Orientation Microscopy (OM) is a technique for determining the crystallites orientations in the automatic way using systems for acquisition and indexing diffraction patterns in grid of points refers to sample coordinate system. In this way, plenty of additional information about examined material can be provided. As a conventional method of orientation visualization, color code map is used; however, data behind each pixel is applicable for further computing. Quantifying parameters and characteristics based on the sets of measured orientations provide essential information about grains, grain boundaries and

OM was introduced in the Scanning Electron Microscope (SEM) in the 1990s [Adams, 1993]. Nowadays OM or Orientation Mapping technique is a very well known and common technique in the SEM. There exist several commercial systems for creating and processing data obtained in the SEM. In order to improve spatial and angular resolution introducing

The study of the microstructure can be carried out using many techniques eg. light and electron microscopy. Most of the classical methods allow only the qualitative analysis. The use of crystallographic information enables more advanced qualitative and quantitative description of microstructure. Of particular importance are information obtained from systematic measurements of crystallographic orientation of the sample in defined areas of the investigated material. Knowledge of the orientation distribution can be linked with the

Crystallographic orientation can be defined as function of coordinates (x,y,z) correlated with points in the sample of investigated material. Since measurements are usually carried out in the plane of the sample, the function depends on coordinates x, y. Then crystallographic orientation at each point with coordinates x, y can be defined as the rotation transforming

the OM in the Transmission Electron Microscope (TEM) was considered.

**2. Crystallographic orientation and measurements methods**

**1. Introduction**

about local crystallographic lattice deformation.

properties characterizing the polycrystalline material.

## **Orientation Microscopy in the Transmission Electron Microscope - Investigations of Small Orientations Changes by Means of Orientation Mapping in TEM**

M. Bieda, K. Sztwiertnia, A. Korneva and J. Kawalko *Institute of Metallurgy and Materials Science PAS, Krakow, Poland*

## **1. Introduction**

50 The Transmission Electron Microscope

[37] Steurer, W. and Kuo, K. H. Acta Cryst., B46, 703 (1990).

Orientation Microscopy (OM) is a technique for determining the crystallites orientations in the automatic way using systems for acquisition and indexing diffraction patterns in grid of points refers to sample coordinate system. In this way, plenty of additional information about examined material can be provided. As a conventional method of orientation visualization, color code map is used; however, data behind each pixel is applicable for further computing. Quantifying parameters and characteristics based on the sets of measured orientations provide essential information about grains, grain boundaries and about local crystallographic lattice deformation.

OM was introduced in the Scanning Electron Microscope (SEM) in the 1990s [Adams, 1993]. Nowadays OM or Orientation Mapping technique is a very well known and common technique in the SEM. There exist several commercial systems for creating and processing data obtained in the SEM. In order to improve spatial and angular resolution introducing the OM in the Transmission Electron Microscope (TEM) was considered.

#### **2. Crystallographic orientation and measurements methods**

The study of the microstructure can be carried out using many techniques eg. light and electron microscopy. Most of the classical methods allow only the qualitative analysis. The use of crystallographic information enables more advanced qualitative and quantitative description of microstructure. Of particular importance are information obtained from systematic measurements of crystallographic orientation of the sample in defined areas of the investigated material. Knowledge of the orientation distribution can be linked with the properties characterizing the polycrystalline material.

Crystallographic orientation can be defined as function of coordinates (x,y,z) correlated with points in the sample of investigated material. Since measurements are usually carried out in the plane of the sample, the function depends on coordinates x, y. Then crystallographic orientation at each point with coordinates x, y can be defined as the rotation transforming

Orientation Microscopy in the Transmission Electron Microscope -

orientation corresponding to the image.

**2.1 Orientation microscopy in SEM**

Exist several types of difraction patterns in SEM eg.:

• MKXD (*Micro Kossel X-ray Diffraction*),

• EBSD (*Electron Back-Scatter Diffraction*).

different crystallographic planes on the pattern.

specific area of the sample.

Investigations of Small Orientations Changes by Means of Orientation Mapping in TEM 53

rapidly in the recent years. Especially in the case of SEM orientation measurements become almost as widely used as X-ray methods. The basic idea of the local orientation measurements is of recording diffraction images, and then finding a crystallographic

Scanning electron microscope has become in the recent years versatile tool to observe microstructure. It can be applied for visualization of the topography of the surface of the sample for analyzing chemical composition or for crystallites orientation determination in

Currently, most developed and most commonly used diffraction patterns for the measurement of local crystallographic orientation in the SEM is EBSD technique. Diffraction pattern is formed under Braggs' law. Kikuchi lines or pairs of lines originating from

Automating the processes of acquisition and indexing diffraction images has allowed the creation maps by systems to measure the orientation in the SEM. The development of such systems was associated with the development of modern research equipment including cameras CCD (Charge Coupled Device) for recording microscopic images, and numerical methods allow for automatic indexing diffraction images based on supplied data. It initiated the development of a new field of electron microscopy, called the orientation microscopy [Adams, 1993]. OM or OIMTM acronym was introduced in the 90s of last century and concerned the distribution of crystallographic orientation in the material in terms of the coordinates of the sample by means of SEM. The best known commercially available systems for measuring the crystallographic orientation is OIMTM TSL - EDAX using the method of indexing diffraction images created by Wright and Adams [Wright, 1992] and HKL Channel 5 - Oxford Instruments based on indexing algorithm developed by Schmidt et al. [Schmidt, 1989]. Own system, named ACOM (Automated Crystal Orientation Mapping) was developed by Schwarzer [Schwarzer, 1997]. The principle of operation of all systems is based on the automatic recording of diffraction images obtained from the particular sample area, indexing and mapping of orientation. The orientation map is a graphical representation of the distribution of orientation on the sample surface. Mapping the orientation is the acquisition of diffraction images in a specific grid of points with a given step [Randle, 2000]. Received orientations are represented on the map by colors matched according to previously defined key colors. In the EBSD system grid step is done by controlling the electron beam position or the stage position. The introduction of a new FIB technique (Focused Ion Beam) in scanning electron microscopes equipped with gallium gun for cutting thin film of a desired size and thickness allowed to extend to three dimensions of orientation maps. With combining FIB and EBSD measurements, it is possible to cut successive samples (with an area of approximately 50x50 microns and a thickness of about 150 nm) of bulk material, and then make orientation measurements [Zeafferer, 2005;

• ECP (*Electron Channeling Pattern*), SAECD (*Selected Area Channeling Diffraction*),

the coordinate system centered at the point (x,y) associated with the sample of the material in a coordinate system associated with a single crystal [Bunge, 1982].

To describe the orientation can be used a number of parameterization. The basic form of presentation of crystallographic orientation is a matrix of rotation. In addition, the orientations can be expressed through the axis and angle of rotation, Euler angles, Rodrigues parameters, quaternions et al. [Bunge, 1982; Morawiec, 2004, Frank, 1988]. The most commonly used are the Euler angles parameterization, where the orientation is expressed by three angles φ1, Φ, φ2, respectively: 0<φ1<2π, 0<Φ<π, 0<φ2<2π (the representation introduced by Euler in 1775) [Wenk, 2004; Morawiec, 2004]. The term crystallographic orientation is also related to the concept of crystallographic texture. Texture is called crystallographic orientation distribution in polycrystalline material [Kocks, 1998]. As defined by the texture of the material can be determined unambiguously from the measured single crystallographic orientation. Collection of individual crystallographic orientation can be used to determine the microstructural characteristics such as orientation distribution functions (ODF), misorientation distribution functions (MDF) of grain boundaries having the same or different phases. Based on the data obtained it is possible to analyze the stereological parameters of microstructures and phase distribution in the material. Proper selection of the data obtained using different measurement methods can provide the full characterization of the microstructure and determine its impact on the important, from a technological point of view, material properties.

To measure the crystallographic orientation can be used various techniques, from optical methods to diffraction methods [Wenk, 2004]. The most commonly used methods are diffraction: X-ray diffraction, neutrons and electrons.

The phenomenon of diffraction on the crystal can be described by the Braggs' equation.

X-ray diffraction methods are widely used to global texture measurements. To advantages of this method belong:

Spatial resolution of diffraction methods by means of the diffractometer depends on the used method and ranging from 50 μm to 0.1 mm [Schwarzer, 1998].

In order to increase spatial resolution instead of the traditional lamp as a source of the x ray radiation can be applied synchrotron. In that way can be obtained spatial resolution about 5 μm. Using the synchrotron radiation it is possible to obtain 3D microstructural characteristic 3DXRD (*Three-dimensional X-ray Diffraction*). This method was developed in Risø National Laboratory (Denmark) [Poulsen, 2004].

For texture characterization of the large volume of bulk materials the most proper is using neutron diffraction. But the access to the neutron diffractometer is limited.

New intensively developed methods are methods of local oreintation measurements by means of electron microscopes.

Due to the progress in equipment and fast data collecting computers methods of orientation measurements in scanning and transmission electron microscopy have been developed rapidly in the recent years. Especially in the case of SEM orientation measurements become almost as widely used as X-ray methods. The basic idea of the local orientation measurements is of recording diffraction images, and then finding a crystallographic orientation corresponding to the image.

#### **2.1 Orientation microscopy in SEM**

52 The Transmission Electron Microscope

the coordinate system centered at the point (x,y) associated with the sample of the material

To describe the orientation can be used a number of parameterization. The basic form of presentation of crystallographic orientation is a matrix of rotation. In addition, the orientations can be expressed through the axis and angle of rotation, Euler angles, Rodrigues parameters, quaternions et al. [Bunge, 1982; Morawiec, 2004, Frank, 1988]. The most commonly used are the Euler angles parameterization, where the orientation is expressed by three angles φ1, Φ, φ2, respectively: 0<φ1<2π, 0<Φ<π, 0<φ2<2π (the representation introduced by Euler in 1775) [Wenk, 2004; Morawiec, 2004]. The term crystallographic orientation is also related to the concept of crystallographic texture. Texture is called crystallographic orientation distribution in polycrystalline material [Kocks, 1998]. As defined by the texture of the material can be determined unambiguously from the measured single crystallographic orientation. Collection of individual crystallographic orientation can be used to determine the microstructural characteristics such as orientation distribution functions (ODF), misorientation distribution functions (MDF) of grain boundaries having the same or different phases. Based on the data obtained it is possible to analyze the stereological parameters of microstructures and phase distribution in the material. Proper selection of the data obtained using different measurement methods can provide the full characterization of the microstructure and determine its impact on the

To measure the crystallographic orientation can be used various techniques, from optical methods to diffraction methods [Wenk, 2004]. The most commonly used methods are

X-ray diffraction methods are widely used to global texture measurements. To advantages

Spatial resolution of diffraction methods by means of the diffractometer depends on the

In order to increase spatial resolution instead of the traditional lamp as a source of the x ray radiation can be applied synchrotron. In that way can be obtained spatial resolution about 5 μm. Using the synchrotron radiation it is possible to obtain 3D microstructural characteristic 3DXRD (*Three-dimensional X-ray Diffraction*). This method was developed in

For texture characterization of the large volume of bulk materials the most proper is using

New intensively developed methods are methods of local oreintation measurements by

Due to the progress in equipment and fast data collecting computers methods of orientation measurements in scanning and transmission electron microscopy have been developed

The phenomenon of diffraction on the crystal can be described by the Braggs' equation.

in a coordinate system associated with a single crystal [Bunge, 1982].

important, from a technological point of view, material properties.

used method and ranging from 50 μm to 0.1 mm [Schwarzer, 1998].

neutron diffraction. But the access to the neutron diffractometer is limited.

diffraction: X-ray diffraction, neutrons and electrons.

