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Sample Preparation Techniques for Electron Microscopy 50 Years of SEM and Beyond!

Syed Nasimul Alam, Lailesh Kumar, Nidhi Sharma, Pallabi Bhuyan and Sivateja Chinnam

Department of Metallurgical and Materials Engineering, National Institute of Technology Rourkela, Rourkela, Orissa, Pin-7690078, India

This chapter pays tribute to the 50 years of commercialization of the scanning electron microscope (SEM) by highlighting the major breakthroughs of the instrument and elaborates in detail the various sample preparation techniques for the SEM and the TEM. In 1965, the first commercial SEM, which was named Stereoscan was built by Cambridge Instrument Company in the UK. Since the commercialization of the SEM 50 years ago, significant improvements have been introduced in the electron microscope which are making them a highly versatile instrument that could be used for characterizing a wide range of materials and solve critical problems in various disciplines. Microscopy is the field of using microscopes to view objects. There are three well-known branches of microscopy, namely, optical, electron and scanning probe microscopy. The key to achieving best results during an examination under a microscope lies in the sample preparation techniques that have been adopted. The required sample preparation techniques depend on the type of samples as well as on the application of the samples. This chapter provides an overview of the various prerequisite sample preparation techniques for both SEM and TEM. Observation of samples under a SEM does not need an elaborate sample preparation process. However, observation of samples under a transmission electron microscope (TEM) requires a sequence of sample preparation steps. Instruments like the TEM need extremely thin sections of the specimens, typically about 100 nm or less. In a TEM the level of reduction of thickness needed for a sample depends on the atomic number of the elements present in the sample. The higher the atomic number, the heavier is the element and the higher is its electron scattering factor. Therefore, such samples need to be thinned down to a larger extent as compared to samples containing lighter elements. In order to polish the sample and to reduce its thickness, it is essential to mount the sample onto bakelite and polish using finer and finer grits or a polishing slurry. An electron microscope uses electron waves. Therefore electrically nonconducting samples need to be coated in order to avoid charging of the sample when under observation to improve the imaging of the sample. Coating a nonconducting sample with a layer of conducting metal can prevent charging of the sample and therefore improve the quality of the image. The coating can also reduce thermal damage and improve the secondary electron signal from the sample which is used for secondary electron imaging of the sample in a SEM. Today it has become possible to image individual atoms and molecules using electron microscopes. Electron microscopes have broken the barrier of resolving power. Their resolving power can be as high as 1 Å and magnifications of upto 2 million can be achieved. The future of microscopy would be the possibility of remotely controlling the microscope which would allow us to use the instrument without the need of being present near the electron microscope itself.

Keywords: Scanning Electron Microscopy; Transmission Electron Microscopy; Sample Preparation Techniques

1. Introduction

"By the help of Microscopes, there is nothing so small, as to escape our inquiry; hence there is a new visible World discovered to the understanding” …Robert Hooke (Micrographia, 1665)

The Electron Microscope Timeline In 1590 Hans Janssen and his son made one of the first compound microscopes. They had put several lenses in a tube and the object near the end of the tube appeared to be greatly enlarged several times more than what any simple magnifying glass could achieve. In 1660 Robert Hooke improved the microscope by adding an oil lamp. However, it was Anton Leeuwenhoek in 1683 who was the first person to make and use a real microscope. He made a simple microscope that could magnify an object 266 times. In 1886 the first modern light compound microscope was invented. The electron microscope was invented by German engineers Max Knoll and Ernst Ruska at the Berlin Technische Hochschule in 1931. This microscope had a resolution much higher than that of light and a magnification of around 12,000 x could be achieved. In the late 1930s electron microscopes having resolutions of 10 nm were designed and produced, and by 1944 the resolution was further reduced to 2 nm. The earliest known working concept of a SEM was given by Max Knoll in 1935. Manfred Von Ardenne in Berlin produced the earliest scanning-transmission electron microscope in 1938 by adding scan coils to a transmission electron microscope. He scanned a very small raster with a demagnified and finely focused electron beam having a diameter of around 10 nm on the target. His first SEM image of was that of ZnO crystal at 8000x. Ernst Ruska at Siemens in Germany developed the first commercial electron microscope in 1938. Cecil Hall, James Hillier, and Albert Prebus, working under Eli Franklin Burton produced an advanced Toronto Model electron microscope at University of Toronto, Canada in 1938. This later became the basis for Radio Corporation of America's Model B, the first commercial electron microscope in North America. Siemens produced the first commercial TEM in 1939. In 1940 Von Ardenne differentiated between the secondary and backscattered electrons. He showed that reducing the beam energy improves contrast at the expense of resolution. The

