AS-LEVEL AQA BIOLOGY NOTES 
TOPIC 3: methods of studying cells

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Light & Electron Microscopy

Using a light microscope:

Light microscopes are the most commonly used microscopes used to examine cells. This is because the equipment used is relatively inexpensive, requires minimal user training, and yields good results in terms of identifying cell structures and activity.
  • A number of lenses are used to produce an image
  • Image viewed directly through eyepiece
  • Light passes from bulb under the stage, through condenser lense, then through specimen
  • Beam of light then focused through objective lens, then through eyepiece lens
  • Four different objective lenses (x4, x10, x40, x100) allow for specimen to be viewed at different magnifications
  • Eyepiece lens magnifies image again, usually x10
  • Total magnification is given by multiplying the magnification of the 2 lenses
  • Magnification possible with light microscope: up to 1500x
Magnification
  • Up to 1500x in total
Resolution
  • 200nm
  • Limited by the wavelength of visible light
Specimen Preparation
  • Staining
  • Applying a coloured stain to the sample which binds to certain chemicals/structures, improving their visibility
  • Acetic orcein stains DNA dark red
  • Gentian violet stains bacterial cell walls
  • Sectioning
  • Specimen is embedded in wax to preserve structure of sample cell walls while cutting them into a thin slice
  •     
To correctly use a light microscope, you also need to calibrate the eye piece graticule using the stage micrometer - this step allows you to measure the size of cells and structures that you are observing. To do this, align the stage micrometer (a microscope slide with a scale on it) with the eye piece graticule, then use the reading from the scales to calculate the calibration factor from the objective lens.
Advantages
Disadvantages
  • Wide range of specimens can be observed
  • Specimens can be alive
  • Specimens can be whole, or embedded in wax ten sectioned
  • Non-coloured specimens must be stained for specific organelles or molecules
  • Relatively low resolution does not give detailed information
Using an electron microscope:
A Transmission Electron Microscope (TEM) emits an electron beam through a very thin prepared sample. Electrons penetrate the denser parts of the sample with greater difficulty and this gives the contrast in the 2D image produced.
A Scanning Electron Microscope (SEM) emits an electron beam directly onto a sample such that none of the electrons penetrate it. Instead, they ‘bounce off’ the sample and are received on a sensor, producing a 3D image.
Magnification
  • TEM: Up to x500, 000
  • SEM: Up to x100, 000


Resolution
  • 0.1nm
  • 2000x times more than light microscope
  • Produces detailed images
Specimen Preparation
    • Specimen needs to be prepared correctly
    • Fixed to make it firm
    • Dehydrated and embedded in resin
    • Stained using metal salts or metal particles
    • Mounted on a copper grid
    • Placed in a vacuum
  • Staining
    • Specimens are stained with metal salts or particles
    • This causes electrons to scatter differently, giving contrast

Magnification
  • The degree to which the size of an image is larger than the object itself
Resolution
  • The degree to which it is possible to distinguish between two points on an object that are very close together
  • The higher the resolution, the greater the detail that you can see
​Advantages
Disadvantages
  • Produces detailed images of the structures inside cells
  • SEM produces detailed 3D images showing contour of cells
  • Electron beams deflected in air, so sample must be in a vacuum
  • Samples must be dead
  • Extremely expensive
  • Large piece of equipment
  • Use requires a high degree of skill and training
1.  Light microscope:
2.  Transmission electron microscope:
3.  ​Scanning electron microscope:
The following formula can help us determine the actual size of the structure we are looking at through the microscope, if we know the magnification and the image size. It can also help us determine the magnification if we know the actual object size, and the image size.
E.g. we look through a light microscope and observe squamous epithelial cells. Through the microscope, they appear 50mm in diameter. The lens piece we are using as a magnification of x1200. What is the actual diameter of the cell?

Magnification = Image size Object size
Object size     = Image size Magnification
            = 50 1200
            = 0.0416mm
            = 41.6ųm


  • Magnification = Number of times larger an image appears compared to the real size of the object
  • Resolution = The ability to distinguish between two adjacent but separate points

Fractionation and Centrifugation
  • Used to separate cell components
  • First, tissues and cells are disrupted - there are several ways of achieving this
    • Lytic reactions
  • Then the products are passed through ultracentrifugation
  • Rapid spinning in a machine
  • Force of gravity separates the cell components for closer observation
EXAM TIP
Get familiar with the units mm, ųm and nm. You’re expected to be confident converting between them.
1mm = 1000ųm
1 ųm = 1000nm