Scanning electron microscopy, or SEM, is a powerful technique used in chemistry and material analysis that uses a scanned electron beam to analyze the surface structure and chemical composition of a sample.
Modern light microscopes are limited by the interaction of visible light waves with an object, called diffraction. The smallest resolvable distance between two objects, or the lateral resolution, varies depending on the size of the diffraction pattern as compared to the object size. As a result, light microscopes have a maximum magnification of up to 1,000X and a lateral resolution of up to 200 nm in ideal situations.
SEM is not limited by diffraction, as it uses a beam of electrons rather than light. Therefore, an SEM can reach magnifications of up to one million X with sub-nanometer lateral resolution. In addition, SEM is not limited to imaging features only in the focal plane, as with light microscopy. Thus, objects outside of the focal plane are resolved, as opposed to light microscopy where they appear blurry. This provides up to 300 times increased depth of field with SEM.
Chemists widely use SEM to analyze surface composition, structure, and shape of nanoscale entities, such as catalyst particles.
This video will outline the principles of the SEM instrument, and demonstrate the basics of SEM sample preparation and operation in the laboratory.
In SEM, samples must be conductive for conventional imaging. Non-conductive samples are coated with a thin layer of metal, such as gold. Images are then generated by scanning a focused beam of high-energy electrons across the sample.
The electron beam used in SEM is generated by an electron gun, fitted with a tungsten filament cathode. The electrons are propelled toward the anode, in the direction of the sample, by an electric field.
The electron beam is then focused at condenser lenses, and enters the objective lens. The objective lens must be calibrated by the user to focus the electron beam on a fixed position on the sample. The focused beam is then raster scanned across the sample.
When the primary electrons interact with the sample, they tunnel to a depth that is dependent on the electron beam energy. This interaction with the surface results in the emission of secondary and backscattered electrons, which are then measured by their respective detectors.
The signal intensity of the emitted secondary electrons varies depending on the angle of the sample. Surfaces perpendicular to the beam release fewer secondary electrons, and therefore appear darker. At the edge of surfaces, more electrons are released and the area appears brighter. This phenomenon produces images with a well-defined 3D appearance, as shown in this SEM scan of asbestos.
In contrast, backscattered electrons are reflected in the opposite direction of the electron beam. Detection intensity increases with increasing atomic number of the sample, enabling the acquisition of compositional information of a surface, as shown in this backscatter image of inclusions in glass.
Now that the principles of the SEM instrument have been outlined, the basic operation of an SEM will be demonstrated in the laboratory.
To begin, sputter coat the sample by placing it onto a sample stub. Make sure that the sample is completely dry and degassed. If necessary, double-sided conductive carbon tape may be used to adhere the sample to the stub. Place the sample into a sputtering system. Sputter a few nanometers of gold onto the sample. The thickness of the gold layer will vary depending on if the coating interferes with the morphology of the sample.
Remove the sample from the sputtering system. Ensure that there is a conductive bridge from the sample surface to the metal stub.
Once the sample has been coated, it is ready to be imaged. To do so, first vent the SEM sample chamber and allow the chamber to reach nominal pressure.
Open the SEM sample compartment, and remove the sample stage. Place the stub onto the sample stage, and tighten the stub in place.
If the distance between the lens and sample, called the working distance, cannot be controlled by the software, ensure that the stage and stub have the proper height to obtain an image.
Put the sample stage into the sample chamber, and close the compartment.
Turn on the vacuum pumps and allow the system to pump down.
To begin imaging, open the SEM software. Select the desired operating voltage ranging from 1–30 kV. With high-density materials, higher acceleration voltages should be used. Select low accelerating voltage for low-density materials.
Most SEM software includes an auto focus feature. This will acquire a focus of the sample to use as a starting point.
Set the magnification to the minimum zoom level of 50X.
SEM has different scan modes such as fast scan, and slow scan. Faster scan mode provides faster refresh rate of the screen with lower quality. Select the fast scan mode to begin, in order to find the sample and begin coarse focusing.
Adjust the course focus until the image becomes sharper. Next, adjust the stage positioning so the region of interest can be seen on the display.
First, focus at the lowest magnification using the coarse focus. Then, increase the magnification level until the desired feature is observed. Adjust the course focus to roughly focus the image at this magnification. If necessary, adjust a coarse focus when the magnification increased.
Then, adjust the fine focus to further improve the image. Repeat these focusing steps every time the magnification is increased.
Asymmetrical beam distortions can cause blurring of the image, called astigmatism, even when the sample is well focused. To diminish this effect, increase the magnification to the maximum level, and focus the image using the fine focus. Then adjust the stigmation in both the x and y direction to reshape the beam.
Keep adjusting the focus and stigmation settings until the image is as focused as possible at the increased magnification level.
Then return to the desired magnification level.
The SEM image can be acquired in either "slow photo" or "fast photo" mode. The "fast photo" mode creates a lower quality image, but is acquired faster. The "slow photo" mode creates a higher quality image, but may saturate the surface with electrons.
To measure features within the captured image, utilize the software's measurement tools.
Most instruments include measurement options such as length, area, and angle.
To determine length, select the distance to be measured on the SEM image. Click on the image to create the points of reference that will be analyzed by the software.
When finished, shut down the SEM according to the manufacturers guidelines.
Scanning electron microscopy is used to image a wide range of samples.
SEM can be used to image complex and highly structured materials, such as a carbon fiber membrane.
The sample showed a high degree of porosity and three dimensional structure; a property that is highly desirable for applications such as catalysis.
SEM can also be used to image biological samples, such as bacteria. In this example, the hair like appendages, or pili, of gut bacteria were imaged with SEM.
Helicobacter pylori were grown on blood agar plates, and the bacteria seeded onto glass cover slips.
Fully dried samples were mounted, and coated with 5 nm of palladium-gold to make the sample conductive.
Finally, the sample was imaged using SEM. H. pylori were easily visible, with measurable nanoscale pili.
This example describes how brain tissue can be embedded into a stable resin, and then imaged in three dimensions using a focused ion beam and SEM.
First, brain tissue was fixed and embedded in resin. Then the region of interest identified and sliced with a microtome.
The sample was then inserted into the focused ion beam scanning electron microscope for three-dimensional imaging. The focused ion beam was then used to sequentially remove thin layers of the sample. Each layer was imaged prior to removal using backscatter SEM.
You've just watched JoVE's introduction to scanning electron microscopy. You should now understand the basic operating principles of SEM and how to prepare and analyze an SEM sample.
Thanks for watching!