Microscopy and imaging science: practical approaches to applied research and education (A. Méndez-Vilas, Ed.)

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introduction of the first commercial SEM in 1965 completely opened up a new world for analyzing the structure of materials and correlating them to their properties. Ultrahigh voltage (up to 3 MeV) TEM instruments at CEMES-LOE/CNRS (Toulouse) and at Hitachi (Tokyo) in the 1960s and 1970s gave electrons higher energy to penetrate more deeply into thick samples. Development of electron sources, such as the lanthanum hexaboride filament (LAB6) and the field emission gun in the 1960s, provided a brighter source of electrons and this led to better imaging and resolution. During 1980s to 1990s, the development of environmental electron microscopes allowed scientists to examine samples under more natural conditions of temperature and pressure. In 1981 the scanning tunneling microscope (STM) was invented by Gerd Binnig and Heinrich Rohrer which cold magnify an object 1,000,000 times [1-4].

Fig. 1 (a) M. von Ardenne's first SEM (b) Manfred von Ardenne (c) Max Knoll and Ernst Ruska working on their first TEM

Fig. 2 (a-e) Parts of a SEM (f) Tungsten filamnet (Electron gun)

2. Sample Preparation Techniques for SEM

Specimens for SEM examination must be prepared by a sequence of steps. The various preliminary sample preparation steps before a sample can be observed under a SEM are listed below. i. Cutting: Sample preparation starts with "Cutting" and good cutting ensures a good beginning to your sample preparation. It is the first step in the overall process of specimen preparation. It enables easy handling of the sample. Correct cutting produces specimens which are in perfect condition for the next preparation steps. Therefore, it is a step that should be given considerable thought and care. Where the sectioning should be placed and what equipment should be used are important factors that need to be considered. Precision cutting is used for precise and deformation-free cutting. Initially bulk specimen should be cut to less than 1.25 inches with a hacksaw or a disc saw. Care must be taken not to heat the sample during cutting. A wide range of diamond impregnated cutting saws is available for precision cutting of a variety of materials. Even highly brittle materials like ceramics and intermetallics can be cut using a diamond saw.

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Fig. 3 Sectioning of a bulk sample using a saw ii. Mounting: Small samples can be difficult to hold safely during grinding and polishing operations, and their shape may not be suitable for observation on a flat surface. In order to polish the sample to and reduce its thickness it can be mounted onto bakelite and polished on finer grits or using polishing slurry. The aim of mounting is to handle small or odd shaped specimens and to protect fragile materials, thin layers or coating during preparation as well as to provide good edge retention. Mounting produces specimens with uniform size so that it is easier to handle in automatic holders for further preparation steps. Two methods are mainly available for mounting, namely, hot mounting and cold mounting. Mounting can be done using a mounting press. Hot mounting in a press with phenolic resin or cold mount in polyester can be done. Hot mounting is faster and more generally used for metals while the cold mounting is favored for ceramic samples.

