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33.12: Scanning Electron Microscopy

JoVE Core
Cell Biology

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Scanning Electron Microscopy

33.12: Scanning Electron Microscopy

A scanning electron microscope (SEM) is used to study the surface features of a sample by using an electron beam that scans the sample surface in a two-dimensional manner. Typically, areas between ~1 centimeter to 5 micrometers in width can be imaged. SEM can be used to image bacteria, viruses, tissues as well as larger samples like insects. Conventional SEM gives a magnification ranging from 20X to 30,000X and spatial resolution of 50 to 100 nanometers.

Fundamental Principles

Accelerated electrons released by the electron gun have high kinetic energy (ranging from 5-30 keV). Electron-sample interactions lead to deceleration of the electrons and dissipation of the energy in the form of different signals. The electrons undergo two types of scattering: elastic and inelastic. Inelastic scattering causes the emission of secondary electrons. These low-energy electrons (~50 eV) are the outer shell electrons of the sample atoms that acquire just enough energy to leave the atom's surface. Only topographical information is provided by the scattering of secondary electrons since the energy level of the electrons leaving from the internal regions of the sample is too low to exit the sample surface. 

X-rays are also generated by inelastic collisions of the incident electrons with electrons in discrete orbitals (shells) of atoms in the sample. As the excited electrons return to lower energy states, they yield X-rays of a fixed wavelength (related to the difference in energy levels of electrons in different shells for a given element). Thus, characteristic X-rays are produced for each element in a mineral excited by the electron beam.

Elastic scattering, on the other hand, is not caused by dislodged electrons from the sample atoms. The principal beam of electrons is backscattered after interaction with the nucleus. These electrons do not change their energy or speed but change their direction based on their interaction with the nucleus. Detection of these electrons provides compositional information, and their varying contrast upon interaction with atoms of different atomic weights allows the user to distinguish differences in sample composition. In biological samples, this can be used to study embedded or attached nanoparticles and nanostructures with heavier atomic weights, such as gold or iron. 

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