Transmission Electron Microscopy (TEM)

The basic principles for image formation using TEM are described in Williams and Carter (2009). Briefly, TEM uses an electron beam to image the sample. This provides a higher resolving power than the visible light in optical microscopy. Because the wavelength of the energized electron beam is very short, the diffraction limit is correspondingly lower. Generally, in electron microscopy, high energy primary electrons hit the specimen and the same or different electrons deflect from the sample to form an image. In TEM, a stationary primary electron beam is transmitted through the ultrathin specimen and transformed into a non-uniform electron intensity after transmission or scattering by the specimen.

This non-uniform electron intensity hits the fluorescent screen or the electron detector and is translated into image contrast on the screen. Either the direct beam or a diffracted beam is used to form bright-field and dark-field images, respectively. Figure 1 illustrates the mechanism of image formation in the bright-field imaging mode using direct beam. In the bright-field mode, scattered electrons are blocked with an objective aperture in order to enhance the contrast. In addition, while interacting with the specimen, a wide range of secondary signals are produced. Many of them are used in analytical electron microscopy, providing the chemical composition and additional information about the specimens. A TEM analysis must be run under an ultra-high vacuum that prevents scattering of the electron beam by the gas molecules so that the electrons can move freely from the gun through the specimen and further to the detector.

Because of the extraneous materials, low crystallinity, and tight association of cell wall materials it has always been challenging to image the structure and morphology of the wood cell wall. Wood specimens are also more susceptible to radiation damage than the highly crystalline Valonia. The direct imaging of cellulose microfibrils in wood using diffraction contrast and low-dose mode has been discussed. A comparison among different cellulose specimens and their sensitivity to the radiation damage at different accelerating voltages. It shows that the radiation damage caused by the energized electron beam can be reduced to an extent using the higher accelerating voltage of the microscope. The unlignified/less-lignified gelatinous layer in tension wood fibers was also found to be a suitable specimen to image with direct beam. The advancement of cryo-TEM also provided the opportunity to image beam sensitive specimens with heavy electron dose. Nowadays, it is possible to keep the specimen temperature below 20 K using liquid helium during the imaging. This way, the electron dose could be increased at least ten times than that at room temperature.

Accelerating voltage and the corresponding dose at beam damage for different cellulose specimens. G-layer: gelatinous layer; S2 layer: middle layer of wood secondary wall.


Sample preparation is a very important step because TEM requires ultrathin specimens. Samples for TEM analysis need to be fit on a specimen support known as a grid. TEM grids are metal mesh screens and are about 3 mm in diameter. The size and shape of the mesh vary. They are mainly made of copper, but gold, nickel, and beryllium grids are also used in special circumstances.

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