Fig. 4 Mounting of a bulk sample. iii. Polishing: Mechanical Grinding – This is a form of rough polishing, which involves using different grades (or grits) of SiC abrasive papers/belts/cloths, with water as a lubricant. There are a number of grades of paper, with 180, 240, 400, 1200, grains of SiC per square inch. 180 grades therefore represent the coarsest particles and this is the grade to begin the grinding operation. Always use light pressure applied at the center of the sample. Wash the sample in water and move to the next grade, orienting the scratches from the previous grade normal to the rotation direction. This makes it easy to see when the coarser scratches have all been removed. After the final grinding operation on 1200 paper, wash the sample in water followed by alcohol and dry it before moving to the polishers. Mechanical Polishing After grinding with abrasive paper, the sample is polished to produce a flat, reasonably scratch-free surface with high reflectivity. The polishers consist of rotating discs covered with soft cloth impregnated with a pre-prepared slurry of hard powdery alumina particles (Al2O3, the size ranges from 0.5 to 0.03 µm). Begin with the coarse slurry and continue polishing until the grinding scratches have been removed. It is of vital importance that the sample is thoroughly cleaned using soapy water, followed by alcohol, and dried before moving onto the final stage. Any contamination of the final polishing disc will make it impossible to achieve a satisfactory polish. Examining the specimen in the microscope after polishing should reveal mirror like surface.

(i) Coarse polishing (30 to 3 μm finish) – Coarse polishing is done on special clothes with diamond paste with some lubricant.

(ii) Fine polishing (≤ 1 μm finish) - Fine polishing is done using diamond paste with lubricant or silica or alumina with water lubricant.

Electropolishing or Electrolytic Polishing – This is a process by which a mechanically polished sample is made smooth and brighter by making the sample as the anode in an electrolytic cell. This is possible only when the correct combination of bath temperature, voltage, current density and time is employed. Chemical Polishing – This procedure can be used instead of electropolishing. It is a method for obtaining a polished surface by immersion in or swabbing with a suitable solution without the need of an external electric current. Finally the polished sample is placed under the SEM for observation. For non-conducting samples the sample surface must be coated with gold (Au) or carbon (C). Carbon coated conducting adhesive tapes are used to stick the SEM samples on the SEM stubs. The stubs are then placed inside the SEM for observation. The carbon coated tapes are electrically conducting and non-porous. As the carbon coated tape is electrically conducting it avoids charging during observation under the SEM and can be used without coating. The thickness of the tapes is around 125 μm. The liner protects the tape from any contamination [5, 6]. These tapes can be very easily removed from the SEM stubs using tweezers or using ethyl acetate, ethanol, isopropanol, alcohols or any other commercial adhesive remover.

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Fig. 5 (a) SEM stubs placed in a SEM pin mount specimen holder (b) Carbon coated conducting adhesive tapes

3. Sample Preparation Techniques for HRTEM

Sample preparation is a key step for the observation of samples in an electron microscope. To achieve the best results during an examination in the electron microscope, perfect sample preparation is a prerequisite. The required techniques depend on the type of samples (biological samples, material samples) as well as on the application. The major disadvantage of the transmission electron microscope (TEM) is the need for extremely thin sections of the specimens, typically about 100 nm. For powder samples the powder size should be less than 0.5 μm. Powder samples which have a size more than this should be ground with mortar and pestle.

i. Sample Preparation of Powder Samples For observing a powder sample under a TEM the powder after grinding should be dispersed using an ultrasonicator. A drop of the dispersed powder could be put on the TEM grid for observation under a TEM. The powder sample is usually dispersed in an ultrasonic tank. An ultrasonic tank is a device that uses high frequency sound waves or ultrasound (from 20-400 kHz) that is transmitted in a liquid medium. It is commonly used for cleaning however it is also used in an electron microscopy laboratory for sample preparation of powder samples. An ultrasound generating transducer present at the bottom of the tank produces ultrasonic waves in the liquid medium with an electrical signal oscillating at an ultrasonic frequency. This creates compression wave in the liquid medium in the tank. The powder sample is dispersed in a suitable liquid medium like distilled water, acetone, alcohol etc.. After putting one or two drops of the powder sample dispersed in a liquid medium on the TEM grid it is then dried and finally placed into the TEM sample holder. The sample holder is then placed in the transmission electron microscope for observation.

