3.7 Microscopy: Magnification & Resolution

Magnification

• Magnification is how many times bigger the image of a specimen observed is in comparison to the actual, real-life size of the specimen
• A light microscope has two types of lens
  • An eyepiece lens which often has a magnification of x10
  • A series of, usually 3, objective lenses, each with a different magnification
• To calculate the total magnification, the magnification of the eyepiece lens and the objective lens are multiplied together

total magnification = eyepiece lens magnification x objective lens magnification

Resolution

• Resolution, or resolving power, is the ability to distinguish between two separate points
  • If two separate points cannot be resolved, they will be observed as one point and the image will be unclear
• The resolution of a microscope limits the magnification that it is capable of; there is no point in magnifying an image at low resolution as this will just result in a big blur rather than a small blur!
• The resolution of a light microscope is limited by the wavelength of light; the wavelength of light is too long to allow for high resolution
  • E.g. the phospholipid bilayer structure of the cell membrane cannot be observed under a light microscope
    • The width of the phospholipid bilayer is about 10 nm
    • The maximum resolution of a light microscope is 200 nm 
    • Any points that are separated by a distance less than 200 nm cannot be resolved by a light microscope and therefore will not be distinguishable as separate points on an image
• Electron microscopes have a much higher resolution, and therefore magnification, than a light microscope as electrons have a much smaller wavelength than visible light

The resolving power of an electron microscope is much greater than that of the light microscope due to the smaller wavelength of electrons

Comparing electron & light microscopes

• Light microscopes are used for specimens larger than 200 nm
  • Light microscopes shine light through the specimen
  • 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, and tissues within organs such as in leaves or skin
• Electron microscopes, both scanning and transmission, are used for specimens larger than 0.5 nm
  • Electron microscopes fire a beam of electrons at the specimen
  • The electrons are picked up by an electromagnetic lens which then shows the image
  • Electron microscopy requires the specimen to be dead; 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
  • Electron microscopes are useful for looking at organelles, viruses, and DNA, as well as looking at whole cells in more detail

Light v Electron Microscope Table

• Specimens to be viewed under a microscope sometimes need to be stained, as the cytoplasm and other cell structures may be transparent or difficult to distinguish
  • Note that most of the colours seen in images taken using a light microscope are the result of added stains
    • Chloroplasts are the exception to this; they show up green, which is their natural colour
• The type of stain used is dependent on what type of specimen is being prepared and what the researcher wants to observe within the specimen
  • Different molecules absorb different dyes depending on their chemical nature
• Specimens or sections are sometimes stained with multiple dyes to ensure that several different tissues within the specimen show up; this is known as differential staining
• Some common stains include
  • Haemotoxylin
    • Stains plant and animal cell nuclei purple, brown or blue
  • Methylene blue
    • Stains animal cell nuclei blue
  • Acetocarmine
    • Stains chromosomes in dividing nuclei of plant and animal cells
  • Iodine
    • Stains starch-containing material in plant cells blue-black
  • Toluidine blue 
    • Stains tissues that contain DNA and RNA blue
  • Phloroglucinol
    • Stains a chemical called lignin found in some plant cells red/pink

Toluidine blue and phloroglucinol have been used to stain this tissue specimen taken from a leaf