Lecture 10: TEM sample preparation
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1 Lecture 10: TEM sample preparation Contents 1 TEM sample holders 1 2 Parts of a TEM holder Types of TEM holder In-situ TEM holders TEM sample preparation Electrolytic polishing Ion milling technique Cross section sample preparation Replica technique Focused ion beam TEM sample preparation 20 1 TEM sample holders The specimen holder is used to insert the sample into the TEM from outside for imaging. The sample is loaded onto the TEM stage for imaging. Now, TEM holder and stage are essentially integrated. The TEM holder plays and important role since it allows for introduction of a sample in ambient conditions into an instrument that is held in vacuum. Usually the holder region is pumped separately before the sample is fully introduced in the TEM. A schematic diagram of some of the pumps used in the TEM is showed in figure 1. There are 2 main types of holder in the TEM 1. Top loading - the sample has no connection to the outside. After delivery of the sample to the stage the holder is withdrawn. Top loading holders were used in earlier instrument version, especially for high resolution since this minimizes external vibrations. A schematic of a top loading holder is shown in figure 2. 1
2 Figure 1: Pumping system in a TEM. Taken from Transmission Electron microscopy - Williams and Carter. 2
3 Figure 2: Top entry loader in (A) cross section and (B) top view. Taken from Transmission Electron microscopy - Williams and Carter. 3
4 Figure 3: Schematic of a side entry holder. Taken from Transmission Electron microscopy - Williams and Carter. 2. Side loading - these are the most commonly used holders now. The advantage of the side loading holders is that it is possible to provide external stimuli (heat, current, load) while imaging. 2 Parts of a TEM holder The focus will be on the side loading holder since it is ubiquitous in use. A schematic of the holder is shown in figure 3. Various parts of the holder can be identified from the figure 1. O-ring - Provides the mechanical link to the TEM column. The o-ring separates the portion of the holder in the ambient from the portion in vacuum. Typically a viton o-ring is used and some holders have more than one. The o-rings are usually greased but the excess grease should be removed before inserting in the TEM since this can affect the vacuum. The way in the holder is inserted in the instrument depends on the specific TEM. 2. Jewel bearing - This provides the other mechanical link of the holder to the TEM. This is used for mechanical translation of the sample (in the x-y plane). 3. The cup - this actually holds the sample. Typical TEM samples are 3 mm discs though bulk holders are available (not very common). 4. Clamping ring - this clamps and holds the sample in place. Both the cup and the clamping ring are exposed to the electron beam and hence 4
5 Figure 4: Some types of side entry holders. From top a rotation holder, heating holder, cooling holder, double-tilt, and single-tilt holder. Taken from Transmission Electron microscopy - Williams and Carter. should be made of a material with low x-ray fluorescence. Be is the material most commonly used. 2.1 Types of TEM holder There are various designs for the side entry holder. Some of them are shown in figure Single tilt holder - most commonly used holder. Tilting around the axis of the holder is possible. Shown in figure Quick change holder - this is a single tilt holder with a clamp instead of screw. Useful for quick change of samples. Philips CM 12 series TEMs have a quick change holder. 3. Multiple specimen holder - some holders can hold more than one sample. 5
6 Figure 5: Single tilt holder with 2 and 5 specimen cups. Taken from Transmission Electron microscopy - Williams and Carter. This is again for multiple sample preparation. These are usually single tilt holders. Multiple tilt holders are shown in figure Bulk specimen holders - These are used for bulk specimens that cannot be made into 3 mm disks. This is not very common. The area of interest should still be thin enough for the electron beam to pass through. A bulk specimen holder is shown in figure Double tilt holder - two tilt angles are possible - in-plane and out-ofplane. This is most commonly used for diffraction and imaging. 6. Tilt-rotate holder - this allows to select the tilt axis and then rotate the specimen. 7. Low-background holder - the cup and clamping ring are made of Be. Mainly used for x-ray studies (EDAX) since Be has low x-ray fluorescence. 2.2 In-situ TEM holders One of the advantages of a side loading holder is that it is possible to have signals from the outside to interact with the sample while imaging. This gives added functionality to the TEM and provides for direct structure-property correlations. Some of the in-situ TEM holders are 6