Risø National Laboratory (Denmark) [Poulsen, 2004].

of this method belong:

means of electron microscopes.

Scanning electron microscope has become in the recent years versatile tool to observe microstructure. It can be applied for visualization of the topography of the surface of the sample for analyzing chemical composition or for crystallites orientation determination in specific area of the sample.

Exist several types of difraction patterns in SEM eg.:

Currently, most developed and most commonly used diffraction patterns for the measurement of local crystallographic orientation in the SEM is EBSD technique. Diffraction pattern is formed under Braggs' law. Kikuchi lines or pairs of lines originating from different crystallographic planes on the pattern.

Automating the processes of acquisition and indexing diffraction images has allowed the creation maps by systems to measure the orientation in the SEM. The development of such systems was associated with the development of modern research equipment including cameras CCD (Charge Coupled Device) for recording microscopic images, and numerical methods allow for automatic indexing diffraction images based on supplied data. It initiated the development of a new field of electron microscopy, called the orientation microscopy [Adams, 1993]. OM or OIMTM acronym was introduced in the 90s of last century and concerned the distribution of crystallographic orientation in the material in terms of the coordinates of the sample by means of SEM. The best known commercially available systems for measuring the crystallographic orientation is OIMTM TSL - EDAX using the method of indexing diffraction images created by Wright and Adams [Wright, 1992] and HKL Channel 5 - Oxford Instruments based on indexing algorithm developed by Schmidt et al. [Schmidt, 1989]. Own system, named ACOM (Automated Crystal Orientation Mapping) was developed by Schwarzer [Schwarzer, 1997]. The principle of operation of all systems is based on the automatic recording of diffraction images obtained from the particular sample area, indexing and mapping of orientation. The orientation map is a graphical representation of the distribution of orientation on the sample surface. Mapping the orientation is the acquisition of diffraction images in a specific grid of points with a given step [Randle, 2000]. Received orientations are represented on the map by colors matched according to previously defined key colors. In the EBSD system grid step is done by controlling the electron beam position or the stage position. The introduction of a new FIB technique (Focused Ion Beam) in scanning electron microscopes equipped with gallium gun for cutting thin film of a desired size and thickness allowed to extend to three dimensions of orientation maps. With combining FIB and EBSD measurements, it is possible to cut successive samples (with an area of approximately 50x50 microns and a thickness of about 150 nm) of bulk material, and then make orientation measurements [Zeafferer, 2005;

Orientation Microscopy in the Transmission Electron Microscope -

of images is performed manually by microscope operator.

by NanoMEGAS company [Rauch, 2008].

calculated database of patterns.

Fig. 1. Spot pattern in TEM.

Investigations of Small Orientations Changes by Means of Orientation Mapping in TEM 55

Exemplary applications of spot patterns for investigation of the materials after severe plastic deformation are presented in [Zeafferrer, 2000]. In this work both geometry and intensity of spot diffraction patterns are used for determination crystallographic orientation. Acquisition

Another system based on spot pattern was presented by Rauch [Rauch, 2006]. Further development of this system resulted in commercially available system called Astar offered

In this case, the main goal of indexing is to match acquired spot pattern to the previously

 (a) (b) Fig. 2. Kikuchi type diffraction patterns: CBED a) and TKP b).

Konrad, 2006]. In this way, based on measured sequence of two-dimensional maps one can obtain three-dimensional image of the microstructure. Modern commercial systems used in SEM, are also equipped with the possibility of a combined analysis of EDS (Energy Dispersive X-ray Spectrometry) and EBSD allowing obtaining information about chemical composition, crystallographic phase and orientation in a certain area of the sample. An important parameter for mapping is the spatial resolution of the method. The spatial resolution can be defined as possibility of distinguishing between two points in which orientation is different. In scanning electron microscopy measurement spatial resolution depends on investigated material and the type of electron source and is limited by the volume at which the beam interacts with the sample. In case of field emission gun (FEG) spatial resolution is the best and is about 30 nm. However, in the case of standard measurements to obtain the resolution of less than 100 nm is very difficult. While the angular resolution of EBSD images is above 0.5 ° [Schwarzer, 1998]. The advantages of EBSD systems are speed, full automation possible and very good quality maps for single-phase materials. Difficulties occur in the analysis of multiphase materials. Due to the slope of sample (>60 °) during the EBSD measurements, the important role plays the good preparation of the sample surface [Wenk, 2004]. Multiphase material, with different susceptibility to polishing and chemical reagents for the various phases may cause difficulties in obtaining equal sample surface, which affects the quality of the diffraction images, and thus the ability to automatically detection lines in the image. In case of materials after severe plastic deformation with nano and submicrocrystalline grains obtain the good quality of orientation map in SEM is challenging. These methods are also not effective for the analysis of local, small changes in orientation (<1 °) on the border of the angular-resolution diffraction EBSD.

#### **2.2 Orientation microscopy in TEM**

There is a strong need for development the method of orientation investigation of nanomaterials with angular and spatial resolution better that can be offered by EBSD/SEM.

This can be attained by means of transmission electron microscopy (TEM) Where the spatial resolution of the measurement is more limited by thickness of thin foils then the parameters of beam (diameter of electron beam can be lower then 2 nm) [Wu ,2009 Zaefferer, 2011]. This allows for analysis materials with ultra crystalline grains.

Automation of the systems in transmission electron microscope (TEM) is difficult. There exist several systems but most of them are not commercial available.

In TEM there are several different types of diffraction patterns [Carter & Williams, 1996]. The most common and wide used for phase discrimination are spot patterns (fig 1.).

Taking into account only geometry of spot patterns, orientation can be determined with accuracy ~5°. Higher accuracy (up to ~0.5°) can be assured by taking into consideration intensity on the diffraction patterns.

Investigation used local orientation measurements in TEM were performed in 1983 by Sztwiertnia and Haessner [Haessner, 1983]. Based on manual collected spot patterns were calculated local partial orientation and misorientation distribution function in cold rolled copper.

Fig. 1. Spot pattern in TEM.

54 The Transmission Electron Microscope

Konrad, 2006]. In this way, based on measured sequence of two-dimensional maps one can obtain three-dimensional image of the microstructure. Modern commercial systems used in SEM, are also equipped with the possibility of a combined analysis of EDS (Energy Dispersive X-ray Spectrometry) and EBSD allowing obtaining information about chemical composition, crystallographic phase and orientation in a certain area of the sample. An important parameter for mapping is the spatial resolution of the method. The spatial resolution can be defined as possibility of distinguishing between two points in which orientation is different. In scanning electron microscopy measurement spatial resolution depends on investigated material and the type of electron source and is limited by the volume at which the beam interacts with the sample. In case of field emission gun (FEG) spatial resolution is the best and is about 30 nm. However, in the case of standard measurements to obtain the resolution of less than 100 nm is very difficult. While the angular resolution of EBSD images is above 0.5 ° [Schwarzer, 1998]. The advantages of EBSD systems are speed, full automation possible and very good quality maps for single-phase materials. Difficulties occur in the analysis of multiphase materials. Due to the slope of sample (>60 °) during the EBSD measurements, the important role plays the good preparation of the sample surface [Wenk, 2004]. Multiphase material, with different susceptibility to polishing and chemical reagents for the various phases may cause difficulties in obtaining equal sample surface, which affects the quality of the diffraction images, and thus the ability to automatically detection lines in the image. In case of materials after severe plastic deformation with nano and submicrocrystalline grains obtain the good quality of orientation map in SEM is challenging. These methods are also not effective for the analysis of local, small changes in orientation (<1 °) on the border of the

There is a strong need for development the method of orientation investigation of nanomaterials with angular and spatial resolution better that can be offered by EBSD/SEM. This can be attained by means of transmission electron microscopy (TEM) Where the spatial resolution of the measurement is more limited by thickness of thin foils then the parameters of beam (diameter of electron beam can be lower then 2 nm) [Wu ,2009 Zaefferer, 2011]. This

Automation of the systems in transmission electron microscope (TEM) is difficult. There

In TEM there are several different types of diffraction patterns [Carter & Williams, 1996].

Taking into account only geometry of spot patterns, orientation can be determined with accuracy ~5°. Higher accuracy (up to ~0.5°) can be assured by taking into consideration

Investigation used local orientation measurements in TEM were performed in 1983 by Sztwiertnia and Haessner [Haessner, 1983]. Based on manual collected spot patterns were calculated local partial orientation and misorientation distribution function in cold rolled

The most common and wide used for phase discrimination are spot patterns (fig 1.).

angular-resolution diffraction EBSD.

**2.2 Orientation microscopy in TEM**

intensity on the diffraction patterns.

copper.

allows for analysis materials with ultra crystalline grains.

exist several systems but most of them are not commercial available.

Exemplary applications of spot patterns for investigation of the materials after severe plastic deformation are presented in [Zeafferrer, 2000]. In this work both geometry and intensity of spot diffraction patterns are used for determination crystallographic orientation. Acquisition of images is performed manually by microscope operator.

Another system based on spot pattern was presented by Rauch [Rauch, 2006]. Further development of this system resulted in commercially available system called Astar offered by NanoMEGAS company [Rauch, 2008].

In this case, the main goal of indexing is to match acquired spot pattern to the previously calculated database of patterns.

Fig. 2. Kikuchi type diffraction patterns: CBED a) and TKP b).

(a) (b)

Orientation Microscopy in the Transmission Electron Microscope -

The system (Fig. 3 and 4) includes the following elements:

connected to a PC via the serial port (RS232-C).

cable card and DMA enabling fast data transfer.

capabilities of Digital Micrograph.

Fig. 3. Elements of orientation mapping system.

University of Metz (France).

to calculate the ODF, etc.

crystallographic orientation set.

Investigations of Small Orientations Changes by Means of Orientation Mapping in TEM 57

hard disk in the form of graphic files using a digital CCD camera from a few to tens thousand of diffraction images and their subsequent indexing and analysis of the

• Philips CM20 microscope - transmission electron microscope with a maximum accelerating voltage 200 kV and spatial resolution (the possibility of distinguishing between two points on the microscopic image) value of ~ 0.2 nm. The microscope is

• CCD camera Gatan Dual Vision Company - 12 bit (4095 gray scale) digital CCD camera with a resolution of 1300 x 1030 pixels and exposure time from 0.01 to 10 s, mounted on the side of the microscope column. The camera is connected to the computer using a

• Software Gatan Digital Micrograph - the software used for communication between a computer and a camera and a microscope. Allows to save images on a computer, has a built-in tools for image analysis, and allows to create own programs in developing

• Software EP for indexing Kikuchi diffraction patterns developed by A. Morawiec [Morawiec, 1999], as the result of cooperation between the IMIM PAS and the

• Software for analysis of data containing information about the distribution of orientation in a sample of material such as Tango (part of Channel 5 system for the analysis data from EBSD in SEM), programs to analyze the components of the texture,

Better angular resolution has Kikuchi types diffraction patterns (Fig. 2). They allow to specify orientation with accuracy 0.1°.

