Microscopes

• Resolution

  The resolution of a microscope is defined as the shortest distance between two points on a specimen that can still be distinguished clearly. The higher the resolution greater the detail you can see.
• Magnification

  The degree to which the size of an image is larger than the object itself. 
• Magnification=

  Image size/ actual size
• There are two types of microscopes:

  • Light Microscopes
  • Electron Microscopes
• Light microscope: light passes through the specimen and is focused by glass lenses. 
• Light Microscopes- Advantages and limitations:  

  • Magnification- most light microscopes are capable of up to x1500. To calculate the magnification in a light microscope = eyepiece lens magnification x objective lens magnification. 
• Light Microscopes- advantages and limitations:

  • Resolution – the maximum resolving power is 200nm. Therefore objects closer together than 200nm will blur together and be seen as one object. This is because of the wavelengths of visible light are from 400-750nm. As light rays of these wavelengths pass through the specimen and the microscope lenses, they are diffracted (spread out) and will overlap each other if they come from points on the specimen that are closer than 200nm.   
• Light Microscopes- advantages and limitations:

  Specimens:

  • A wide range of specimens can be viewed in colour, including living organisms such as daphnia and euglena.
  • Can view thin sections of larger plants and animals and smear preparations of blood and cheek cells.
  • Can’t use it for ultra-structure (small structures in the cell below the resolution limit in size) but can use it for lab analysis of tissues (groups of cells) and research and education. 
• The preparation of specimens for light microscopes involves:

  • Sectioning
  • staining
• Preparation of specimens for light microscopes- sectioning

  • specimens are embedded in wax and then thin sections are cut without distorting the structure of the specimen. 
• Preparation of specimens for light microscopes- Staining

  • Light passes through the specimen and many structures in the cell are transparent so cannot be seen.
  • Coloured stains are chemicals that bind to chemicals in or on the specimen.
  • Some structures take up the stain more than others do, this allow you to see contrast between components. 
• An example of a stain is Acetic Orcein, which stains DNA dark red.
• An example of a stain is Gentian Violet, which stains bacterial cell walls.
• Electron microscopes- Use beams of electrons which are focused by magnets. The wavelength of electrons is smaller so electron microscopes have better resolution. 
• The higher resolution of electron microscopes allows you to see in detail the structure of the internal components of cells which are not clear when using a light microscope. This is called the ultrastructure.  
• The two types of electron microscopes:

  • Transmission electron microscopes
  • Scanning electron microscopes
• Transmission electron microscopes (TEM):

  • Electron beam passes through very thin section of sample.
  • The sample is prepared by being dipped in heavy metals such as lead. Some structures absorb more than others therefore appear dark. Other parts of the specimen allow the electrons to pass through so appear bright 
  • Final image is produced on a screen and can be photographed to give a photomicrograph. It is 2D and black and white- Colour can be added to the image by computer 
  • The useful magnification possible is x500,000 because… 
  • the resolution is 0.1nm 
• TEMs:

  • magnification= x500,000
• TEMs:

  • resolution= 0.1nm
• TEMs:

  • Final image is black and white.
  • Final image is 2D
• Light microscopes:

  • magnification= x1500
• Light microscopes:

  • resolution= 200nm
• Scanning Electron Microscopes (SEMs):

  • Electron beam directed on to the surface of the sample (not through). 
  • Electrons bounce off sample and are detected by several sensors 
  • Final computed image is 3D and black and white - colour can be added to the image by computer. The image will be of the surface detail of the specimen.  
  • Magnification possible is x100,000 
  • The possible resolution is 20nm.
• SEMs:

  • magnification= x100,000
• SEMs:

  • resolution= 20nm
• SEMs:

  • Final image is black and white.
  • Final image is 3D.
• Limitations of electron microscopes:

  • The whole system must be in a vacuum so living specimens cannot be observed. 
• Limitations of Electron Microscopes:

  • A complex staining process is required and even then the image is not in colour. 
• Limitations of electron microscopes:

  • The specimen must be extremely thin (for a TEM). 
• Limitations of electron microscopes:

  • The image may contain artefacts. Artefacts are things that result from the way the specimen is prepared. They may then appear on the finished photomicrograph but are not part of the natural specimen. 
  • There was a considerable period of time before scientists distinguished between artefacts and cell organelles