7 Taken from Transmission Electron mi- Figure 6: Bulk specimen holder. croscopy - Williams and Carter. 1. Heating holder - for heating samples in-situ. Usually a resistive heater is used with temperatures as high as 1000 C possible. Care should be taken not to weld the sample to the holder. A heating holder is shown in figure Cooling holder - holders can go up to liquid nitrogen (77 K) or helium temperatures (4 K). This is mainly used for biological samples, where beam damage is minimized by cooling the sample. A cooling holder is shown in figure Cryo-transfer holder - cooling holder with cryo-transfer so that the sample is never heated to room temperature. 4. Straining holder - holder is used for applying external strain to the TEM sample. Usually piezoelectric actuators are used for precise strain application. They can be combined with other in-situ holders, like heating holder, like figure 5. EBIC and CL holders - these can supply electrical current to the sample. There are a number of in-situ holders developed by specific research groups. For example, in-situ thermal evaporation is possible in the TEM. Similarly dynamic TEM holders are available for driving in-situ reactions at high heating rates combined with diffraction measurement. 7
8 Figure 7: Strain holder with heating. microscopy - Williams and Carter. Taken from Transmission Electron 8
9 3 TEM sample preparation Sample preparation is an important part of TEM analysis. There are two main criteria for TEM sample preparation 1. The sample should be electron transparent. If not the whole sample at least the ROI should be thin. Typical thickness values for metallic samples should be nm. Usually, 100 nm is a upper limit for the sample thickness. 2. The sample should be mechanically robust for handling. Usually, the sample to be investigated is directly prepared from the underlying specimen. Sometimes for bulk specimens replica samples are prepared. This is mainly for studying surface topographies and precipitates. TEM samples are either self-supported or mounted on a grid for analysis. Copper grids are the most commonly used, though for high temperature work Mo grids are used. For nanoparticles and thin films a-c film is used as support. a-c has low contrast in the TEM and will not obscure the contrast arising from the specimen. Some typical TEM grids are shown in figure 8. The TEM preparation technique depends on the type of sample and also on the property of interest. Mechanical damage during sample preparation might be of an issue for studying dislocation densities in a specimen. Similarly cross sectional TEM preparation might be necessary for some samples, which can be a time consuming process. For most samples we need to prethin the sample to an initial thickness of µm. Cut a 3 mm disk from the sample (for bulk specimens). Thin the central disk to a few µm and then thin further (by different techniques) till sample is electron transparent. Sample sizes in the different stages of TEM preparation are shown in figure Electrolytic polishing Electrolytic polishing is used for conducting samples like metals/alloys in order to produce samples that are electron transparent. The initially sheet thickness can be around a few hundred µm. This can be prepared by rolling or grinding bulk specimens. Similarly, metal coatings on substrates can be peeled off and used for the final thinning. Thin discs can also be cut from bulk specimens. This process is called coring. Some of the different equipment for coring is shown in figure 10. There will be some mechanical damage at the surface due to coring but these can be removed during polishing. These discs are thinned by electrolytic polishing. The most common elec- 9
10 Figure 8: Typical TEM grids. Taken from Transmission Electron microscopy - Williams and Carter. Figure 9: Stages in the TEM sample preparation from a bulk specimen. Taken from TEM sample preparation - Sridhara Rao et al, Microscopy: Science, Technology, Applications and Education. 10
11 Figure 10: Different coring tools (A) mechanical punch (B) Abrasive-slurry disc cutter (B) Ultrasonic cutter (D) Spark-erosion cutter. Taken from Transmission Electron microscopy - Williams and Carter. 11
