Microscopy

• Use of microscopes 


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


  How much bigger an image appears compared to the original object calculated using the following formula:

  Image size=Actual size x Magnification
• Resolution 


  the ability to distinguish between objects that are close together

  • The resolution of a light microscope is limited by the wavelength of light
• Microscope diagram

  
• Light Microscopes

  -use light

  -lower resolution than electron microscopes-they have a maximum resolution of around 0.2 micrometres.

  -2D image

  -usually used to look at whole cells or tissues (eukaryotic cells, their nuclei and possibly mitochondria and chloroplasts)

  -living or dead specimens

  -cannot be used to observe smaller organelles such as ribosomes, the endoplasmic reticulum or lysosomes

  -maximum magnification of x1500
• Laser scanning Microscope (Confocal)

  -uses laser beams to scan a specimen usually tagged with fluorescent dye

  -laser causes the dye to fluoresce-give off light. This light is then focused through a pinhole onto a detector. The detector is hooked up to a computer, which generates an image (can be 3D).

  -pinhole=any out-of-focus light is blocked, so it produces a clearer image than light microscopes
• Laser Scanning Microscope Advantages


  • can be used on thick or 3-D specimens
  • allow the external, 3-D structure of specimens to be observed
  • high resolution structure of the cytoskeleton in cells
  • structure of the cytoskeleton in cells visible
• Laser Scanning Microscope Disadvantages


  • a slow process and takes a long time to obtain an image
  • laser has the potential to cause photodamage to the cells
• Electron Microscopes

  • use electrons instead of light to form an image
  • higher resolution than light microscope so give more detailed images
• Transmission Electron Microscope


  • use electromagnets to focus a beam of electrons that are transmitted through the specimen
  • 2D or 3D image
  • have a maximum resolution of around 0.0002 micrometres
  • have a maximum magnification x1000000 or more
  • Denser parts of the specimen absorb more electrons
  • makes the denser parts appear darker on the final image produced
• Transmission Electron Microscope Advantages


  • high-resolution images
  • allows the internal structures within cells (or even within organelles) to be seen
• Transmission Electron Microscope Disadvantages



  • can only be used with very thin specimens or thin sections of the object being observed
  • cannot be used to observe live specimens
  • lengthy treatment required to prepare specimens means that artefacts can be introduced
  • do not produce a colour image 
• Scanning Electron Microscopes


  • scan a beam of electrons across the specimen
  • bounces off the surface of the specimen and the electrons are detected, forming an image
  • =SEMs can produce 3D images that show the surface of specimens
  • have a maximum resolution of around 0.002 micrometres
  • have a maximum magnification < x500000
• Scanning Electron Microscopes Advantages


  • can be used on thick or 3-D specimens
  • show the surface of the specimen
  • allow the external, 3D structure of specimens to be observed
• Scanning Electron Microscopes Disadvantages


  • give lower resolution images than TEMs
  • cannot be used to observe live specimens 
  • do not produce a colour image 
• How to use a Light Microscope


  
1)Start by clipping the slide containing the specimen you want to look at onto the stage.

  2)Select the lowest-powered objective lens

  3)Use the coarse adjustment knob to bring the stage up to just below the objective lens

  4)Look down the eyepiece. Use the coarse adjustment knob to move the stage downwards, away from the objective lens until the image is roughly in focus.

  5)Adjust the focus with the fine adjustment knob, until you get a clear image of what's on the slide.

  6)Swap to a higher-powered objective lens and refocus.
• What is an eyepiece graticule?



  It is a transparent ruler with no units that is fitted onto the eyepiece.
• What is a stage micrometer?


  It is a microscope slide with an accurate scale(with units) and it's used to work out the value of divisions on the eyepiece graticule at a specific magnification.
• How to use a stage micrometer and eyepiece graticule? 


  1)Line up the eyepiece graticule and the stage micrometer.

  2)Each division on the stage micrometer is 0.1mm long.

  3)At this magnification, 1 division on the stage micrometer is the same as 4.5 divisions on the eyepiece graticule.

  4)To work out the size of 1 division on the eyepiece graticule you do 0.1/number of divisions on the eyepiece graticule.

  5)Then you multiply this by how many eyepiece divisions a specimen is under the microscope to get its' length.
• How to use a Microscope

  
• Limitations


  • The size of cells or structures of tissues may appear inconsistent in different specimen slides
  • Cell structures are 3D and the different tissue samples will have been cut at different planes resulting in this inconsistencies when viewed on a 2D slide
  • Optical microscopes do not have the same magnification power as other types of microscopes and so there are some structures that can not be seen
  • The treatment of specimens when preparing slides could alter the structure of cells
• Why are stains used?


  • Many tissues that are used in microscopy are naturally transparent, they let both light and electrons pass through them
  • This makes it very difficult to see any detail in the tissue when using a microscope
  • Stains are often used to make the tissue coloured/visible
• Staining for light microscopy


  • Coloured dyes are used when staining specimens
  • dyes used absorb specific colours of light while reflecting others
  • Certain tissues absorb certain dyes, which dye they absorb depends on their chemical nature
  • Specimens or sections are sometimes stained with multiple dyes to ensure the different tissues within the specimen show up - this is known as differential staining
  • important to remember that most of the colours seen in photomicrographs are not natural
  • Chloroplasts don't need stains
• Staining for electron microscopy


  • When using TEMs the specimen must be stained in order to absorb the electrons
  • Unlike light, electrons have no colour
  • The dyes used for staining cause the tissues to show up black or different shades of grey
  • Heavy-metal compounds are commonly used as dyes because they absorb electrons well
  • Osmium tetroxide and ruthenium tetroxide are examples
  • Any of the colour present in electron micrographs is not natural and it is also not a result of the staining
  • Colours are added to the image using an image-processing software