3.2 - The electron microscope

• Magnification of an object is how many times bigger the image is when compared to the object
• Resolution is the minimum distance apart that two objects can be in order for them to appear as separate items
• The resolving power of a microscope depends on the wavelength or form of radiation used
• increasing the magnification increases the size of the object but does not always increase the resolution
• Light microscope have a poor resolution as a result of the relatively long wavelength of light
• in the 1930’s the electron microscope was developed, which uses a beam of electrons instead of light
• Two main advantages of an electron microscope: an electron beam has a relatively short wavelength so has a high resolving power, and electrons are negatively charged so the beam can be focussed using electromagnets
• The best modern electron microscopes can resolve objects that are just 0.1 nm apart, which is 2000 times better than a light microscope
• Disadvantage of an electron microscope: had to be operated in a near-vacuum to work effectively as electrons are absorbed of deflected by molecules in air
• There are two types of electron microscope: transmission electron microscope and scanning electron microscope
• A transmission electron microscope consists of an electron gun that produces a beam of electrons that is focused onto the a thin section of the specimen by a condenser electromagnet. the beam passes through a thin section of the specimen - parts of the specimen absorbing the electrons and therefore appearing dark while other parts allow the electrons to pass through so appear bright
• Before examining a cell, it is stained. this causes the cel surface membrane to appear as two dark lines because the membrane has a phospholipid bilayer and the stain binds to the phosphate in the inside and the outside of the membrane
• How do you make a temporary mount of a piece of plant tissue?

  add a drop of water to the glass slide

  obtain a thin section of the plant tissue and place it in the slide

  stain the tissue with iodine and lower a cover slip using a mounted needle
• the resolving power of a TEM is 0.1 nm, however this may not be achieved due to difficulty in preparing the specimen and the need for the a higher energy electron beam (which if used could destroy the specimen)
• Main limitations of the TEM: whole system must be in a vacuum so living specimens cannot be observed, complex staining process, image produced is not in colour, specimen must be very thin, image may contain artifacts, 2D image produced
• In the scanning electron microscope all the same limitations occur as the TEM, other than the need for the specimen to be extremely thin - the electrons do not penetrate for this microscope
• the SEM directs a beam of electrons on to the surface of the specimen from above. the beam is then passed back and forth across a portion of the specimen in a regular pattern - the electrons are scattered by the specimen, the pattern of this scattering depending on the contours of the specimen surface. this enables us to build a £D image of the specimen
• the resolving power of the SEM is lower than the resolving power of the TEM but greater than that of the light microscope
• the resolving power of the SEM is 20 nm