12 trolytic polishing technique is the window technique. The sample is made the anode and a thin stainless sheet is made the cathode. The sample is immersed in the electrolyte, which is usually cooled by water or liquid nitrogen. Perchloric acid is usually used as the electrolyte. The sample edges are covered by lacquer to expose a window, hence the name. The experimental setup and the hole generation are shown in figure 11. When a current is applied the material is dissolved from the anode (sample) and deposits on the cathode. The rate of dissolution depends on the current and applied voltage. The I V characteristics are shown in figure 12. Depending on the current and voltage, there are three regimes - etching, polishing, and pitting. The edges are coated so that material removal will start within the window. Once a hole is formed within the window the sample is pulled out. The region around the hole is usually electron transparent and can be mounted on a TEM grid. The problem with this technique is that it is hard to control the location of the hole in the window. A modification to the window technique is the Bollmann technique. A pointed cathode is used to form an initial hole in the location of choice and this is then replaced by flat electrodes to expand the electron transparent region. A variation of the Bollmann technique, which is very commonly used, is the Jet polishing technique. A schematic of the jet polishing technique is shown in figure 13. Here the electrolyte is locally sprayed on the sample, using pointed electrodes. The jet helps in polishing from both ends of the sample and hence it is faster. The method also reduces electrolyte use and can produce large electron transparent areas. The electrolyte polishing technique can be used for samples that are conducting - mostly metals and alloys. For non-conducting samples like ceramics, semiconductors, deposited films, other TEM sample preparation techniques are needed. 3.2 Ion milling technique For non-conducting samples usually grinding and polishing steps are used in order to reduce sample thickness. Some an ultramicrotome is used in order to generate thin samples. These can be either electron transparent or can be used as the starting material for further thinning. The schematic of the technique is shown in figure 14. For samples, where ultramicrotome cannot be used then a standard tripod polisher is used in order to thin the sample. This produces samples that are a few µm thick. The final polishing step is done by an ion beam miller. The schematic of the ion beam miller and an actual instrument are shown in figure 15. The sample is bombarded by high energy ions or neutral atoms. 12
13 Figure 11: Window polishing technique. Taken from Transmission Electron microscopy - Williams and Carter. 13
14 Figure 12: I V characteristics during polishing. Taken from Transmission Electron microscopy - Williams and Carter. Figure 13: Schematic of the jet polishing technique. Taken from Transmission Electron microscopy - Williams and Carter. 14
15 Figure 14: Schematic of ultramicrotome technique. Taken from Transmission Electron microscopy - Williams and Carter. 15
16 Figure 15: Schematic of ion beam milling technique. Taken from Transmission Electron microscopy - Williams and Carter. 16
17 Figure 16: Penetration vs. thinning as a function of beam incidence. Taken from Transmission Electron microscopy - Williams and Carter. Usually Ar ions are used and they are formed by passing the Ar gas though a high voltage (4-6 kev ). The sample is held in vacuum and also usually cooled by liquid nitrogen. The ions are incident on the sample and sputter material away. To minimize ion penetration the beam is usually incident at a low angle ( 20 ) though if the angle is very small the sputter rate is small. The trade off between ion penetration and thinning rate is shown in figure 16. Ion beam is highly controlled and a localized process but it is time consuming. Sputter rates are usually a few Å per second so that creating an electron transparent sample can take hours, especially if the initial thickness is high. 3.3 Cross section sample preparation Cross sectional samples are routinely interrogated in the TEM, especially for studies of interface. The schematic of the cross section process is shown in figure 17. Slices from the sample are cut using a diamond slicer. These slices are placed between spacer layers and then glued on to a grid. The slices are glued in such a way that the interface is parallel to the slot in the grid. This sample is then thinned by standard tripod polishing until it is a few µm thick. The final sample is thinned using a ion beam miller to create an electron transparent sample. 17