To creating orientation map can be applied Kikuchi types patterns obtained in transmission beam (TKP – Transmission Kikuchi Diffraction Patterns) and convergent beam (CBED – Convergent Beam Electron Diffraction Patterns).

Method for automatic measurement of local crystallographic orientations in the TEM in defined areas of thin film using diffraction CBED images developed as one of the first Haessner and Sztwiertnia [Sztwiertnia, 1991; Pospiech, 1991; Haessner, 1992]. This method has been used to study recrystallization textures in the alloys with cubic symmetry. The first semi-automatic system for mapping the orientation of the TEM was created by Schwarzer and Zeafferer [Zeafferer, 1994] and then developed by Zeafferer [Zeafferer, 2000]. System operation is based on direct analysis of the collected diffraction images: spot patterns and Kikuchi type patterns. Besides the automatic indexing of images of diffraction system also contains procedures for computer processing of images and to determine the crystallographic dependencies such as: disorientation between grains, Burgers vector, slip systems and twinning systems.

Since systems for measuring the orientation maps in transmission microscopy are much harder to automate, most of these systems are created without the automation of image acquisition diffraction [Zeafferer, 1994; Schwarzer, 1997a]. System relies on recording diffraction in automatic way was built at the University of Metz [Funderberger, 2003; Fundenberger, 2003a]. These systems are not commercially available and must be individually adapted to a specific laboratory.

In addition to the different diffraction image analysis technique in TEM orientation map are created using Dark Field Image technique [Dingley, 2006]. This technique was introduced as a commercial system in 2000 by TSL-EDAX [Dingley, 2000]. For the measurements are used large series of dark field images recorded at different diffraction conditions. Intensity analysis is made on each dark field image. This allows the reconstruction of diffraction images and calculation the orientation at that point. The resolution of such measurements is dependent on the magnification used and the resolution of the microscope in dark field images [Zeafferer, 2002]. The advantage of this system is a fast measurement time of about 15 - 20 minutes. However, there are problems associated with drift of the sample and the need to use a large range of intensity (required for high-quality CCD camera).

All these methods and the system built in the IMIM PAS, whose description will be presented in the following subchapter, are used in various aspects of research in materials science. Together with X-ray difractometry and EBSD measurements techniques are complementary methods that allow for a detailed description of the microstructure of materials.

#### **3. Elements of the system**

In IMIM PAS the system for orientation mapping on TEM was built [Bieda, 2008]. The main idea of this system is automatic recording of diffraction images and save them on computer hard disk in the form of graphic files using a digital CCD camera from a few to tens thousand of diffraction images and their subsequent indexing and analysis of the crystallographic orientation set.

The system (Fig. 3 and 4) includes the following elements:

56 The Transmission Electron Microscope

Better angular resolution has Kikuchi types diffraction patterns (Fig. 2). They allow to

To creating orientation map can be applied Kikuchi types patterns obtained in transmission beam (TKP – Transmission Kikuchi Diffraction Patterns) and convergent beam (CBED –

Method for automatic measurement of local crystallographic orientations in the TEM in defined areas of thin film using diffraction CBED images developed as one of the first Haessner and Sztwiertnia [Sztwiertnia, 1991; Pospiech, 1991; Haessner, 1992]. This method has been used to study recrystallization textures in the alloys with cubic symmetry. The first semi-automatic system for mapping the orientation of the TEM was created by Schwarzer and Zeafferer [Zeafferer, 1994] and then developed by Zeafferer [Zeafferer, 2000]. System operation is based on direct analysis of the collected diffraction images: spot patterns and Kikuchi type patterns. Besides the automatic indexing of images of diffraction system also contains procedures for computer processing of images and to determine the crystallographic dependencies such as: disorientation between grains, Burgers vector, slip

Since systems for measuring the orientation maps in transmission microscopy are much harder to automate, most of these systems are created without the automation of image acquisition diffraction [Zeafferer, 1994; Schwarzer, 1997a]. System relies on recording diffraction in automatic way was built at the University of Metz [Funderberger, 2003; Fundenberger, 2003a]. These systems are not commercially available and must be

In addition to the different diffraction image analysis technique in TEM orientation map are created using Dark Field Image technique [Dingley, 2006]. This technique was introduced as a commercial system in 2000 by TSL-EDAX [Dingley, 2000]. For the measurements are used large series of dark field images recorded at different diffraction conditions. Intensity analysis is made on each dark field image. This allows the reconstruction of diffraction images and calculation the orientation at that point. The resolution of such measurements is dependent on the magnification used and the resolution of the microscope in dark field images [Zeafferer, 2002]. The advantage of this system is a fast measurement time of about 15 - 20 minutes. However, there are problems associated with drift of the sample and the need to use a large range of intensity (required

All these methods and the system built in the IMIM PAS, whose description will be presented in the following subchapter, are used in various aspects of research in materials science. Together with X-ray difractometry and EBSD measurements techniques are complementary methods that allow for a detailed description of the microstructure of

In IMIM PAS the system for orientation mapping on TEM was built [Bieda, 2008]. The main idea of this system is automatic recording of diffraction images and save them on computer

specify orientation with accuracy 0.1°.

systems and twinning systems.

for high-quality CCD camera).

**3. Elements of the system**

materials.

individually adapted to a specific laboratory.

Convergent Beam Electron Diffraction Patterns).

Fig. 3. Elements of orientation mapping system.

Orientation Microscopy in the Transmission Electron Microscope -

distinguished Brass and Goss components.

components.

**5. Results and discussion**

Investigations of Small Orientations Changes by Means of Orientation Mapping in TEM 59

represented characteristic layered microstructure for the deformed material consisting of grains and subgrain bands lying almost parallel to the rolling plane, Figure 5. In previously published works such as [Pospiech, 1998; Sztwiertnia, 2006a] was presented the study of the microstructure of aluminum rolled on the basis of that single crystallographic orientation manually recorded along the direction normal to the plane of rolling (ND). The results obtained (about 1000 crystallographic orientation) were analyzed due to the contribution of individual components of texture in the plane perpendicular to the direction of the transverse plate (TD). An example of the orientation distribution function calculated from the measured orientation is presented in Figure 6. Pure aluminum after cold rolling to 90% deformation texture is characterized by copper with fiber type α and β. In the main β fiber are S and Copper components, and in the fiber α can be

Fig. 6. Exemplary local orientation distribution function (ODF) for cold rolled up to 90%

Local crystal orientation measurements using the system to create orientation maps, allowed the registration of several thousand orientation at one measurement with a fixed step controlling electron beam displacement. This allowed obtaining the orientation maps in areas with a width from a few to several microns. With the ability to measure diffraction at the measuring points distant from each other by several nanometers, could be observed

pure aluminium counted on the base of 1106 of orientation with marked texture

subtle changes in the crystallographic orientation in very small distances.

Fig. 4. Schema of orientation mapping system.

#### **4. Application of the system**

One of the possible applications of the OM measurements in the TEM are presented. Deformed pure (4N) aluminum was chosen as an exemplary material. The new quality and quantity of supplied data made possible better microstructural characterization and observation of very small orientation changes produced characteristic subgrains structure (fig. 5.).

Fig. 5. TEM bright field image of cold-rollling up to 90% pure alluminium

Measurements of the local crystallographic orientation were carried out in aluminum with a purity of 99.99%. Thin foils were prepared from cross-rolled up to 90% sheet of metal. That

Microscope

Indexing

scripts

program

Controlling beam position

Diffraction image acquisition

Image filtering

Hough transform – Line detection

Image indexing

Data analysis

One of the possible applications of the OM measurements in the TEM are presented. Deformed pure (4N) aluminum was chosen as an exemplary material. The new quality and quantity of supplied data made possible better microstructural characterization and observation of very small orientation changes produced characteristic subgrains structure

Fig. 5. TEM bright field image of cold-rollling up to 90% pure alluminium

Measurements of the local crystallographic orientation were carried out in aluminum with a purity of 99.99%. Thin foils were prepared from cross-rolled up to 90% sheet of metal. That

Fig. 4. Schema of orientation mapping system.

**4. Application of the system**

(fig. 5.).

represented characteristic layered microstructure for the deformed material consisting of grains and subgrain bands lying almost parallel to the rolling plane, Figure 5. In previously published works such as [Pospiech, 1998; Sztwiertnia, 2006a] was presented the study of the microstructure of aluminum rolled on the basis of that single crystallographic orientation manually recorded along the direction normal to the plane of rolling (ND). The results obtained (about 1000 crystallographic orientation) were analyzed due to the contribution of individual components of texture in the plane perpendicular to the direction of the transverse plate (TD). An example of the orientation distribution function calculated from the measured orientation is presented in Figure 6. Pure aluminum after cold rolling to 90% deformation texture is characterized by copper with fiber type α and β. In the main β fiber are S and Copper components, and in the fiber α can be distinguished Brass and Goss components.

Fig. 6. Exemplary local orientation distribution function (ODF) for cold rolled up to 90% pure aluminium counted on the base of 1106 of orientation with marked texture components.

## **5. Results and discussion**

Local crystal orientation measurements using the system to create orientation maps, allowed the registration of several thousand orientation at one measurement with a fixed step controlling electron beam displacement. This allowed obtaining the orientation maps in areas with a width from a few to several microns. With the ability to measure diffraction at the measuring points distant from each other by several nanometers, could be observed subtle changes in the crystallographic orientation in very small distances.

Orientation Microscopy in the Transmission Electron Microscope -

Investigations of Small Orientations Changes by Means of Orientation Mapping in TEM 61

Fig. 8. Bright field image (TEM) of aluminum cold rolled up to 90% and a) orientation map with colors from inverse pole figure, the black lines are misorientations angles > 1°.

Unresolved diffraction patterns are presented as white points and b) misorientation profile

across three subgrains.

Orientation maps were measured for the longitudinal section (perpendicular to transverse direction TD) in such a way as to include as much information about the boundaries lying parallel to the plane of the aluminum sheet, Figure 7, 8 and 9. The analyzed areas of the samples were above 10 microns of thickness (along normal direction ND). In such areas piles of elongated subgrains in rolling direction RD were observed, which boundaries in the ND were characterized by disorientation angles generally less than 5 °.

Fig. 7. Bright field image (TEM) of aluminum cold rolled up to 90% and orientation map with colors from inverse pole figure, the black lines are misorientations angles > 5° and corresponding texture components. Unresolved diffraction patterns are presented as white points.

