스캐닝 전자 현미경 검사법 (SEM)
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Source: Laboratory of Dr. Andrew J. Steckl — University of Cincinnati
A scanning electron microscope, or SEM, is a powerful microscope that uses electrons to form an image. It allows for imaging of conductive samples at magnifications that cannot be achieved using traditional microscopes. Modern light microscopes can achieve a magnification of ~1,000X, while typical SEM can reach magnifications of more than 30,000X. Because the SEM doesn’t use light to create images, the resulting pictures it forms are in black and white.
Conductive samples are loaded onto the SEM’s sample stage. Once the sample chamber reaches vacuum, the user will proceed to align the electron gun in the system to the proper location. The electron gun shoots out a beam of high-energy electrons, which travel through a combination of lenses and apertures and eventually hit the sample. As the electron gun continues to shoot electrons at a precise position on the sample, secondary electrons will bounce off of the sample. These secondary electrons are identified by the detector. The signal found from the secondary electrons is amplified and sent to the monitor, creating a 3D image. This video will demonstrate SEM sample preparation, operation, and imaging capabilities.
Princípios
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.
Procedimento
1. Preparation of the Sample
- Place sample onto sample stub. If necessary, carbon tape may be used to adhesively bond the sample to the stub.
- Place the sample into a gold sputtering system. Using a mini-gold sputter, sputter gold for 30 s at ~ 70 mTorr pressure. A different gold layer thickness may be necessary depending on the geometry of the sample. More rough or porous surface requires a longer sputtering time.
- Remove stub from gold sputtering system.
2. Sample Insertion and SEM Startup
- Vent the SEM chamber, allowing the chamber to reach nominal pressure.
- Open the SEM sample compartment and take out the sample stage.
- Insert the sample stub containing the sample onto the stage. Tighten the stub into place.
- If the z-distance cannot be controlled by software, make sure the sample stage with sample stub has proper height to obtain the better image.
- Put the sample stage into sample chamber. Close the sample compartment.
- Turn on the pumps and allow system to reach vacuum. The system will notify the user when this is completed.
- Open the SEM software. Select desired operating voltage ranging from 1–30 kV. Higher operating voltage gives better image contrast, but can yield lower resolution if charges accumulate in the sample.
3. Capturing the SEM Image
- Begin ‘Auto Focus’ in the SEM software by clicking on the key icon. This will acquire a focused image of the sample to use as a starting point.
- Make sure the magnification is set to the minimum zoom level of 50X.
- Select ‘fast scan’ mode.
- Adjust the focus in coarse mode until a rough focus is acquired.
- Adjust the stage manually using the exterior knobs so that the region of interest is visible on the display.
- Increase the magnification level until the desired feature is observed. Adjust the coarse focusing knob to roughly focus the image at this magnification. Then, improve the focus using the fine focusing knob to get a focused image at the desired magnification level. This step will be repeated whenever the magnification level is increased.
- Once the desired magnification is reached, adjust the fine focusing knob to improve clarity.
- To optimize image clarity, increase the magnification close to the maximum level, and then focus the image using the fine focus knob. If a clear image cannot be obtained, adjust the stigmation in both the x and y direction. Keep adjusting the focus and stigmations until the clearest image is obtained at the exaggerated magnification level.
- After reaching a quality image of the sample, go back to the desired magnification level. The image can be taken by pressing the photo button in either ‘slow photo’ or ‘fast photo’ mode. The ‘slow photo’ mode gives better quality and high resolution of the image.
4. Making Measurements Using the SEM Software
- In the ‘Panels’ dropdown list, select ‘M. Tools’.
- Various measurements such as length, area, and angle can be measured directly in the SEM software. To perform one of these measurements, click on the desired icon in the M. Tools window.
- Scroll to the measurement site on the SEM image. Measurements are done by clicking on the image to create points of reference that will be analyzed by the software. Data points measured can be inserted directly onto the image if desired by the user.
- Images are then saved to the computer.
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!
Resultados
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.
Referências
- 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).
Transcrição
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!