A Beginners Guide to Scanning Electron Microscopy Jake Rabinowitz

Figure 1: Scanning Electron Microscope

Foreword Scanning electron microscopy is a powerful tool that originated in 1933.1 Since that time, it has been and continues to be improved upon to push the limits of what we can view under a microscope. Currently, scanning electron microscopes (SEMs) can magnify images up to 500,000x,2 allowing users to view samples in the nanoscale. This functionality has become increasingly crucial as scientists have made promising advancements in all areas of nanoscience. Looking forward, the growing importance of nanotechnology will make the SEM a vital cog in the wheel of technological development. This guide will first inform readers on how an SEM operates, and then will explain key variable parameters and the consequences of manipulating them. Then, it will conclude by providing step-by-step instructions for imaging a sample using an SEM. How the Image Forms A scanning electron microscopes operation starts with the electron gun. This gun is a metallic filament that builds up a concentrated negative charge. Once heated, this potential difference causes the filament to literally shoot a beam of electrons down the column. As seen to the right, the anode absorbs most of these electrons, leaving only a small circular opening through which a focused beam passes. The purpose behind all this is to generate a concentrated, columnated beam of light; the magnetic lens serves to further focus the beam of electrons passing through the column. The scanning coils move the beam across the specimen in a raster pattern, traversing from left to right and then moving down before traversing back to a point just below the starting point. This is where the scanning in SEM comes from. Once electrons hit the specimen, most retain their energy (elastic scattering) and are recovered by the backscattered electron detector. Those that lose a significant portion of their energy (inelastic scattering) are attracted to the positive charge on the secondary electron detector. This detector is connected to a TV and provides most of the information used to generate the image.2

Figure 2: Simple View of SEM Column Chamber3

Generating a Vacuum If near-vacuum conditions are not achieved within the microscope column, the contaminants in the chamber will cloud the array of lenses and disrupt the focus of the beam. Users typically image specimens at a pressure of 5 * 10-5 torr; in contrast, atmospheric pressure is 760 torr. An SEM attains such a low pressure through a series of increasingly sensitive pumps mechanical, diffusion, and ion. Mechanical pumps lower pressure through a rotor connected to an engine. As this device rotates, it pushes air from the chamber through an outlet while sealing off any new air from coming in. Though mechanical pumps can attain very low pressures if allowed enough time, they become inefficient at 1 * 10-2 torr. Thus, around this level the diffusion pump takes over. The diffusion pump lowers chamber pressure by vaporizing oil through a chimney and then releasing this oil back towards the bottom of the chimney. This base is connected to the microscope column, and the oil vapor creates a pressure gradient through which air in the column diffuses to an outlet. Diffusion pumps are highly effective and can generate the necessary pressure of 5 * 10 -5 torr. Ion pumps, though not always necessary, can bring pressure down even further by introducing highly reactive ions that convert air molecules to solid compounds.2 This vacuum is relevant to the user for several reasons. It is the first thing that the user should check if imaging is unsuccessful; often times, attaining a lower pressure will solve the problem. Additionally, it is crucial for understanding the process behind loading a sample onto the stage. When loading, there is a secondary chamber that has to be in equilibrium with whatever environment it will be open to. Thus, the chamber will need to be pumped down to a vacuum or purged of said vacuum before it can interact with the environment or the SEM column.

Figure 3: Side view of (closed) secondary SEM chamber4

Optimizing the Image Now that readers have an understanding of how the SEM generates an image and how the vacuum system works, it is time to discuss the different parameters that affect this image. There are two key components of image generation, both of which are multifaceted and vary according to the users preferences. They are electron beam focusing and electron gun power. Electron Beam Focusing Apertures are the series of holes that the electron beam passes through. These holes are crucial to the formation of a columnated beam. Users will always keep the smallest aperture size below 50 m; otherwise, it would be impossible to focus the beam. At this size, users will have the highest beam current. This allows them to penetrate the sample to the greatest degree, while also generating the brightest images. Moving smaller, however, generates more resolution and an increased depth of field. Thus, when viewing smaller samples, a smaller aperture is more effective. Stigmation coils allow the user to manipulate the cross-sectional beam area by inducing a magnetic field. This is vital because the beam needs to be circular but often comes through the magnetic lenses with an elliptical cross-section. Users can induce magnetic fields in both the x and y direction to correct for this error, and image quality will noticeably improve when users adjust the stigmation to achieve a circular beam. Working distance is the distance from the top of the sample to the lowest aperture. Users need to optimize this distance in order to generate good images. Furthermore, users must operate within a proper working distance in order to calibrate the lowest aperture and the stigmation coils. Besides determining the resolution of the image, the working distance determines the depth of the field that a user can view. If the user is viewing relatively tall structures, he or she Figure 3: Working Distance and Effect on Users Depth of Field2 will need a greater working distance to be able to focus on the base of the structures.