Cumulative disorientation angles, less than 5 °, occurred between the orientations of subgrains, corresponding to one component of the texture. In Fig.7 on orientation map is marked boundary misorientation angle of > 12 ° occurs between the areas representing the type of texture components, respectively Copper and Brass. In the area of Copper component, we can distinguish the boundary disorientation angle of approximately 5 °. Since the border between the two components of the texture observed a gradual shift in orientation towards the ideal position of component-type Copper. Larger changes in the angle of disorientation identify the extent of occurrence of individual components of the texture (Fig. 7 and 10), while only minor changes were observed within the same component.

Orientation maps were measured for the longitudinal section (perpendicular to transverse direction TD) in such a way as to include as much information about the boundaries lying parallel to the plane of the aluminum sheet, Figure 7, 8 and 9. The analyzed areas of the samples were above 10 microns of thickness (along normal direction ND). In such areas piles of elongated subgrains in rolling direction RD were observed, which boundaries in the

Fig. 7. Bright field image (TEM) of aluminum cold rolled up to 90% and orientation map with colors from inverse pole figure, the black lines are misorientations angles > 5° and corresponding texture components. Unresolved diffraction patterns are presented as white

Cumulative disorientation angles, less than 5 °, occurred between the orientations of subgrains, corresponding to one component of the texture. In Fig.7 on orientation map is marked boundary misorientation angle of > 12 ° occurs between the areas representing the type of texture components, respectively Copper and Brass. In the area of Copper component, we can distinguish the boundary disorientation angle of approximately 5 °. Since the border between the two components of the texture observed a gradual shift in orientation towards the ideal position of component-type Copper. Larger changes in the angle of disorientation identify the extent of occurrence of individual components of the texture (Fig. 7 and 10), while only minor changes were observed within the same

points.

component.

ND were characterized by disorientation angles generally less than 5 °.

Fig. 8. Bright field image (TEM) of aluminum cold rolled up to 90% and a) orientation map with colors from inverse pole figure, the black lines are misorientations angles > 1°. Unresolved diffraction patterns are presented as white points and b) misorientation profile across three subgrains.

Orientation Microscopy in the Transmission Electron Microscope -

direction of another stable orientation Brass (Fig. 10).

Investigations of Small Orientations Changes by Means of Orientation Mapping in TEM 63

Bands shown in the orientation maps (Fig. 8 and 9) are composed of bundles of parallel, highly elongated in the RD subgrains, between which there are relatively small (between 1 ° and 10 °), cumulative disorientation angles. In addition, in Figure 9c, is showed that the color gradation is related to a gradual change from the initial orientation. On this map red are marked boundaries, where the orientation changes were> 5 °. Typical course of changes in the angle of disorientation between adjacent subgrains along a line drawn parallel to the length of the orientation map is shown in Fig. 10a. Again, it can be seen that the differences in orientation are often very small on the boundaries of two subgrains, and may be cumulative, with the angle of disorientation between the first and last subgrains of the band may exceed 20 °. For example, total disorientation angle across the transition band width of about 12 microns, Fig. 10c, is ~ 20 °. Identifying the orientation of the corresponding components of the texture, it appears a transition from a stable orientation of S in the

Fig. 10. Misorientation profiles across the line marked on the orientation map b) between particular grains a) and relative to the first orientation c) with marked texture components,

the numbers in bracket means deviation from ideal orientation.

Fig. 9. Bright field image (TEM) of aluminum cold rolled up to 90% b) and orientation map with colors from inverse pole figure a), the black lines are misorientations angles > 1°. Unresolved diffraction patterns are presented as white points and c) color map where gradient of color means deviation from the first orientation up to 20°, the red lines are misorientations angles > 5°.

Fig. 9. Bright field image (TEM) of aluminum cold rolled up to 90% b) and orientation map with colors from inverse pole figure a), the black lines are misorientations angles > 1°. Unresolved diffraction patterns are presented as white points and c) color map where gradient of color means deviation from the first orientation up to 20°, the red lines are

misorientations angles > 5°.

Bands shown in the orientation maps (Fig. 8 and 9) are composed of bundles of parallel, highly elongated in the RD subgrains, between which there are relatively small (between 1 ° and 10 °), cumulative disorientation angles. In addition, in Figure 9c, is showed that the color gradation is related to a gradual change from the initial orientation. On this map red are marked boundaries, where the orientation changes were> 5 °. Typical course of changes in the angle of disorientation between adjacent subgrains along a line drawn parallel to the length of the orientation map is shown in Fig. 10a. Again, it can be seen that the differences in orientation are often very small on the boundaries of two subgrains, and may be cumulative, with the angle of disorientation between the first and last subgrains of the band may exceed 20 °. For example, total disorientation angle across the transition band width of about 12 microns, Fig. 10c, is ~ 20 °. Identifying the orientation of the corresponding components of the texture, it appears a transition from a stable orientation of S in the direction of another stable orientation Brass (Fig. 10).

Fig. 10. Misorientation profiles across the line marked on the orientation map b) between particular grains a) and relative to the first orientation c) with marked texture components, the numbers in bracket means deviation from ideal orientation.

Orientation Microscopy in the Transmission Electron Microscope -

the same type of orientation.

Investigations of Small Orientations Changes by Means of Orientation Mapping in TEM 65

On the basis of 56 000 measured orientation orientation distribution function (ODF) was calculated (Fig.11) and texture components as S, Brass and Copper were identified. Their respective percentage share was: 35.3%, 29.7% and 24%. Transitional areas between the bands in which there are no large misorientation are called deformation bands [Dillamore, 1972]. An example of such spectrum is shown in Fig. 12. Here we do not observe a smooth change of orientation angle combined with the accumulation of misorientaton angles, but only the fluctuations around a certain position. Thickness of the deformation bands having the same orientation was a few micrometers, and the angles of misorientation between adjacent bands were not large. Distribution of orientation along the ND was not accidental. Cells or subgrains were often concentrated in clusters containing up to 15 crystallites having

Fig. 12. Bright field image (TEM) of aluminum cold rolled up to 90% and orientation map with colors from inverse pole figure, the black lines are misorientations angles > 1°.

Detailed maps obtained at higher magnification allowed to show the microstructure of subgrains in bands around 100 nm in widith (Fig. 13) with an angle of disorientation ~ 1 °.

Unresolved diffraction patterns are presented as white points.

Accumulation of disorientation angles between subgrains across the spectrum is characteristic for the transition band [Hjelen, 1991], which distinguishes it from other elements of the microstructure of similar morphology. Transition band separates the fragments of the original grains, in which, during deformation, act various combinations of slip systems as a result of what they turned toward different, stable end orientations [Hu, 1963a]. With increasing strain - as the fragments of deformed grains are close to the stable end orientation - total disorientation angle increases, e.g. [Hjelen, 1991]. It should be emphasized that the transition band is often observed in deformed single crystals. In polycrystalline materials, their identification has been difficult because there were no methods to study small changes in orientation at short distances. In earlier work, in which orientation measurement was performed [Pospiech, 1998; Sztwiertnia, 2006a], the transition bands in deformed polycrystalline microstructure could not be noticed, because the diffraction at certain points of measurement were recorded only when followed their apparent change in that way it was easy to "lose" their subtle changes within the band.

Fig. 11. Local orientation distribution function (ODF) for cold rolled up to 90% pure aluminium counted on the base of 56 000 of orientation measured in TEM with marked texture components.

Accumulation of disorientation angles between subgrains across the spectrum is characteristic for the transition band [Hjelen, 1991], which distinguishes it from other elements of the microstructure of similar morphology. Transition band separates the fragments of the original grains, in which, during deformation, act various combinations of slip systems as a result of what they turned toward different, stable end orientations [Hu, 1963a]. With increasing strain - as the fragments of deformed grains are close to the stable end orientation - total disorientation angle increases, e.g. [Hjelen, 1991]. It should be emphasized that the transition band is often observed in deformed single crystals. In polycrystalline materials, their identification has been difficult because there were no methods to study small changes in orientation at short distances. In earlier work, in which orientation measurement was performed [Pospiech, 1998; Sztwiertnia, 2006a], the transition bands in deformed polycrystalline microstructure could not be noticed, because the diffraction at certain points of measurement were recorded only when followed their apparent change in that way it was easy to "lose" their subtle changes within the band.

Fig. 11. Local orientation distribution function (ODF) for cold rolled up to 90% pure aluminium counted on the base of 56 000 of orientation measured in TEM with marked

texture components.

On the basis of 56 000 measured orientation orientation distribution function (ODF) was calculated (Fig.11) and texture components as S, Brass and Copper were identified. Their respective percentage share was: 35.3%, 29.7% and 24%. Transitional areas between the bands in which there are no large misorientation are called deformation bands [Dillamore, 1972]. An example of such spectrum is shown in Fig. 12. Here we do not observe a smooth change of orientation angle combined with the accumulation of misorientaton angles, but only the fluctuations around a certain position. Thickness of the deformation bands having the same orientation was a few micrometers, and the angles of misorientation between adjacent bands were not large. Distribution of orientation along the ND was not accidental. Cells or subgrains were often concentrated in clusters containing up to 15 crystallites having the same type of orientation.

Fig. 12. Bright field image (TEM) of aluminum cold rolled up to 90% and orientation map with colors from inverse pole figure, the black lines are misorientations angles > 1°. Unresolved diffraction patterns are presented as white points.

Detailed maps obtained at higher magnification allowed to show the microstructure of subgrains in bands around 100 nm in widith (Fig. 13) with an angle of disorientation ~ 1 °.

Orientation Microscopy in the Transmission Electron Microscope -

D. Dingley Proceedings of the Electron Crystallography School (2005)

F. Haessner, K. Sztwiertnia, Scripta metall. mater.27 s. 1545 (1992) J. Hjelen, R. Ørsund, E. Nes Acta metall. Mater. 39 s. 1337 (1991)

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R.A. Schwarzer Ultramicroscopy 67 p.19 (1997)

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p.13 (2010)

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H. Hu J. Wiley and Sons New York s. 311 (1963)

A. Morawiec J. Appl. Cryst. 32 p. 788 (1999)

A. Morawiec J. Appl. Cryst. 32 p. 788 (1999)

A. Morawiec Springer-Verlag (2004)

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V. Randle, O. Engler CRC Press (2000)

R.A. Schwarzer Micron 28 s.249 (1997)

Investigations of Small Orientations Changes by Means of Orientation Mapping in TEM 67

M. Bieda, K. Sztwiertnia, A. Korneva, T. Czeppe, R. Orlicki Solid State Phenomena Vol. 163

D.J. Dingley, S.I. Wright. 7th Asia-Pacific Electron Microscopy Physical Sciences, Singapore,

J.J. Fundenberger, A. Morawiec, E. Bouzy, J.S. Lecomte Mater. Chem. Phys. 81, p.535 (2003) J.J. Fundenberger, A.Morawiec, E. Bouzy, J.S. Lecomte Ultramicroscopy 96 p. 127 (2003)

N. C. Krieger Lassen Proc. of 16th Riso Int. Symp. on "Microstructural and Crystallographic

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Aspects of Recrystallization" Riso National Laboratory, Roskilde, Denmark s. 405

F. Haessner, J. Pospiech, K. Sztwiertnia Materials Science and Engineering 57 s.1 (1983)

Fig. 13. Bright field image (TEM) of aluminum cold rolled up to 90% and orientation map with colors from inverse pole figure, the black lines are misorientations angles > 1°. Unresolved diffraction patterns are presented as white points.