Fig. 6 An ultrasonication tank used for dispersing powder samples

Fig.7 (a, b) SEM images of a TEM grid (c) Schematic diagram of the TEM grid After properly dispersing the powder sample in liquid medium using an ultrasonicator one/two drops of the dispersion is then put on a 3 mm diameter TEM Cu grid. The thickness of the TEM grids could vary from 10-25 μm. The mesh size of the grid tells us about the number of holes per inch in the grid. So approximately a 200 mesh grid would have 20 holes along the diameter and a 400 mesh grid would have about 40 holes along the diameter of the grid.

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A hole in th 100 mesh TEM grid would be around ∼200 μm whereas the hole in a 200 mesh grid would be around 97 μm and in 400 mesh TEM grid it could be as low as ∼42 μm. Fig. 7(a) is the SEM image of a TEM Cu grid. The high magnification SEM image of the Cu grid in Fig,.7(b) shows that the size of the square hole in the TEM grid is around 34.7 μm. A TEM grid could be made of Cu , Ni or Au. Formvar coated grids without a carbon layer could be used for upto an operating voltage of 100 kV. Formvar is the commercial name of polyvinyl formal. For many applications the mesh sizes of the standard TEM grids are too large to support the sample. For such cases a holey carbon film on the TEM grid acts as a suitable alternative. A holey carbon film has holes of various sizes up to 100 µm. A continuous carbon film is also used for few applications. However, a continuous carbon film can have a problem as the thin film can inelastically scatter electrons and contribute to background noise. This is why a holey carbon film is used as it is capable of providing support as well as higher image contrast in the regions of the holes as compared to other regions of the carbon film. Apart from this the high electrical and thermal conductivity the carbon film helps to stabilize the formavar films when exposed to the electron beam. The carbon film could be around ~15 nm thick and is mounted on either Cu, Ni or Au grids. The holes could be either square or round in shape and have different sizes. The holes could have a size of around of 0.2 to 8 μm. These grids can be used at high magnifications and high beam intensities and are stable in the electron microscope under almost all operating conditions. They can also withstand vigorous specimen preparation techniques. Sometimes lacey carbon films are also used to coat the HRTEM grids. They are very similar to the holey carbon films however they provide a greater open area as compared to the holey carbon films. They act as support film and provide adequate strength to the TEM grids without adding to any background interference. HRTEM grids coated with lacey carbon films are often used for studying nanostructured materials like nanotubes, graphenes, biological samples, etc. Due to a large open area without support in the case of HRTEM grids coated with lacey carbon films, the sample under observation remains unsupported and this is why they are preferred where background interference is not desired [7-9]. ii. Sample Preparation of Bulk Samples The various steps involved in the sample preparation of bulk TEM samples are as follows:

• Initial thinning to make a slice of material between 100-200 mm thick • Cut a 3-mm disk from the bulk sample. • Prethin the central region from one or both faces of the disk to a few micrometers • Final thinning of the disk to electron transparency. In order to achieve electron transparency the thickness of the thickness should be brought down to below 100 nm.

Fig. 8 Schematic diagram showing the various stages of reduction of sample thickness during TEM sample preparation [10]

Initial thinning Initial thinking for ductile materials can be done by using a wafering saw. Chemical wire/string can also be used which works by passing the string through a solvent and then across the sample until the string cuts the sample. Materials like Si, GaAs, NaCl, MgO can be cleaved with a razor blade. Ultramicrotomy is also used for sectioning which operates by moving the specimen past a knife blade. The blade can be a glass for soft materials and but will be diamond for harder materials. Ultramicrotomy provides an easy sample preparation technique for by which we can create ultrathin sections of a material having highly smooth surfaces for observation under a HRTEM. A microtome is an instrument used for


