be observed

• Disadvantages of SEMs:

  • They give lower resolution images (less detail) than TEMs

  • They cannot be used to observe live specimens (unlike optical microscopes that can be used to observe live specimens)

  • They do not produce a colour image (unlike optical microscopes that produce a colour image)