## **6. Conclusions**

Principles of diffraction patterns used for orientation measurements in the TEM and short survey of existed systems for orientation mapping in the TEM was introduced. Pros and cons of TEM systems and comparison with SEM systems was presented in this chapter including spatial and angular resolution.

Difficulties in automation and variety of diffraction patterns used in the TEM made the existing systems, with some exceptional, hard to commercialize. Generally, methods in the TEM and the SEM can be used as complementary methods for investigating microstructure in nano and micro scale.

In spite of better spatial resolution in the TEM than in the SEM, the more important is better angular resolution in the TEM (about 0.1°), which can be assured of using Kikuchi type diffraction patterns and make possible investigation of small orientation changes in material in micro and nano-scale.

### **7. References**

B. L. Adams, D. J. Dingley Materials Science Forum 157-62 p. 31 (1994) M. Bieda PhD. thesis IMIM PAS (2008)

Fig. 13. Bright field image (TEM) of aluminum cold rolled up to 90% and orientation map with colors from inverse pole figure, the black lines are misorientations angles > 1°.

Principles of diffraction patterns used for orientation measurements in the TEM and short survey of existed systems for orientation mapping in the TEM was introduced. Pros and cons of TEM systems and comparison with SEM systems was presented in this chapter

Difficulties in automation and variety of diffraction patterns used in the TEM made the existing systems, with some exceptional, hard to commercialize. Generally, methods in the TEM and the SEM can be used as complementary methods for investigating microstructure

In spite of better spatial resolution in the TEM than in the SEM, the more important is better angular resolution in the TEM (about 0.1°), which can be assured of using Kikuchi type diffraction patterns and make possible investigation of small orientation changes in material

B. L. Adams, D. J. Dingley Materials Science Forum 157-62 p. 31 (1994)

Unresolved diffraction patterns are presented as white points.

**6. Conclusions**

in nano and micro scale.

in micro and nano-scale.

**7. References**

including spatial and angular resolution.

M. Bieda PhD. thesis IMIM PAS (2008)

**4**

Jian Li

*Canada*

**Advanced Techniques in**

**TEM Specimen Preparation**

*CANMET-Materials Technology Laboratory, Hamilton, Ontario,*

Since it was invented about 30 years ago, focused ion beam microscopes (FIB) has mostly been used in semiconductor industry for mask repair, lithography, doping and circuit edit (Melngailis, 1987). The number of FIB systems increased significantly in recent years, and their applications have extended into materials and biological sciences (Phaneuf et al.,, 1998,

The recent development of Dual-Beam*TM* or Cross-Beam*TM* FIB systems has gradually taken over the traditional single beam FIB systems. A typical FIB column contains a liquid metal ion source that produces a finely focused Ga ion beam. The primary Ga ion beam is accelerated by 30-50 kV, and directed towards the features of interest on the specimen. The incident ion beam will sputter atoms from the specimen surface, producing both secondary ions and secondary electrons. Depending on the application, the beam current can be set to high (e.g. over 60 nA) for rapid ion beam milling. Aside from electron beam imaging, operator can perform high-resolution ion beam imaging (up to 2.5 nm imaging resolution in some FIB systems). Site-specific micro-depositions (e.g., Pt, W, C, and SiO2) and microetching can also be achieved by the interaction of the primary ion beam (or electron beam) with the deposition (or etching) gas introduced into the system. Figure 1 shows a schematic

Similar to a typical SEM, FIB microscopes can be used to produce high-resolution images directly from almost any kind of samples including fine powder particles. The primary Ga ion beam can produce images with enhanced crystallographic orientation contrast using secondary electrons (SE) generated from the specimen surface. The ion beam induced secondary electron yield is strongly dependant on the crystallographic orientation of the specimen. This is attributed to the very small ion beam-specimen interaction volume. The secondary ion (SI) particles can also be collected by the detector to produce secondary ion images that can sometimes provide valuable information related to the local chemistry. In addition to high-resolution imaging on sample surfaces, ion beam cross sectioning is the most important feature of FIB microscopes. The stress-free FIB cross-sections produce valuable subsurface information. In dual-beam FIB systems equipped with EDS and EBSD, subsurface chemistry and crystallographic orientation of specific subsurface features can be

**1. Introduction**

obtained.

1999; Li et al., 2001, 2006a).

diagram of ion-specimen interaction in FIB microscopes.

## **Advanced Techniques in TEM Specimen Preparation**

Jian Li *CANMET-Materials Technology Laboratory, Hamilton, Ontario, Canada*

### **1. Introduction**

68 The Transmission Electron Microscope

S. Zaefferer, R. Schwarzer Z. Metallkd. 85 (1994)

S. Zaefferer Advanced in imaging and electron physics 125 s.355 (2002)

S. Zaefferer Advanced engineering materials 5(10) s.745 (2003)

S. Zaefferer J. Appl. Cryst. 33 s.10 (2000)

S. Zaefferer Cryst. Res. Technol., 1-22 (2011)

Since it was invented about 30 years ago, focused ion beam microscopes (FIB) has mostly been used in semiconductor industry for mask repair, lithography, doping and circuit edit (Melngailis, 1987). The number of FIB systems increased significantly in recent years, and their applications have extended into materials and biological sciences (Phaneuf et al.,, 1998, 1999; Li et al., 2001, 2006a).

The recent development of Dual-Beam*TM* or Cross-Beam*TM* FIB systems has gradually taken over the traditional single beam FIB systems. A typical FIB column contains a liquid metal ion source that produces a finely focused Ga ion beam. The primary Ga ion beam is accelerated by 30-50 kV, and directed towards the features of interest on the specimen. The incident ion beam will sputter atoms from the specimen surface, producing both secondary ions and secondary electrons. Depending on the application, the beam current can be set to high (e.g. over 60 nA) for rapid ion beam milling. Aside from electron beam imaging, operator can perform high-resolution ion beam imaging (up to 2.5 nm imaging resolution in some FIB systems). Site-specific micro-depositions (e.g., Pt, W, C, and SiO2) and microetching can also be achieved by the interaction of the primary ion beam (or electron beam) with the deposition (or etching) gas introduced into the system. Figure 1 shows a schematic diagram of ion-specimen interaction in FIB microscopes.

Similar to a typical SEM, FIB microscopes can be used to produce high-resolution images directly from almost any kind of samples including fine powder particles. The primary Ga ion beam can produce images with enhanced crystallographic orientation contrast using secondary electrons (SE) generated from the specimen surface. The ion beam induced secondary electron yield is strongly dependant on the crystallographic orientation of the specimen. This is attributed to the very small ion beam-specimen interaction volume. The secondary ion (SI) particles can also be collected by the detector to produce secondary ion images that can sometimes provide valuable information related to the local chemistry. In addition to high-resolution imaging on sample surfaces, ion beam cross sectioning is the most important feature of FIB microscopes. The stress-free FIB cross-sections produce valuable subsurface information. In dual-beam FIB systems equipped with EDS and EBSD, subsurface chemistry and crystallographic orientation of specific subsurface features can be obtained.

Advanced Techniques in TEM Specimen Preparation 71

Similar to conventional SEMs, FIB microscopes can produce high-resolution secondary electron and secondary ion images. Chemical etching, although necessary to reveal metallurgical features in many microscopy routines, are not preferred since it can introduce many forms of artifacts. A typical SEM study would require metallographic etching that result in a modified surface topography. The FIB secondary ion imaging could provide both chemical and crystallographic details from the as-polished surface. Careful metallographic polishing is required to obtain high-quality FIB images (Figure 2). Apart from potential surface/subsurface mechanical damage introduced during sample polishing, any surface oxidation or contamination will also have a significant effect on FIB imaging. Some FIB systems are equipped with plasma cleaners to remove surface contaminants and thin oxide films in-situ. In addition, the gallium ions can also be used to sputter off the surface oxide film prior to imaging. Figure 3 shows a stress corrosion crack (SCC) tip imaged using an optical microscope, a SEM and a FIB (ion beam induced secondary electron image) microscope. The optical and SEM images were taken from a chemically etched surface, while the FIB image was taken from an as-polished surface. The significance of these images

Fig. 2. FIB images of an IF steel (a) annealed and (b) deformed. Images taken from a

Fig. 3. Images taken from the same crack tip (a) optical, (b) SEM secondary electron image

**2.1 FIB imaging and cross sectioning**

was published elsewhere (Li et al., 2008a, 2008b).

metallographic polished surface

and (c) FIB secondary ion image

Fig. 1. Schematic diagram of a typical FIB system

Another major application of modern FIB microscopes is to prepare TEM specimens. FIB-TEM specimen preparation techniques have been reported (Giannuzzi & Steve, 1999; Anderson, 2002; Shankar et al., 2003). In this chapter, we will briefly review and compare the currently available techniques, and provide examples using our practical experiences.

#### **2. Focused-ion beam techniques and associated TEM specimen preparation routines**

Modern FIB systems are used to perform the following functions: high-resolution imaging using electron- and ion-beam, TEM specimen preparation (where STEM imaging can be performed in-situ), micro-machining, micro-deposition, and microanalysis using EDS and EBSD. Most of FIB microscopes are mainly used to prepare TEM specimens using semiautomatic routines provided by microscope vendors, while the unique imaging capability using the primary Ga ion beam has frequently been overlooked. In addition, the highresolution SEM column tends to take over the "imaging job" while the Ga ion beam in the FIB columns are often regarded as the dedicated "milling machine". Although the modern FEG SEM columns could, no doubt, achieve higher ultimate imaging resolution, the unique FIB images are still beneficial in many aspects (Li et al., 2004, 2006a). The heavy Ga ions, although accelerated by 30 kV, can only penetrate a few nanometers into the specimen (depending on the material's properties). This makes FIB imaging extremely surface sensitive. Strong crystallographic contrast can be obtainable directly from the metallographic polished surface.

#### **2.1 FIB imaging and cross sectioning**

70 The Transmission Electron Microscope

Another major application of modern FIB microscopes is to prepare TEM specimens. FIB-TEM specimen preparation techniques have been reported (Giannuzzi & Steve, 1999; Anderson, 2002; Shankar et al., 2003). In this chapter, we will briefly review and compare the currently available techniques, and provide examples using our practical experiences.