18 Figure 17: Cross sectional sample preparation. Electron microscopy - Williams and Carter. Taken from Transmission 3.4 Replica technique Replica technique is used for studying bulk specimens which cannot be destroyed to prepare electron specimens. It is also useful for studying surface topography features and precipitates though SEM techniques have gradually replaced replica sample preparation. The schematic of the replica technique is shown in figure 18. A replica of the sample surface is prepared using a plastic mold. The mold is then removed from the surface and the surface of the specimen is replicated by the surface of the plastic. A thin film of carbon or metal like Cr, Pt is evaporated on the surface of the plastic. Sometimes the evaporation is done from an oblique angle, shadow evaporation, to enhance the contrast. The plastic is removed by dissolving in a suitable solvent and the film is then floated on to a grid for analysis. A variation of the replica technique is the extraction replica technique. As the name implies, it is used to extract and study precipitates embedded in a matrix. The schematic of the process is shown in figure 19. The bulk sample is etched in order to expose the precipitates. A C film is then evaporated on the sample and then etching is continued until the bulk material is removed leaving behind the precipitates. A TEM image of precipitates removed by the extraction technique is shown in figure
19 Figure 18: Replica technique for sample preparation. Taken from Transmission Electron microscopy - Williams and Carter. Figure 19: Extraction replica technique for sample preparation. Taken from Transmission Electron microscopy - Williams and Carter. 19
20 Figure 20: TEM image of sample prepared by extraction replica technique. Taken from Transmission Electron microscopy - Williams and Carter. 4 Focused ion beam TEM sample preparation Focused ion beam (FIB) instrument along with SEM is becoming increasingly important in TEM sample preparation. The advantage of FIB-SEM is that it is possible to prepare TEM specimens from precise locations in the sample. This is not possible in other techniques since there is always some uncertainty from where the final TEM sample will be obtained. One area where the FIB is used extensively is in the microelectronics industry for evaluation of defects in parts of the micro-chip. Here the area to be investigated is of the order of tens of nms, so that conventional TEM preparation techniques cannot be used. The schematic of the 2 beam FIB-SEM is shown in figure 21. The FIB can be considered as an ion beam miller except that Ga ions are used instead of Ar ions in a conventional ion beam miller. The Ga ions are originated from a liquid metal source (Ga melting point is 30 C). The beam is accelerated and scanned over the specific portion of the specimen for milling. Modern FIB-SEM also have gas-injection systems (GIS) for Pt deposition and a micromanipulator (Omniprobe) for removing the specimen from the sample and attaching to the TEM grid. The steps involved in sample preparation are 1. Identification of the ROI. 2. Deposition of a Pt layer on top of this region. Pt deposition is done 20
21 Figure 21: Schematic of the 2 beam FIB-SEM. Taken from Transmission Electron microscopy - Williams and Carter. using the GIS. The Pt deposition is done over a region few µm long and wide and a few nm thick. 3. The Ga ions are used to ion mill the region around the Pt layer to create a trench. Sample can be manipulated in-situ with three dimensional translation and rotation possible. The Pt layer is also cut from below to create a lamellar. 4. The Omniprobe is used to lift this lamellar from the sample and then attach it to the TEM grid. The Pt metal is used to weld the lamellar to the grid. 5. The final cleaning of the lamellar is done using the Ga ions. Final thinning to electron transparency is also done. The various stages in making the lamellar are shown in figure 22. The entire TEM preparation is done in-situ and the SEM is used for imaging the sample preparation. The FIB can also be used for imaging. The FIB-SEM instrument and the electron and ion beam column is shown in figure 23. The TEM sample before lift-off and the final sample attached to the grid is shown in figure 24. The final sample has dimensions of the order of µm but its thickness is of the order of nm, so that it is electron transparent. The FIB-SEM technique is the most versatile of the TEM sample preparation techniques since it can be used for both conducting and insulating samples. It also allows for the preparation of TEM samples from specific areas of the sample. But sample preparation and manipulation using the FIB-SEM is technically challenging. 21
22 Figure 22: Stages in creation of the lamella from a specimen are shown. Taken from Transmission Electron microscopy - Williams and Carter. 22
23 Figure 23: Dual beam FIB-SEM instrument with the close-up of the electron and ion beam column. Taken from Transmission Electron microscopy - Williams and Carter. 23
24 Figure 24: TEM lamellar before lift-off and the final sample attached to the TEM grid. From P. Swaminathan - unpublished. 24
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