Microscopia Eletrônica de Varredura (MEV)
English
Share
Overview
Fonte: Laboratório do Dr. Andrew J. Steckl — Universidade de Cincinnati
Um microscópio eletrônico de varredura, ou SEM, é um poderoso microscópio que usa elétrons para formar uma imagem. Permite imagens de amostras condutoras em ampliações que não podem ser alcançadas usando microscópios tradicionais. Microscópios de luz modernos podem alcançar uma ampliação de ~1.000X, enquanto o MEI típico pode atingir ampliações de mais de 30.000X. Como o SEM não usa luz para criar imagens, as imagens resultantes que ele forma estão em preto e branco.
As amostras condutoras são carregadas no estágio amostral do SEM. Uma vez que a câmara de amostra atinja o vácuo, o usuário passará a alinhar a arma eletrônica no sistema ao local adequado. A arma eletrônica dispara um feixe de elétrons de alta energia, que viajam através de uma combinação de lentes e aberturas e eventualmente atingem a amostra. À medida que a arma eletrônica continua a disparar elétrons em uma posição precisa na amostra, elétrons secundários saltarão da amostra. Estes elétrons secundários são identificados pelo detector. O sinal encontrado a partir dos elétrons secundários é amplificado e enviado para o monitor, criando uma imagem 3D. Este vídeo demonstrará as capacidades de preparação, operação e imagem da sem.
Principles
Procedure
Results
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.
Applications and Summary
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.
Disclosures
References
- Goldstein, J., Newbury, D., Joy, D., Lyman, C., Echlin, P., Lifshin, E., Sawyer, L., Michael, J. Scanning Electron Microscopy and X-ray Microanalysis. 3rd Ed. Springer, New York, NY. (2003).
- Purandare, S., Gomez, E.F., Steckl, A.J. High brightness phosphorescent organic light emitting diodes on transparent and flexible cellulose films. Nanotechnology. 25, 094012 (2014).
- Masuda, Y., Yamanaka, N., Ishikawa, A., Kataoka, M., Aral, T., Wakamatsu, K., Kuwahara, N., Nagahama, K., Ichikawa, K., Shimizu, A. Glomerular basement membrane injuries in IgA nephropathy evaluated by double immunostaining for a5(IV) and a2(IV) chains of type IV collagen and low-vacuum scanning electron microscopy. Clinical and Experimental Nephrology. 1-9. (2014).
- Kang, J.H., Lee, Y.J., Oh, B.K., Lee, S.K. Hyun, B.R. Lee, B.W, Choi, Y.G., Nam, K.S., Lim, J.D. Microstructure of the water spider (Argyroneta aquatic) using the scanning electron microscope Journal of Asia-Pacific Biodiversity. 7 484-488 (2014).
Transcript
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!