**2. Focused-ion beam techniques and associated TEM specimen preparation**

Modern FIB systems are used to perform the following functions: high-resolution imaging using electron- and ion-beam, TEM specimen preparation (where STEM imaging can be performed in-situ), micro-machining, micro-deposition, and microanalysis using EDS and EBSD. Most of FIB microscopes are mainly used to prepare TEM specimens using semiautomatic routines provided by microscope vendors, while the unique imaging capability using the primary Ga ion beam has frequently been overlooked. In addition, the highresolution SEM column tends to take over the "imaging job" while the Ga ion beam in the FIB columns are often regarded as the dedicated "milling machine". Although the modern FEG SEM columns could, no doubt, achieve higher ultimate imaging resolution, the unique FIB images are still beneficial in many aspects (Li et al., 2004, 2006a). The heavy Ga ions, although accelerated by 30 kV, can only penetrate a few nanometers into the specimen (depending on the material's properties). This makes FIB imaging extremely surface sensitive. Strong crystallographic contrast can be obtainable directly from the

Fig. 1. Schematic diagram of a typical FIB system

**routines**

metallographic polished surface.

Similar to conventional SEMs, FIB microscopes can produce high-resolution secondary electron and secondary ion images. Chemical etching, although necessary to reveal metallurgical features in many microscopy routines, are not preferred since it can introduce many forms of artifacts. A typical SEM study would require metallographic etching that result in a modified surface topography. The FIB secondary ion imaging could provide both chemical and crystallographic details from the as-polished surface. Careful metallographic polishing is required to obtain high-quality FIB images (Figure 2). Apart from potential surface/subsurface mechanical damage introduced during sample polishing, any surface oxidation or contamination will also have a significant effect on FIB imaging. Some FIB systems are equipped with plasma cleaners to remove surface contaminants and thin oxide films in-situ. In addition, the gallium ions can also be used to sputter off the surface oxide film prior to imaging. Figure 3 shows a stress corrosion crack (SCC) tip imaged using an optical microscope, a SEM and a FIB (ion beam induced secondary electron image) microscope. The optical and SEM images were taken from a chemically etched surface, while the FIB image was taken from an as-polished surface. The significance of these images was published elsewhere (Li et al., 2008a, 2008b).

Fig. 2. FIB images of an IF steel (a) annealed and (b) deformed. Images taken from a metallographic polished surface

Fig. 3. Images taken from the same crack tip (a) optical, (b) SEM secondary electron image and (c) FIB secondary ion image

Advanced Techniques in TEM Specimen Preparation 73

"lift-out" technique has shown significant advantages over the "original" H-bar technique that TEM specimens can be made directly from a bulk specimen (Giannuzzi and Steve, 1999). The original lift-out technique does not allow foil re-thinning once prepared. The liftout technique was later improved that much thicker specimens (typically 5 µm in thickness) are lifted out from the bulk and then transferred to TEM grids using a micromanipulator (insitu or ex-situ) followed by final FIB thinning. This technique can be used to produce almost all types of TEM specimens. However they are ineffective in some special cases. In this section, FIB TEM specimen preparation techniques are reviewed and compared using

The conventional H-bar technique was the only available FIB TEM preparation method in the mid-1990s (Longo et al., 1999). Figure 5 shows a schematic diagram of the H-bar

1. Small samples, containing the feature of interest, (about 2.5x1.0x0.5 mm in size) are cut

2. Both sides of the small specimen are then carefully polished using a "tripod polisher" (Anderson et al., 1997) in order to produce parallel surfaces and minimize the mechanical damage introduced during diamond saw cutting. The polished sample

3. The specimens are then mounted onto a half TEM grid using epoxy adhesive and

4. The mounted specimens are loaded into a FIB microscope for precise ion-beam

Fig. 5. Schematic diagram of conventional FIB-TEM specimen preparation technique

Some thought should be given as to the sample's thickness before FIB thinning. The thinner the sample, the less the FIB milling time will be needed. However, if the sample is too thin, it may have insufficient mechanical strength, and residual mechanical damage from the mechanical polishing could lead to problems and artifacts. This technique has found many applications, especially for non-site-specific TEM specimens. Once the sample is mounted onto a TEM grid, FIB sectioning and imaging can be used to identify the area of interest, as shown in Figure 6. An electron-transparent TEM sample can subsequently be made by thinning the backside of the specimen. Care must be taken during diamond saw cutting and various stages of mechanical polishing to ensure the integrity of the area of interest. With

out from bulk specimens using a precision diamond cut-off wheel.

practical examples in our recent research.

technique, which includes the following steps:

should have a thickness of less than 100 µm.

allowed sufficient time to cure.

thinning.

**2.2.1 Conventional H-bar technique**

Focused-ion beam microscopes are also used to produce high quality cross sections. Microscopic features appearing on the sample surfaces can be cross-sectioned using the primary Ga ion beam. High-resolution images of the cross sections can be obtained by tilting the sample in single beam FIB systems, or more conveniently using the electron beam in modern dual-beam systems. The ion beam milling process can be monitored in real-time using images generated by the electron beam. Prior to FIB sectioning, a strip of coating is deposited in the FIB to prevent the surface features from ion beam erosion during milling. Figure 4 shows the corrosion of a 316L stainless steel test coupons under supercritical water condition (Li et al., 2011a, 2011b). The typical sizes of FIB cross-sections are usually less than 100 µm. Very small cross-sections usually suffer from re-deposition, while large FIB sections not only need extensive FIB milling time but are also generally difficult to produce highquality cross-sections. The primary reason for this difficulty is the relatively coarse ion beams that are frequently used to minimize milling time usually result in cross-sections with a "curtain" effect. Smaller beam current can result in good cross-sections, but when milling in large scale which needs the extensive milling time, the system instability (beam- and stage-drift) could diminish the advantage. The largest cross-section accomplished by the author was 500 µm in width on a galvanealed steel sheet to assess the quality of the Zn coating. In this case, the major milling job was performed using high current (40 nA) and consecutive small sections of ~100 µm in width were polished using a lower beam current (1.5 nA) in a relatively old Micrion-2500 FIB system owned by FIBICS Inc. If larger crosssections are needed, a combination of mechanical polishing and FIB imaging could be used. Topics related to FIB sectioning and imaging have been published extensively in the past years (Su et al., 2002; Schmidt et al., 2011; Li et al., 2006c).

Fig. 4. SCW corrosion of stainless 316L. (a) layer of fine recrystallized grains prevented extensive corrosion, (b) severe corrosion due to lack of Cr2O3 passive film formation

#### **2.2 TEM sample preparation**

FIBs are only used to produce TEM specimens in many laboratories. FIB microscopes have become powerful tools in TEM specimen preparation (Phaneuf, 1999; Giannuzzi & Steve, 1999; Anderson, 2002; Shankar et al., 2003), and the techniques have evolved rapidly. The

Focused-ion beam microscopes are also used to produce high quality cross sections. Microscopic features appearing on the sample surfaces can be cross-sectioned using the primary Ga ion beam. High-resolution images of the cross sections can be obtained by tilting the sample in single beam FIB systems, or more conveniently using the electron beam in modern dual-beam systems. The ion beam milling process can be monitored in real-time using images generated by the electron beam. Prior to FIB sectioning, a strip of coating is deposited in the FIB to prevent the surface features from ion beam erosion during milling. Figure 4 shows the corrosion of a 316L stainless steel test coupons under supercritical water condition (Li et al., 2011a, 2011b). The typical sizes of FIB cross-sections are usually less than 100 µm. Very small cross-sections usually suffer from re-deposition, while large FIB sections not only need extensive FIB milling time but are also generally difficult to produce highquality cross-sections. The primary reason for this difficulty is the relatively coarse ion beams that are frequently used to minimize milling time usually result in cross-sections with a "curtain" effect. Smaller beam current can result in good cross-sections, but when milling in large scale which needs the extensive milling time, the system instability (beam- and stage-drift) could diminish the advantage. The largest cross-section accomplished by the author was 500 µm in width on a galvanealed steel sheet to assess the quality of the Zn coating. In this case, the major milling job was performed using high current (40 nA) and consecutive small sections of ~100 µm in width were polished using a lower beam current (1.5 nA) in a relatively old Micrion-2500 FIB system owned by FIBICS Inc. If larger crosssections are needed, a combination of mechanical polishing and FIB imaging could be used. Topics related to FIB sectioning and imaging have been published extensively in the past

years (Su et al., 2002; Schmidt et al., 2011; Li et al., 2006c).

**2.2 TEM sample preparation**

Fig. 4. SCW corrosion of stainless 316L. (a) layer of fine recrystallized grains prevented extensive corrosion, (b) severe corrosion due to lack of Cr2O3 passive film formation

FIBs are only used to produce TEM specimens in many laboratories. FIB microscopes have become powerful tools in TEM specimen preparation (Phaneuf, 1999; Giannuzzi & Steve, 1999; Anderson, 2002; Shankar et al., 2003), and the techniques have evolved rapidly. The "lift-out" technique has shown significant advantages over the "original" H-bar technique that TEM specimens can be made directly from a bulk specimen (Giannuzzi and Steve, 1999). The original lift-out technique does not allow foil re-thinning once prepared. The liftout technique was later improved that much thicker specimens (typically 5 µm in thickness) are lifted out from the bulk and then transferred to TEM grids using a micromanipulator (insitu or ex-situ) followed by final FIB thinning. This technique can be used to produce almost all types of TEM specimens. However they are ineffective in some special cases. In this section, FIB TEM specimen preparation techniques are reviewed and compared using practical examples in our recent research.

#### **2.2.1 Conventional H-bar technique**

The conventional H-bar technique was the only available FIB TEM preparation method in the mid-1990s (Longo et al., 1999). Figure 5 shows a schematic diagram of the H-bar technique, which includes the following steps:

Fig. 5. Schematic diagram of conventional FIB-TEM specimen preparation technique

Some thought should be given as to the sample's thickness before FIB thinning. The thinner the sample, the less the FIB milling time will be needed. However, if the sample is too thin, it may have insufficient mechanical strength, and residual mechanical damage from the mechanical polishing could lead to problems and artifacts. This technique has found many applications, especially for non-site-specific TEM specimens. Once the sample is mounted onto a TEM grid, FIB sectioning and imaging can be used to identify the area of interest, as shown in Figure 6. An electron-transparent TEM sample can subsequently be made by thinning the backside of the specimen. Care must be taken during diamond saw cutting and various stages of mechanical polishing to ensure the integrity of the area of interest. With

Advanced Techniques in TEM Specimen Preparation 75

Fig. 7. Schematic diagram of traditional lift-out TEM specimen preparation technique

challenging and rewarding.

the specimen onto the TEM grid.

The preparation of a lift-out TEM specimens affect only a relatively small local region typically on the order of 50 x 50 x 50 µm in size, where other part of the surface is virtually unaffected. The lift-out technique enables rapid TEM specimen preparation with minimal mechanical damage. The TEM specimens produced by this technique have reduced spurious bulk X-ray signal due to significantly reduced bulk material around the electrontransparent membrane. This is particularly beneficial for TEM investigations of magnetic materials as there is less mass of the specimen to be attracted to the TEM pole pieces. In addition, samples that are fragile or sensitive to contamination can be prepared using this technique. In the early stage of the development of lift-out technique, we undertook a challenging case of precision specimen preparation - preparing TEM sections through controversial features in ALH84001, the NASA Mars meteorite that reportedly contains evidence of former Martian life. On that small and extremely fragile surface of the meteorite, small features of 20-30 nm (Figure 8) in the smallest dimension was lifted out (using an exsitu lift-out instrument), and studied in TEM (Li et al., 2003). The lift-out process was

The conventional lift-out technique proved to be an efficient and versatile TEM specimen preparation method where the conventional H-bar technique can not be used. However, TEM foil thickness cannot be precisely measured in FIB systems, and the degree of final FIB thinning is very dependant on the operator's experience. Once the TEM specimen is made and transferred onto the TEM grid, it is not possible to perform any further FIB re-thinning.

