Microscopy

• What is a light microscope used for?

  Observing living and dead specimens
• What are the pros and cons of a light microscope?


  Pros: Cheap, portable, easy to use, can study living specimens. Cons: Limited magnification, poor resolution.
• What is the formula to calculate magnification?


  Magnification = Image size / Actual size
• What is the formula to calculate actual object size?


  Actual size = Image size / Magnification
• How do we work out image size?


  Use a ruler and measure the image.
• What is magnification?


  A measure of how much larger the image of a specimen looks under the microscope
• What is resolution?


  The ability to distinguish between to adjacent individual points as separate.
• What resolution are light microscopes limitied to?


  0.2 micrometers
• Why do electron microscopes give a clearer image than light microscopes?


  The wave length of light limits resolution to 0.2 micrometers. The wavelength of electrons allows a resolution of 0.1 nanometers.
• What is a transmission electron microscope be used for?


  Observing the internal ultrastructure of cells under high magnification and resolution
• What is a scanning electron microscope used for?


  Viewing the surface of objets under high magnification and resolution
• What are the limitations of a TEM?


  Subject must be in a vacuum, so no living specimens; Complex preparation; No colour; Specimen must be extremely thin; Preparation of specimen may create artifacts.
• What are TEM artifacts?


  Things created during sample preparation. They can be mistaken for cellular components.
• What is the difference between light and electron microscopes?


  Light uses lenses to focus a beam of light. Electron uses a beam of electrons, focused by magnets.
• What is the resolving power of a TEM compared to a SEM?


  TEM = 0.1nm; SEM = 2.0nm
• What is the difference in images between TEM and SEM?


  TEM gives a 2D image of the contents of a cell. SEM gives a 3D image of the surface of an object.
• What is an eye piece graticule?


  A small ruler fitted to a light microscope's eyepiece. It must be calibrated using a stage micrometer before being used to measure specimens. Each lens needs to be calibrated individually.
• What is a stage micrometer?


  A millimeter long ruler etched onto a slide. it has 100 divisions, each of 0.01mm or 10 micrometers. It is used to calibrate the eyepiece graticule
• How is the eye piece graticule calibrated?

  The eyepiece graticule and stage micrometer are lined up. And the number of divisions that match up are compared. This allows the length of 1 eyepiece division to be calculated.
• What is the formula to calculate magnification?


  Magnification = Image size / Actual size
• What are the maximum resolutions of the different microscopes?


  Light: 200nm; SEM: 20nm; TEM: 0.1nm.
• How does a Transmission Electron Microscope (TEM) work?

  It shoots a beam of electrons at a thin slice of a sample and detects those electrons that pass through.
• What is the primary advantage of using a TEM?

  It allows us to look at a thin section of a sample in very high resolution.
• Why is the TEM particularly useful for studying cells?


  It helps learn about the internal structure of cell components, such as organelles.
• What type of beam does a Scanning Electron Microscope (SEM) use?


  A focused beam of high-energy electrons.
• What type of image does a SEM produce?


  A 3D image of the sample's surface.
• What is a key requirement for using electron microscopes?


  They require a vacuum to prevent air particles from interfering with the electron beam.
• Why must specimens be dead when using electron microscopes?


  Because air particles would interfere with the beam of electrons.
• What are the main differences between Transmission Electron Microscopes (TEM) and Scanning Electron Microscopes (SEM)?


  • TEM uses a beam of electrons to look at thin sections, providing high resolution.
  • SEM uses a focused beam of electrons to reflect off surfaces, giving 3D images.
  • TEM requires thin samples, while SEM does not.