2.6: Overview of Microscopy Techniques
The early pioneers of microscopy opened a window into the invisible world of microorganisms. In 1830, Joseph Jackson Lister created an essentially modern light microscope. The 20th century saw the development of microscopes that leveraged nonvisible light, such as fluorescence microscopy that uses an ultraviolet light source and electron microscopy that uses short-wavelength electron beams. These advances significantly improved magnification, image resolution, and contrast. By comparison, the relatively rudimentary microscopes of van Leeuwenhoek and his contemporaries were far less powerful than even the most basic microscopes in use today.
Light Microscopy
Many types of microscopes fall under the category of light microscopes, which use light to visualize images. Examples of light microscopes include brightfield microscopes, darkfield microscopes, phase-contrast microscopes, differential interference contrast microscopes, fluorescence microscopes, confocal scanning laser microscopes, and two-photon microscopes. These various types of light microscopes can complement each other in diagnostics and research.
Electron Microscopy
The wavelengths of visible light 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. However, this does not begin to approach the magnifying power of an electron microscope (EM), which uses short-wavelength electron beams rather than light to increase magnification and resolution.
Electrons, like electromagnetic radiation, can behave as waves, but with wavelengths of 0.005 nm, they can produce much better resolution than visible light. An EM can produce a sharp image that is magnified up to 100,000x. EMs can resolve subcellular structures and some molecular structures (e.g., single strands of DNA); however, electron microscopy cannot be used on living material because of the methods needed to prepare the specimens.
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 from above the specimen that is focused using a magnetic lens (rather than a glass lens) and projected through the specimen onto a detector. Electrons pass through the specimen, and then the detector captures the image.
Scanning Probe Microscopy
A scanning probe microscope does not use light or electrons but rather very sharp probes that are passed over the surface of the specimen and interact with it directly. This produces information that can be assembled into images with magnifications up to 100,000,000x. Such large magnifications can be used to observe individual atoms on surfaces. To date, these techniques have been used primarily for research rather than for diagnostics. There are two types of scanning probe microscopes: the scanning tunneling microscope (STM) and the atomic force microscope (AFM).
This text is adapted from Openstax, Microbiology, Section 2.3: Instruments of Microscopy
