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3/25/2020 Introduction to Electron Microscopy - Advanced Microscopy - Imaging Facilities - The University of Utah
https://advanced-microscopy.utah.edu/education/electron-micro/index.html 1/
Electron Microscopy Tutorial
Overview
Electron microscopes use electrons to illuminate a sample. In Transmission Electron Microscopy (TEM), electrons pass through the sample and illuminate film or a digital camera. Electron dense material in the sample casts shadows on the camera face and thereby produces a two-dimensional projection of material in the section.
Wavelength and Resolution
Resolution in microscopy is limited to about ½ of the wavelength of the illumination source used to image the sample (see Super-resolution Tutorial). Because our eyes can only detect photons with wavelengths greater than ~400 nm, the best resolution that can be achieved by light microscopes is about ~200 nm. One way to beat the diffraction limit of light is to use an illumination source with a shorter wavelength than photons – electrons (see Box 1).
Box 1: The de Broglie relationship
Louis de Broglie showed that every particle or matter propagates like a wave. The wavelength of a particle or a matter can be calculated as follows.
where λ is the wavelength of a particle, h is Planck’s constant (6.626 x 10-34^ J seconds), and p is the momentum of a particle. Since the momentum is the product of the mass and the velocity of a particle,
Because the velocity of the electrons is determined by the accelerating voltage, or electron potential where
The velocity of electrons can be calculated by
Therefore, the wavelength of propagating electrons at a given accelerating voltage can be determined by
Since the mass of an electron is 9.1 x 10-31^ kg and e = 1.6 x 10-19,
3/25/2020 Introduction to Electron Microscopy - Advanced Microscopy - Imaging Facilities - The University of Utah
https://advanced-microscopy.utah.edu/education/electron-micro/index.html 2/
Thus, the wavelength of electrons is calculated to be 3.88 pm when the microscope is operated at 100 keV, 2.74 pm at 200 keV, and 2.24 pm at 300 keV.
However, because the velocities of electrons in an electron microscope reach about 70% the speed of light with an accelerating voltage of 200 keV, there are relativistic effects on these electrons. These effects include significant length contraction, time dilation, and an increase in mass. By accounting for these changes,
where c is the speed of light, which is ~3 x 10^8 m/s. Therefore, the wavelength at 100 keV, 200 keV, and 300 keV in electron microscopes is 3.70 pm, 2.51 pm and 1.96 pm, respectively.
The wavelength of electrons is much smaller than that of photons (2.5 pm at 200 keV). Thus the resolution of an electron microscope is theoretically unlimited for imaging cellular structure or proteins. Practically, the resolution is limited to ~0. nm due to the objective lens system in electron microscopes. Thus, electron microscopy can resolve subcellular structures that could not be visualized using standard fluorescences microscopy, such as the microvilli of intestinal cells or the internal structure of a bacterium (Figure 1).
Figure 1: E. coli bacteria being digested in the intestine of a nematode, C. elegans. The microvilli of the intestinal cells project into the lumen of the gut; the bacteria are lodged in the lumen. The cross-section of E. coli is about 400 nm. (photo Shigeki Watanabe and Erik Jorgensen)
Electron microscopes can even resolve molecular structure in a cell, for example the plasma membrane is only about 5nm in diameter. The membrane is composed of a lipid bilayer, each layer is a single molecule thick. These fatty acids are about 20 carbons long with a hydrophilic head group. These individual lipid layers can be distinguished in an electron micrograph (Figure 2).
