Microscopes (SL IB Biology)

• Microscopes can be used to analyse cell components and observe organelles
• Magnification and resolution are two scientific terms that are very important to understand and distinguish between when answering questions about microscopy (the use of microscopes):
  • Magnification tells you how many times bigger the image produced by the microscope is than the real-life object you are viewing
  • Resolution is the ability to distinguish between objects that are close together (i.e. the ability to see two structures that are very close together as two separate structures)

• There are two main types of microscopes:
  • Optical microscopes (sometimes known as light microscopes)
  • Electron microscopes

Optical (light) microscopes

• Optical microscopes use light to form an image
• This limits the resolution of optical microscopes
  • Using light, it is impossible to resolve (distinguish between) two objects that are closer than half the wavelength of light
  • The wavelength of visible light is between 500-650 nanometres (nm), so an optical microscope cannot be used to distinguish between objects closer than half of this value
• This means optical microscopes have a maximum resolution of around 0.2 micrometres (µm) or 200 nm
  • Optical microscopes can be used to observe eukaryotic cells, their nuclei and possibly mitochondria and chloroplasts
  • They cannot be used to observe smaller organelles such as ribosomes, the endoplasmic reticulum or lysosomes
• The maximum useful magnification of optical microscopes is about ×1500

Electron microscopes

• Electron microscopes use electrons to form an image
• This greatly increases the resolution of electron microscopes compared to optical microscopes, giving a more detailed image
  • A beam of electrons has a much smaller wavelength than light, so an electron microscope can resolve (distinguish between) two objects that are extremely close together
• This means electron microscopes have a maximum resolution of around 0.0002 µm or 0.2 nm (i.e. around 1000 times greater than that of optical microscopes)
  • This means electron microscopes can be used to observe small organelles such as ribosomes, the endoplasmic reticulum or lysosomes
• The maximum useful magnification of electron microscopes is about ×1,500,000
• There are two types of electron microscopes:
  • Transmission electron microscopes (TEMs)
  • Scanning electron microscopes (SEMs)

Transmission electron microscopes (TEMs)

• TEMs use electromagnets to focus a beam of electrons
• This beam of electrons is transmitted through the specimen
• Denser parts of the specimen absorb more electrons
  • This makes these denser parts appear darker on the final image produced (produces contrast between different parts of the object being observed)
• Advantages of TEMs:
  • They give high-resolution images (more detail)
  • This allows the internal structures within cells (or even within organelles) to be seen
• Disadvantages of TEMs:
  • They can only be used with very thin specimens or thin sections of the object being observed
  • They cannot be used to observe live specimens
    • As there is a vacuum inside a TEM, all the water must be removed from the specimen and so living cells cannot be observed, meaning that specimens must be dead. Optical microscopes can be used to observe live specimens
  • The lengthy treatment required to prepare specimens means that artefacts can be introduced
    • Artefacts look like real structures but are actually the results of preserving and staining
  • They do not produce a colour image
    • Unlike optical microscopes that produce a colour image

Scanning electron microscopes (SEMs)

• SEMs scan a beam of electrons across the specimen
• This beam bounces off the surface of the specimen and the electrons are detected, forming an image
  • This means SEMs can produce three-dimensional images that show the surface of specimens
• Advantages of SEMs:
  • They can be used on thick or 3-D specimens
  • They allow the external, 3-D structure of specimens to be observed
• Disadvantages of SEMs:
  • They give lower resolution images (less detail) than TEMs
  • They cannot be used to observe live specimens
  • They do not produce a colour image

Comparison of the electron microscope & light microscope

• Light microscopes are used for specimens above 200 nm
  • Light microscopes shine light through the specimen, this light is then passed through an objective lens (which can be changed) and an eyepiece lens (x10) which magnify the specimen to give an image that can be seen by the naked eye
  • The specimens can be living (and therefore can be moving), or dead
  • Light microscopes are useful for looking at whole cells, small plant and animal organisms, tissues within organs such as in leaves or skin

• Electron microscopes, both scanning and transmission, are used for specimens above 0.5 nm
  • Electron microscopes fire a beam of electrons at the specimen either a broad static beam (transmission) or a small beam that moves across the specimen (scanning)
  • Due to the higher frequency of electron waves (a much shorter wavelength) compared to visible light, the magnification and resolution of an electron microscope is much higher than a light microscope
  • Electron microscopes are useful for looking at organelles, viruses and DNA as well as looking at whole cells in more detail
  • Electron microscopy requires the specimen to be dead however this can provide a snapshot in time of what is occurring in a cell e.g. DNA can be seen replicating and chromosome position within the stages of mitosis are visible

Diagram of the comparison of resolution of microscopes

The resolving power of an electron microscope is much greater than that of the light microscope, as structures much smaller than the wavelength of light will interfere with a beam of electrons

Light Microscope vs Electron Microscope Table

Electron Microscope	Light Microscope
Large and installation means it cannot be moved	Small and easy to carry
Vacuum needed	No vacuum needed
Complicated sample preparation	Simple sample preparation
Over x500000 magnification	Up to x2000 magnification
Resolution 0.5nm	Resolution 200nm
Specimens need to be dead	Specimens can be living or dead

• The microscope has undergone many developments since the first one used in the 1600s by Robert Hooke
• Every advancement in microscopy technologies has improved our understanding of cells and their structures

Optical (light) microscopes

• Optical (light) microscopes have made advancements in their ability to to view living cells and their internal structures
• Condenser lenses have been developed to direct light from the light source through the specimen
  • Light rays pass from the specimen through the objective lens to the eyepiece
  • Different types of condensers give different features to the microscope
• The use of fluorescent stains and immunofluorescence can be used in optical microscopes which have made it possible to view cellular structures such as RNA
  
  • Fluorescent dyes and stains are used to combine with specific cell structures and organelles which, when exposed to UV rays, gives a more detailed view of the specimen
  • Immunofluorescence involves the use of antibodies that have been prepared with fluorescent dyes which can bind with target molecules complimentary to the antibody. This allows specific molecules to be detected such as virus proteins 

Electron microscopes

• Electron microscopes bring us many advantages to studying cells
  • High magnification and resolution meaning that great detail can be seen in a range of cells and structures within cells, and including viruses
  • 3D images can be produced using a scanning electron microscope
• Electron microscopes have also undergone developments in their abilities
  • Cryogenic electron microscopy
    • This involves flash-freezing solutions containing proteins or other biological molecules
    • The frozen solution is then exposed to electrons to produce images of individual molecules
    • Computer software is used to reconstruct a 3D representation of a cell's proteins using the images of individual molecules
    • Our understanding of virus structure and composition, cell membrane arrangement and protein synthesis have improved thanks to this technique
  • Freeze fracture
    • A sample is rapidly frozen using liquid nitrogen and then physically broken apart (fractured) in a vacuum
    • It can be used to provide a unique planar view of the internal organisation of cell membranes