The lift-out process described in the previous section can be very risky. The potential risk of losing a TEM specimen is quite high. The electrostatic charge at the tip of the glass needle (used in ex-situ lift-out process) could repel the tiny TEM foil and cause it to "fly-off" the needle tip. Also, too much charge at the needle tip could make it very difficult to "unload"

**2.2.3 The "H-bar lift-out" and "Plan-view lift-out" techniques**

the invention of lift-out technique, especially the perfection of in-situ lift-out instruments, the H-bar FIB-TEM specimen preparation technique has become obsolete. However, it remains as a powerful method that can still be used in some special cases.

Fig. 6. An example of the H-bar technique to make a TEM specimen from galvannealed steel. Stress relief cut could be made for specimens with high residual stress.

#### **2.2.2 The "Lift-out" technique**

The invention of the lift-out technique in FIB-TEM specimen preparation was revolutionary (Giannuzzi & Steve, 1999; Shankar et al., 2003; Prenitzer et al., 1998; Langford et al., 2002; Gasser et al., 2004). The main advantage of this technique is that TEM specimens can be made directly from bulk samples, without the need of cutting and polishing. This is particularly valuable for those samples that are sensitive to contamination and fragile that cannot be prepared by any mechanical means. The traditional lift-out processes are illustrated in Figure 7, and described as follows:

the invention of lift-out technique, especially the perfection of in-situ lift-out instruments, the H-bar FIB-TEM specimen preparation technique has become obsolete. However, it

Fig. 6. An example of the H-bar technique to make a TEM specimen from galvannealed

The invention of the lift-out technique in FIB-TEM specimen preparation was revolutionary (Giannuzzi & Steve, 1999; Shankar et al., 2003; Prenitzer et al., 1998; Langford et al., 2002; Gasser et al., 2004). The main advantage of this technique is that TEM specimens can be made directly from bulk samples, without the need of cutting and polishing. This is particularly valuable for those samples that are sensitive to contamination and fragile that cannot be prepared by any mechanical means. The traditional lift-out processes are

1. Once the feature of interest is identified on the surface, a protective metal (e.g. tungsten

2. FIB milling around the targeted area to create an electron transparent membrane with a typical thickness of less than 100 nm depending on the material, type of analysis and

3. This thin-membrane (TEM foil) containing the feature of interest is then cut free from

4. The thin membrane is transferred to carbon-coated TEM grid using an in-situ or ex-situ

steel. Stress relief cut could be made for specimens with high residual stress.

or platinum) is used to cover that local area (using FIB deposition).

**2.2.2 The "Lift-out" technique**

the TEM.

illustrated in Figure 7, and described as follows:

micromanipulator for TEM observation.

the bulk using the ion beam.

remains as a powerful method that can still be used in some special cases.

Fig. 7. Schematic diagram of traditional lift-out TEM specimen preparation technique

The preparation of a lift-out TEM specimens affect only a relatively small local region typically on the order of 50 x 50 x 50 µm in size, where other part of the surface is virtually unaffected. The lift-out technique enables rapid TEM specimen preparation with minimal mechanical damage. The TEM specimens produced by this technique have reduced spurious bulk X-ray signal due to significantly reduced bulk material around the electrontransparent membrane. This is particularly beneficial for TEM investigations of magnetic materials as there is less mass of the specimen to be attracted to the TEM pole pieces. In addition, samples that are fragile or sensitive to contamination can be prepared using this technique. In the early stage of the development of lift-out technique, we undertook a challenging case of precision specimen preparation - preparing TEM sections through controversial features in ALH84001, the NASA Mars meteorite that reportedly contains evidence of former Martian life. On that small and extremely fragile surface of the meteorite, small features of 20-30 nm (Figure 8) in the smallest dimension was lifted out (using an exsitu lift-out instrument), and studied in TEM (Li et al., 2003). The lift-out process was challenging and rewarding.

The conventional lift-out technique proved to be an efficient and versatile TEM specimen preparation method where the conventional H-bar technique can not be used. However, TEM foil thickness cannot be precisely measured in FIB systems, and the degree of final FIB thinning is very dependant on the operator's experience. Once the TEM specimen is made and transferred onto the TEM grid, it is not possible to perform any further FIB re-thinning.

#### **2.2.3 The "H-bar lift-out" and "Plan-view lift-out" techniques**

The lift-out process described in the previous section can be very risky. The potential risk of losing a TEM specimen is quite high. The electrostatic charge at the tip of the glass needle (used in ex-situ lift-out process) could repel the tiny TEM foil and cause it to "fly-off" the needle tip. Also, too much charge at the needle tip could make it very difficult to "unload" the specimen onto the TEM grid.

Advanced Techniques in TEM Specimen Preparation 77

meteorites is their concentration and grain size, with grains typically < 1 to 30 microns in longest dimension. The identified baddeleyite island was lifted out (planview), and fully

The commonly used FIB-TEM specimen preparation techniques present difficulties when dealing with small and delicate samples, such as fine powders and fine and/or fragile fibers. Once the sample dimensions approach the size of the usual lift-out specimen, the application of conventional lift-out techniques becomes difficult. Early work by Cairney and Munroe (Cairney & Munroe, 2001) prepared TEM specimens from fine FeAl and WC powders by embedding the powder into Epoxy resin. A TEM specimen was prepared using the conventional H-bar technique by treating the hardened resin (embedded with the powder particles) as a bulk specimen. TEM specimens prepared using this method contains significant amount of epoxy, and can be problematic in many ways during TEM examination. In addition, residual stress introduced to the particles during the resin-curing

Fig. 10. Planview lift out of a baddeleyite phase from a polished section of Martian

the baddeleyite phase cut free, lifted out and mounted on TEM grid, (d) FIB thinning

specimen showing the retained baddeleyite phase.

meteorite. (a). baddeleyite phase identified on the polished thin section, (b) FIB deposited W to protect the baddeleyite phase from ion beam damaging, (c) The protected zone containing

characterized in TEM (Li et al., 2010; Herd et al. 2010).

process could be unfavorable.

**2.2.4 The "Direct Lift-out" technique for ultra-fine specimens**

Patterson combined the "H-bar" and "lift-out" techniques (Patterson et.al., 2002). Using this combined method, a much thicker "slab" on the order of ~5 µm in thickness is transferred to a TEM grid (similar to that shown in Figure 7) before final FIB thinning to electron transparency. This is by far the most versatile and widely used lift-out technique under almost all circumstances.

Fig. 8. High-resolution SEM image showing the small feature of interest found on a piece of Martians meteorite, from which a TEM specimen was made.

Fig. 9. H-bar lift out TEM sample preparation from a fine Mg alloy particle.

The H-bar lift-out technique is particularly powerful when used to prepare plan-view TEM specimens. In some TEM investigations, plan-view samples are desired to characterize areas of interest. For example, a TEM foil containing the crack tip would be extremely valuable to understand the mechanism of SCC (Li et al. (2004, 2005, 2006d; Elboujdaini et al. 2004). Preparing site-specific plan-view TEM samples are tedious and time consuming. Figure 10 shows a baddeleyite (ZrO2) grain identified on a small piece of Martian meteorite, which is to be used to date the mineral. The largest challenge posed by baddeleyite in Martian

Patterson combined the "H-bar" and "lift-out" techniques (Patterson et.al., 2002). Using this combined method, a much thicker "slab" on the order of ~5 µm in thickness is transferred to a TEM grid (similar to that shown in Figure 7) before final FIB thinning to electron transparency. This is by far the most versatile and widely used lift-out technique under almost all circumstances.  High-resolution SEM image showing the small feature of interest found on a piece of

The H-bar lift-out technique is particularly powerful when used to prepare plan-view TEM specimens. In some TEM investigations, plan-view samples are desired to characterize areas of interest. For example, a TEM foil containing the crack tip would be extremely valuable to understand the mechanism of SCC (Li et al. (2004, 2005, 2006d; Elboujdaini et al. 2004). Preparing site-specific plan-view TEM samples are tedious and time consuming. Figure 10 shows a baddeleyite (ZrO2) grain identified on a small piece of Martian meteorite, which is to be used to date the mineral. The largest challenge posed by baddeleyite in Martian

Martians meteorite, from which a TEM specimen was made.

Fig. 9. H-bar lift out TEM sample preparation from a fine Mg alloy particle.

particle to be lifted out for final FIB thinning.

meteorites is their concentration and grain size, with grains typically < 1 to 30 microns in longest dimension. The identified baddeleyite island was lifted out (planview), and fully characterized in TEM (Li et al., 2010; Herd et al. 2010).

### **2.2.4 The "Direct Lift-out" technique for ultra-fine specimens**

The commonly used FIB-TEM specimen preparation techniques present difficulties when dealing with small and delicate samples, such as fine powders and fine and/or fragile fibers. Once the sample dimensions approach the size of the usual lift-out specimen, the application of conventional lift-out techniques becomes difficult. Early work by Cairney and Munroe (Cairney & Munroe, 2001) prepared TEM specimens from fine FeAl and WC powders by embedding the powder into Epoxy resin. A TEM specimen was prepared using the conventional H-bar technique by treating the hardened resin (embedded with the powder particles) as a bulk specimen. TEM specimens prepared using this method contains significant amount of epoxy, and can be problematic in many ways during TEM examination. In addition, residual stress introduced to the particles during the resin-curing process could be unfavorable.

Fig. 10. Planview lift out of a baddeleyite phase from a polished section of Martian meteorite. (a). baddeleyite phase identified on the polished thin section, (b) FIB deposited W to protect the baddeleyite phase from ion beam damaging, (c) The protected zone containing the baddeleyite phase cut free, lifted out and mounted on TEM grid, (d) FIB thinning specimen showing the retained baddeleyite phase.