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cutting extremely thin slices from a sample. Various types of materials ranging from very hard to soft can be sliced and can be used as an alternative to electropolishing and ion-milling. Slices having thickness in the range of 50-100 μm can be made. Disc grinding can be done to produce parallel-sided thin samples. A Disc Punch System A disc punch system is used for cutting 3 mm discs for the TEM from metals or alloys or any other ductile material after the initial material is brought down to a thickness of around 200 μm. A 3 mm disc can be cut from this material using an ultrasonic disc cutter or a disc punch system. The ultrasonic cutter is ideal for cutting brittle materials like ceramics and semiconductors whereas the disc punch system is ideal for ductile materials like metals and alloys. Dimpling The Dimpler provides with the easiest and most reliable means to produce many different types of samples for TEM and can be used for ceramics, many semiconductors, carbons, carbon composites, oxides, borides, silicides, glasses and many others. During the final thinning stages of polishing it is not desirable to intruduce any damage to the sample. Therefore, care should be take while reducing the thickness during these stages. This is why mechncial grinding techniques are not practical during the final stages of sample preparation. Dimpling can very effectively reduce the thickness of the sample form 100-200 μm to few microns in a very short time without producing relatively little damage. Thereafter, chemical polishing or particle beam thinning techniques like ion milling could be used for producing electron transparent areas in the sample. The use of dimpler is to create a dimple having a thickness of few microns in a 3 mm diameter parallel sided slice of the sample. A schematic diagram of the dimpler is shown in the Fig.9. Dimpling is based on the mechanical action of abrasion. The grinding wheel made of a hard material rotates on the sample. During the abrasion process a fluid is used which acts both as a lubricant and as a cooling agent. The objective is to obatin a scratch free dimple having low mechanical damage. After dimpling the final thinning for obtaining an electron transparent sample for obseravtion in a TEM can be done by electropolishing, chemical polishing or ion beam thinning techniques. One of the major advantages of dimpling is that creates a large smooth and polished area. Dimpling reduces the chances of developing surface irregularities during the final thinning operation and thereby increases the yield of electron transparent material. Dimpling can reduce the thickness of the sample to less than 3 μm. This thickness is actually sufficient for observation of the sample using intermediate voltage TEMs. It should be noted that only materials which are resistant to mechanical damage like metals, ceramics etc. can be used for this sample preparation technique. The grinding speed can be controlled and should be kept low to avoid damage on the sample [11]. During the final few microns special polishing wheel can also be used.

Fig.9 Schematic diagram of a dimpler grinder

Final thinning: Electropolishing and Jet polishing Electrochemistry and the fundamental principles of electrolysis (Faraday's Law) replace traditional mechanical finishing techniques, including grinding, milling, blasting and buffing as the final finish. In basic terms, the metal object to be electropolished is immersed in an electrolyte and subjected to a direct electrical current. The object is maintained anodic, with the cathodic connection being made to a nearby metal conductor. During electropolishing, the polarized surface film is subjected to the combined effects of gassing (oxygen), which occurs with electrochemical metal removal, saturation of the surface with dissolved metal and the agitation and temperature of the electrolyte [9]. Electro polishing can only be used for electrically conductive samples such as metals and alloys. Thinning is accomplished by electropolishing at a high current density. Jet polishing is a technique in electrolytic polishing. In this technique, a jet-like polishing solution is sprayed onto a specimen to make a hole at its center. Spraying the solution makes it possible to keep the solution at the specimen surface fresh, thus a quick and smooth polishing is achieved. In a jet-polishing machine the 3 mm disk TEM sample is kept vertical and spins rapidly around its axis. A nozzle spouts a thin stream of solution on the disk. The solution could be an acid as many metals are very vulnerable to acids. The acid corrodes the


