Microscopes

• What is the definition magnification?

  Magnification is how many times bigger the image is compared to the actual object.
• What does resolution refer to in microscopy?

  Resolution is the minimum distance apart two objects can be for them to appear separate.
• What factors affect resolution?

  Resolution depends on the wavelength of light or radiation used.
• How should you ensure the units are consistent when calculating magnification?

  You need to make sure the object and image are in the same unit, such as not mixing mm and nm.
• What are the equivalent units for length in microscopy?

  - Kilometre (km) = x103

  - Metre (m) = 1

  - Millimetre (mm) = x10-3

  - Micrometre (µm) = x10-6

  - Nanometre (nm) = x10-9
• What are the steps to use a light microscope effectively?

  1. Raise condenser to maximum position
  2. Adjust light to maximum
  3. Move scanning (4x) into position
  4. Centre specimen on stage
  5. Adjust binocular lens distance
  6. Bring course focus into adjustment
  7. Bring fine focus into adjustment
  8. Adjust condenser (iris) position for optimum illumination
  9. Adjust and focus low power and high dry lens to view specimen
• What is the resolution limit of light microscopes?

  Light microscopes cannot resolve structures closer than 200 nm.
• Why can light microscopes view living specimens?

  Light microscopes can view living specimens because they use visible light and do not require a vacuum.
• What is the maximum magnification of light microscopes?

  Cheek cells can be magnified up to 1500 times with a light microscope.
• What is the wavelength range of electrons used in electron microscopy?

  Electrons have wavelengths of 0.01 nm to 0.001 nm.
• What are the two types of electron microscopes?

  The two types of electron microscopes are Transmission Electron Microscopes (TEM) and Scanning Electron Microscopes (SEM).
• How does a Transmission Electron Microscope (TEM) work?

  The beam passes through the specimen, with parts absorbing electrons appearing dark and others lighter.
• What are the advantages and disadvantages of using a TEM?

  Advantages:

  - Small objects can be seen

  - High resolution (0.1 nm)

  - Wavelength of electrons is shorter

  
  

  Disadvantages:

  - Cannot look at living cells

  - Must be in a vacuum

  - Requires extremely thin specimens

  - Preparation may create artefacts

  - Does not produce color images
• How does a Scanning Electron Microscope (SEM) create images?

  SEM directs a beam on the surface of the specimen, building a 3D image from scattered electrons.
• What are the advantages and disadvantages of using a SEM?

  Advantages:

  - Higher resolution than light microscopes (20 nm)

  - Produces 3D images

  
  

  Disadvantages:

  - Cannot view living specimens

  - Requires extremely thin specimens

  - Complex staining technique
• Compare the light microscope and electron microscope.

  Light Microscope:

  • Uses light to view specimens
  • Can view living specimens
  • Useful for whole cells and tissues

  
  

  Electron Microscope:

  • Uses electron beams
  • Cannot view living specimens
  • Higher magnification and resolution
  • Useful for organelles and viruses
• What is a limitation of electron microscopy regarding specimen condition?

  Electron microscopy requires specimens to be dead.
• What is the process of cell fractionation?

  Cell fractionation involves breaking up cells and separating the different organelles.
• What is the purpose of using a buffered solution in cell fractionation?

  The buffered solution maintains the same water potential as the cell.
• How are cells broken up during homogenation?

  Cells are broken up by a homogenizer (blender) to release the organelles.
• What is the homogenate in cell fractionation?

  The homogenate is the mixture obtained after breaking up the cells.
• What is done to the homogenate after homogenation?

  The homogenate is filtered to remove complete cells and large pieces of debris.
• What are the advantages and limitations of using a TEM to investigate cell structure?


  Advantages:

  - High resolution

  - Can see small objects

  - Shorter wavelength of electrons

  
  

  Limitations:

  - Cannot observe living cells

  - Requires a vacuum

  - Must cut thin sections

  - Preparation may create artefacts

  - Does not produce color images