Scanning Electron Microscopy (SEM)

Electrons are generated by heating by the electron gun, which acts like a cathode. These electrons are propelled towards the anode, in the same direction as the sample, due to a strong electric field. After the beam of electrons is condensed, it enters the objective lens, which is calibrated by the user to a fixed position on the sample. (Figure 1)

Once the electrons strike the conductive sample, two things can happen. First, the primary electrons that hit the sample will tunnel through it to a depth which is dependent on the energy level of those electrons. Then, the secondary and backscattered electrons will hit the sample and reflect outwards from it. These reflected electrons are then measured either by the secondary electron (SE) or backscattered (BS) detector. After signal processing takes place, an image of the sample is formed on the screen.¹

In SE mode, secondary electrons are attracted by positive bias on the front of detector because of their low energy. The signal intensity is varied depending on the angle of the sample. Therefore, SE mode provides highly topographical images. On the other hand, in BS mode, the direction of electrons is almost directly opposite of the e-beam direction and the detection intensity is proportional to the atomic number of the sample. Therefore, it is less topographical, but useful for compositional images. BS mode is also less affected by the charging effect on the sample, which is beneficial for non-conductive samples.¹

Figure 1. Schematic of the SEM.

The SEM, seen in Figure 2a, has been used for making measurements and acquiring sample photos. The sample consisted of sodium chloride (NaCl) salt. It was placed onto the stub as seen in Figure 2b, then a few nanometers of gold was sputtered onto it to make it conductive. The conductive sample was then placed into the SEM sample area as seen in Figure 2c.

SEM images were obtained at 50X, 200X, 500X, 1,000X, and 5,000X magnification levels as seen in Figure 3. Figure 3a shows a birds-eye view of the salt sample at 50X magnification. Figure 3b then zooms in to an individual salt particle at a magnification of 200X. Figure 3c shows this same magnification level but includes area and diameter measurements made within the SEM software. Figure 3d then zooms to 500X, showing the area of interest on the salt particle. Figure 3e shows a magnification of 1,000X, allowing one to observe the corner of the salt particle that has been damaged. Figure 3f shows a magnification of 5,000X, allowing the user to view the structure of the salt particle.

Figure 2. (a) Image of SEM. (b) NaCl salt placed onto sample stub with carbon tape. (c) Sample stub placed into SEM sample stage after it was treated with gold coating.

Figure 3. SEM images of sample at various magnification levels: (a) 50X, (b) 200X, (c) 200X with measurements, (d) 500X, (e) 1,000X, and (f) 5,000X.

The SEM is a very powerful tool that is common in most research institutions because of its ability to image any object that is conductive, or has been treated with a conductive coating. The SEM has been used to image objects such as semiconductor devices,² biological membranes,³ and insects,⁴ among others. We have also used the SEM to analyze nanofibers and paper-based materials, biomaterials, micropatterned structures. Of course, there are materials, such as liquids, that can’t be placed into a standard SEM for imaging, but continuous development of Environmental Scanning Electron Microscopes (ESEM) allows for such functionality. ESEM is similar to SEM in that it uses an electron gun and analyzes the electron interaction with the sample. The main difference is that the ESEM is split into two separate chambers. The top chamber consists of the electron gun and goes into a high vacuum state, while the lower chamber contains the sample and enters a high pressure state. Because the sample area does not need to enter a vacuum, wet or biological samples can be used during the imaging process. Another ESEM benefit is that the sample does not need to be coated with a conductive material. However, ESEM has some disadvantages of low image contrast and small working distance due to gaseous environment in the sample chamber. . The general rule of thumb is that if you are able to coat a sample with a conductive layer, then it can be imaged in an SEM, allowing for almost all solid objects to be analyzed.