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disk in the centre until a small hole is made. The disk becomes thin in the middle around the hole and thick near the edges. The sample is now reedy for observation under a TEM as around the edge of the hole the material is thin enough to be electron transparent. Ion Milling Ion milling thins samples until they are transparent to electrons by firing ions at the surface at an angle and sputtering materials from the surface of the sample. In ion milling ions contained within plasma formed by an electrical discharge are accelerated by a pair of optically aligned grids. The highly collimated beam is focused on a tilted work plate that rotates during the milling operation. A neutralization filament prevents the buildup of positive charge on the work plate. Two argon (Ar) ion guns can be used to sputter from both sides of the sample. The sample is rotated about the axis perpendicular to the plane of assembly. This ensures uniform removal of waste material resulting in straight side walls. By making the sample electron transparent it can be imaged using a TEM. The standard sample preparation method of mechanical polishing often requires a relatively long ion milling time upto several hours, which can induce defects into the sample. Smaller angle of incidence can cause less damage to the sample. On the other hand high ion current can cause more damage to the sample [12].

Cross Section TEM sample Preparation For cross section TEM sample preparation first a stack has to be made by gluing together two or more substrate fragments film-to-film.

Fig.10 Diagram of a multilayer in a sandwich form for cross section TEM observation. Finally the sample is placed in a TEM sample holder and placed in the TEM for observation. Fig. 11 shows the various parts of a TEM sample holder.

Fig.11 (a) Schematic diagram showing the parts of a TEM sample holder (b-d) TEM sample holder

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Fig. 12 shows SEM and HRTEM images of nanomaterials.

Fig. 12 (a, b) SEM images of graphite nanoplatelets and (c, d) HRTEM images of MWCNTs

Acknowledgements We gratefully acknowledge the support provided by the SEM, FESEM and HRTEM laboratories of Metallurgical and Materials Engineering Department, Ceramic Engineering Department and Chemical Engineering Department at NIT Rourkela. We also thank the support provided by Central Research Facility, IIT Kharagpur.

References [1] Sample Preparation Techniques in Analytical Chemistry, Edited by Somenath Mitra, John Wiley & Sons, Inc., 2003. [2] Advances in high-resolution transmission electron microscopy for materials science Angus Kirkland and Neil Young Microscopy

and Analysis, 25th Anniversary Issue September 2012 19-24. [3] Sample Preparation for Microscopic and Spectroscopic Characterization of Solid Surfaces and Films Sharmila M.

Mukhopadhyay. [4] David B. Williams and C. Barry Carter, Transmission Electron Microscopy, Plenum Press, New York, 1996. [5] G. F. Vander Voort, ed., "Chemical and Electrolytic Polishing," ASM Handbook, Vol. 9: Metallography and

Microstructures, ASM International, 2004, p 281-293, ISBN 978-0-87170-706-2. [6] J.W. Edington, Practical Electron Microscopy in Materials Science, Vols. 1-5, 1974, Philips, Eindhoven. [7] Thomson-Russell K.C. and Edington J.W. (1977) Electron Microscope preparation techniques in material science. Macmillan Ed. [8] TEM specimen preparation techniques D. V. Sridhara Rao, K. Muraleedharan, and C. J. Humphreys Microscopy: Science,

Technology, Applications and Education, Volume-2, A. Méndez-Vilas and J. Díaz (Eds.), 2010. [9] Gareth Thomas and Michael J. Goringe, Transmission Electron Microscopy of Materials, John Wiley & Sons, New York, 1979. [10] Electron Microscopy and Analysis, John Humphreys, Richard Beanland, Peter J. Goodhew, CRC Press, 2000. [11] S.D. Walck, J.P. McCaffrey, in: R.M. Anderson, S.D.Walek (Eds.),Specimen Preparation for Transmission Electron Microscopy

of Materials IV, Vol. 480, Materials Research Society, Pittsburgh, PA, 1997, p. 149. [12] N.G. Chew, A.G. Cullis, The preparation of transmission electron microscope specimens from compound semiconductors by ion

milling,., Ultramicroscopy, Volume 23, Issue 2, 1987, Pages 175-198.

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