Electron Gun Power EHT stands for extra high tension and is a term used to describe a high electrical voltage. When operating the SEM, a high electron voltage is necessary to propel the electrons through the chamber. The voltage, however, varies according to the sample that the user is viewing. A higher voltage will have electrons with more energy and a smaller wavelength, allowing them to penetrate the sample to a greater extent. This is beneficial when viewing dense materials such as metals, but it can be harmful to more sensitive samples like polymers or biological cells. EHT is typically measured in keV and most samples can be viewed within 10 30 keV.

Filament Current describes the current measured at the metal filament that powers the electron gun. There is a direct relationship between filament current and electron beam strength up until the point of saturation. Past this point, the beam strength will increase nominally, but the user begins to incur the risk of blowing out the current. Consequently, it is an important value to monitor and should be kept within 2.5 3 A. Operating the SEM

1. Open the Gun Monitor This will allow you to monitor the system pressure, the EHT, and the filament current. You should refer back to this monitor periodically because many errors that can harm the equipment will show themselves here. 2. Load the Sample As mentioned above, the sample is loaded through the secondary chamber. To insert a sample, you will have to put it on the stage and then equilibrate the secondary chamber with the SEM column chamber. To do this, you will have to: a) Purge the vacuum in the secondary chamber (you will hear a click when it reaches atmospheric pressure) b) Open the secondary chamber door and load the sample onto the stage c) Close the secondary chamber door d) Pump the secondary chamber down to a vacuum e) Open the door from the secondary chamber to the SEM column chamber f) Push the stage from the secondary chamber into the SEM column chamber g) Close the door from the secondary chamber to the SEM column chamber 3. Set the EHT and Aperture Use information above to determine appropriate values. 4. Rotate the Stage Under the Gun This is done using a joystick and watching on the TV monitor of the SEM chamber. You will be able to see on the monitor when the sample is under the gun. 5. Turn on the EHT This creates the electron beam and allows you to view the sample under the microscope. 6. Focus Lenses This is the most crucial and complex step. Focusing involves a series of adjustments, first to the working distance, then to the stigmation, then to the aperture. This process is made easier if you start at a large contaminant on your sample and then focus on the smaller scale after you are focused on the larger scale. This technique is a universal one, developed because a blank region of a sample will not have any distinct features, making it difficult to know how well-focused the microscope is. One final note is that all focusing can be done through coarse adjustments and fine adjustments. Use coarse adjustments when approaching a focused image and then use fine adjustments to maximize image clarity.

a) Find contaminant or other large, distinct feature to assist in focusing b) Set scan speed to four Scan speed determines how fast the electron beam traverses the sample. Lower scan speeds lead to faster scanning, which refreshes the image more frequently but shows less definition. For focusing, a medium scan speed provides the best balance between image refreshment and image definition. c) Adjust zoom to 1,000x or lower d) Adjust working distance until feature is as focused as possible e) Adjust stigmation until feature is as focused as possible. Stigmation can be adjusted in the x and y direction. You will have to experiment with each direction and carefully look to determine what manipulations most clarify the image. f) Adjust aperture until feature is as focused as possible g) Set scan speed to eight to determine if image is sufficiently focused h) Increase magnification and repeat steps a-g The amount to increase magnification depends largely on experience. Gaining experience will allow you to focus easier after making larger changes to magnification. i) Repeat steps a-h until you have reached your desired magnification 7. View the Sample You have already done the heavy lifting. Now is the time to determine how your experiments went. 8. Unload the Sample Like earlier, you will have to equilibrate the secondary chamber with the SEM column chamber to remove the stage from the column chamber. Then, the same logic applies for removing the sample from the secondary chamber. a) Pump the secondary chamber down to a vacuum b) Open the door from the SEM column chamber to the secondary chamber c) Pull the stage from the SEM column chamber into the secondary chamber d) Close the door from the SEM column chamber to the secondary chamber e) Purge the vacuum in the secondary chamber (you will hear a click when it reaches atmospheric pressure) f) Open the door to the secondary chamber and remove the sample g) Close the door to the secondary chamber Conclusion Operating a scanning electron microscope is quite difficult; the most experienced microscopy experts have spent tens of thousands of hours perfecting their craft. Generating clear images at magnifications of 10,000x, 100,000x, and beyond will require a keen eye for subtle details and a deft ability to manipulate the aperture, stigmation, and working distance in exactly the right ways. Some of the more humble experts would even admit that their magnification limitation comes from their ability to manipulate the SEM, as opposed to the capabilities of the SEM itself.

Persevere and know that after gaining some skill, it will be possible to characterize all kinds of research in ways that are no other device can.

References
[1] D. McCullen, "Scanning Electron Microscopy 1928 - 1965," in 51st Annual Meeting of the Microscopy Society of America, Cincinnati, 1993. [2] M. T. Postek, K. S. Howard, A. H. Johnson and K. L. McMichael, "Scanning Electron Microscopy," 1980, pp. 13-81; 461-494. [3] Hitachi, "Hitachi's system solution for sample preparation and inter-instrument transfer under a protecting atmosphere," [Online]. Available: http://www.hht-eu.com/cms/39574.html. [Accessed 9 October 2013]. [4] J. Schweitzer, "Scanning Electron Microscope," Purdue University, [Online]. Available: http://www.purdue.edu/rem/rs/sem.htm. [Accessed 9 October 2013].