33.11: Overview of Electron Microscopy
The wavelengths of visible light ultimately limit the maximum theoretical resolution of images created by light microscopes. Most light microscopes can only magnify 1000X, and a few can magnify up to 1500X. Electrons, like electromagnetic radiation, can behave like waves, but with wavelengths of 0.005 nm, they produce significantly greater resolution up to 0.05 nm as compared to 500 nm for visible light. An electron microscope (EM) can create a sharp image that is magnified up to 2,000,000X. Thus, EMs can resolve subcellular structures and some molecular structures (for example, single strands of DNA).
There are two basic types of EM — the transmission electron microscope (TEM) and the scanning electron microscope (SEM). The TEM is somewhat analogous to the brightfield light microscope in terms of the way it functions. However, it uses an electron beam focused on the sample from above using a magnetic lens (rather than a glass lens) and projected through the sample onto a detector. Electrons pass through the specimen, and then the detector captures the image.
For electrons to pass through the specimen in a TEM, the sample must be extremely thin (20–100 nm thick). The image is produced because of varying opacity in various parts of the sample. This opacity can be enhanced by staining the specimen with materials such as heavy metals, which are electron-dense. TEM requires that the beam and sample be in a vacuum and that the sample be ultrathin and dehydrated.
SEMs form images of surfaces of specimens, usually from electrons that are knocked off the sample by a beam of electrons. This can create highly detailed images with a three-dimensional appearance displayed on a monitor. Typically, specimens are dried and prepared with fixatives that reduce artifacts before being sputter-coated with a thin layer of metal such as gold. Whereas transmission electron microscopy requires very thin sections and allows one to see internal structures such as organelles and the interior of membranes, scanning electron microscopy can be used to view the surfaces of larger objects (such as a pollen grain) as well as the surfaces of very small samples.
This text is adapted from Openstax, Microbiology, Section 2.3: Instruments of Microscopy