Advanced Techniques in TEM Specimen Preparation 79

TEM images of the fiber made using the direct lift-out technique. There are several

1. The entire cross-section of the fiber can be made electron transparent for TEM

2. Minimum mechanical damage is introduced during the TEM specimen preparation

3. Lower risk of mechanical damage by the micromanipulator compared to other lift-out

This lift-out technique has also been used to make TEM samples from fine powders. The powder particles are spread onto a clean surface, and small particles are lifted-out and mounted directly onto the edge of a copper grid (as that shown in Figure 12). These particles

The conventional H-bar FIB-TEM specimen preparation technique is a simple and straightforward technique. With the invention of lift-out technique, this relatively "old" technique becomes unpopular. The lift-out technique not only provides the capability to prepare TEM samples with minimum mechanical damage and minimal contamination, but is also capable of producing site-specific TEM specimens. TEM specimens can be prepared either perpendicular or parallel to the sample surface (plan-view). The "direct lift-out" technique further facilitates TEM sample preparation of small and/or fragile specimens

Fig. 12. Surface oxidation of pure Mg particles, (a) A Mg particle lifted out, (b) FIB image of showing thin oxide film on the surface, and (c) STEM work confirming thin MgO layer of

In the past years, almost every major micro-beam vendors started to offer FIB systems. The number of FIB owners and users increased exponentially, and applications of FIB techniques extended into various field of materials engineering. FIB users ranging from university students to reputable research institutes start to produce TEM specimens like in massive production mode without thorough understanding of potential artifact. Throughout the years in the author's materials research, in both private sectors and the Canadian national materials research lab, the author encountered a wide range of engineering materials and processes. His FIB experience started about 15 years ago in the early days when FIB was just beginning to be noticed by materials researchers. The author has gradually experienced,

can be made electron transparent in the FIB with minimal milling effort.

advantages of this new technique:

such as fine fibers and powders.

about 20 nm in thickness.

**2.3 Potential artifacts**

observation.

process.

processes.

In a recent study (Li et al. 2003, 2006b), the author needs to evaluate the coating integrity of nickel-coated carbon fibers (about 10 µm in diameter). The entire cross-section of the fiber was to be made electron transparent. The project was to investigate the coating integrity produced using various coating processes. Some of the coatings were extremely fragile or even flaky. The "resin embedding" technique described in the previous section could have caused unacceptable mechanical damage in resin solidification process. None of the lift-out techniques described in the previous section was deemed likely to provide artifact-free TEM samples from these coated fibers. Even the very versatile technique of diamond-knife ultramicrotomy, normally excellent for cross-sectional TEM specimens, would have produced mechanically damaged or "shattered" cross-sections, and coating delamination would have been highly likely. The authored discovered the fine glass needle used for exsitu lift-out process can pick up much larger pieces than previously reported (Li et al., 2006b). The fibers were first carefully cut to 4-5 mm in length and transferred onto the inner edge of a TEM copper grid using an ex-situ micromanipulator. A minimal amount of lowshrinkage epoxy was used to stabilize the fiber to the grid. The fiber that is fixed onto the grid was then directly cut and thinned in a FIB microscope. Figure 11 shows the FIB and

Fig. 11. FIB and TEM images of successfully prepared TEM specimens using the direct lift-out technique. (a) FIB low magnification image of a bundle of fine fibers. (b) FIB image of a mounted fiber on a copper grid. (c) A low-magnification TEM image of a fiber with good coating quality. (d) Low-magnification TEM image showing a fiber with poor coating quality.

In a recent study (Li et al. 2003, 2006b), the author needs to evaluate the coating integrity of nickel-coated carbon fibers (about 10 µm in diameter). The entire cross-section of the fiber was to be made electron transparent. The project was to investigate the coating integrity produced using various coating processes. Some of the coatings were extremely fragile or even flaky. The "resin embedding" technique described in the previous section could have caused unacceptable mechanical damage in resin solidification process. None of the lift-out techniques described in the previous section was deemed likely to provide artifact-free TEM samples from these coated fibers. Even the very versatile technique of diamond-knife ultramicrotomy, normally excellent for cross-sectional TEM specimens, would have produced mechanically damaged or "shattered" cross-sections, and coating delamination would have been highly likely. The authored discovered the fine glass needle used for exsitu lift-out process can pick up much larger pieces than previously reported (Li et al., 2006b). The fibers were first carefully cut to 4-5 mm in length and transferred onto the inner edge of a TEM copper grid using an ex-situ micromanipulator. A minimal amount of lowshrinkage epoxy was used to stabilize the fiber to the grid. The fiber that is fixed onto the grid was then directly cut and thinned in a FIB microscope. Figure 11 shows the FIB and

Fig. 11. FIB and TEM images of successfully prepared TEM specimens using the direct lift-out technique. (a) FIB low magnification image of a bundle of fine fibers. (b) FIB image of a mounted fiber on a copper grid. (c) A low-magnification TEM image of a fiber with good coating quality. (d) Low-magnification TEM image showing a fiber with poor coating quality. TEM images of the fiber made using the direct lift-out technique. There are several advantages of this new technique:

This lift-out technique has also been used to make TEM samples from fine powders. The powder particles are spread onto a clean surface, and small particles are lifted-out and mounted directly onto the edge of a copper grid (as that shown in Figure 12). These particles can be made electron transparent in the FIB with minimal milling effort.

The conventional H-bar FIB-TEM specimen preparation technique is a simple and straightforward technique. With the invention of lift-out technique, this relatively "old" technique becomes unpopular. The lift-out technique not only provides the capability to prepare TEM samples with minimum mechanical damage and minimal contamination, but is also capable of producing site-specific TEM specimens. TEM specimens can be prepared either perpendicular or parallel to the sample surface (plan-view). The "direct lift-out" technique further facilitates TEM sample preparation of small and/or fragile specimens such as fine fibers and powders.

Fig. 12. Surface oxidation of pure Mg particles, (a) A Mg particle lifted out, (b) FIB image of showing thin oxide film on the surface, and (c) STEM work confirming thin MgO layer of about 20 nm in thickness.

#### **2.3 Potential artifacts**

In the past years, almost every major micro-beam vendors started to offer FIB systems. The number of FIB owners and users increased exponentially, and applications of FIB techniques extended into various field of materials engineering. FIB users ranging from university students to reputable research institutes start to produce TEM specimens like in massive production mode without thorough understanding of potential artifact. Throughout the years in the author's materials research, in both private sectors and the Canadian national materials research lab, the author encountered a wide range of engineering materials and processes. His FIB experience started about 15 years ago in the early days when FIB was just beginning to be noticed by materials researchers. The author has gradually experienced,

Advanced Techniques in TEM Specimen Preparation 81

ion dose increases (Shuman et al., 2001; Phaneuf et al., 2002). The appearance of such Garich phase is also found to appear on low-carbon steel as shown in Figure 15. There have been little reports on how to reduce Ga phase formation during FIB milling. It is not known if changing milling parameters (reduce beam energy etc.) will have any positive effect.

However, one should always be aware of these gallium phases in any FIB milling.

Fig. 13. FIB drills on silicon using a 670 pA beam. (a) a well-aligned beam, (b) a poorly

Fig. 14. FIB cross-section of a galvanealed Zn coating on IF steel. (a) polished with a wellaligned beam of 210 pA and imaged with a 32 pA beam, (b) a few more imaging passes

using 32 pA causes more damage to the coating.

aligned beam.

learned, and start to understand and tried hard to cope with most of the commonly occurring FIB-induced artifacts, and in many cases, after hard lessons learnt.

Common FIB induced artifacts include:

The root causes of FIB-generated artifacts are often complex. Aside from the nature of the materials and the type of FIB work to be performed, the proper control of the Ga ion beam is of paramount importance. However, the control of the ion beam has frequently been overlooked by many FIB users. Different FIB systems are designed with different milling routines. It is nearly impossible to create a universal recipe that is optimal for both efficient milling and minimize artifacts. In general, controlling the ion beam includes: acceleration voltage, beam current, beam dwell time, pixel spacing (beam overlap), re-trace, refresh time etc. In addition, one of the most of important factors is the actual beam shape. When performing fine milling especially during the final stage of the TEM specimen preparation, the beam shape should be checked carefully (with each aperture change). One should definitely eliminate "beam tail". A tight, stable and well-aligned beam is essential to reduce most of the FIB-induced artifacts on the TEM foil. Figure 13 shows examples of FIB holedrills on silicon wafer using (a) a nearly perfectly aligned, and (b) a poorly aligned ion beam. A well-aligned beam with minimum beam spread is essential to produce high-quality ion beam milling and polishing, while a beam tail of the poorly aligned beam will result in a noticeable "curtain effect". In some materials with hexagonal crystal symmetry (e.g. Zn and Zr), artifacts resulting from ion-beam milling are almost inevitable. Figure 14 shows a FIB cross-section of a galvanealed Zn coating on an interstitial-free (IF) steel. Ion-beam damage appears as dark speckles in the coating microstructure. It seems that fine grains at the Fe-Zn interface (Г phase) and the Zn-rich phase (ξ phase) near the coating surface are less prone to ion-beam damage and can be resolved as shown in Figure 14(a). The majority of the δ phase in the coating suffers noticeable damage. A few more imaging passes using a very small ion beam current (only 32 pA) worsens the damage, as shown in Figure 14(b).

FIB IC chip circuit modification of Cu-based interconnect is generally problematic (Malis et al., 2002; Herschbein et al., 1998; Casey et al., 2000) due to the significantly different sputter rate of Cu metal interconnects. The sputter rate variation is related to Cu grain crystallographic orientations. Measurements of FIB sputter rates on single crystal Cu specimens (Phaneuf et al., 2003) show a sputter rate variation of about four times between fast milling orientations, such as (111), and slow milling orientations, such as (110). This difference in sputter rate is not limited only to Cu, but has also been observed in FIB milling of Au and other alloys with cubic structures. The slow sputtering of the Cu (110) orientation is attributed to the of Ga ion beam channelling, and the formation of an anomalous metal– gallium (MxGay) phase during FIB milling under conditions in which the incident FIB beam hits the specimen at angles far from glancing and closer to normal incidence. During FIB milling of Cu, some grains become dark in color; these dark islands grow and spread as the

learned, and start to understand and tried hard to cope with most of the commonly

1. "curtain effect": due to insufficient FIB metal deposition and improper milling

The root causes of FIB-generated artifacts are often complex. Aside from the nature of the materials and the type of FIB work to be performed, the proper control of the Ga ion beam is of paramount importance. However, the control of the ion beam has frequently been overlooked by many FIB users. Different FIB systems are designed with different milling routines. It is nearly impossible to create a universal recipe that is optimal for both efficient milling and minimize artifacts. In general, controlling the ion beam includes: acceleration voltage, beam current, beam dwell time, pixel spacing (beam overlap), re-trace, refresh time etc. In addition, one of the most of important factors is the actual beam shape. When performing fine milling especially during the final stage of the TEM specimen preparation, the beam shape should be checked carefully (with each aperture change). One should definitely eliminate "beam tail". A tight, stable and well-aligned beam is essential to reduce most of the FIB-induced artifacts on the TEM foil. Figure 13 shows examples of FIB holedrills on silicon wafer using (a) a nearly perfectly aligned, and (b) a poorly aligned ion beam. A well-aligned beam with minimum beam spread is essential to produce high-quality ion beam milling and polishing, while a beam tail of the poorly aligned beam will result in a noticeable "curtain effect". In some materials with hexagonal crystal symmetry (e.g. Zn and Zr), artifacts resulting from ion-beam milling are almost inevitable. Figure 14 shows a FIB cross-section of a galvanealed Zn coating on an interstitial-free (IF) steel. Ion-beam damage appears as dark speckles in the coating microstructure. It seems that fine grains at the Fe-Zn interface (Г phase) and the Zn-rich phase (ξ phase) near the coating surface are less prone to ion-beam damage and can be resolved as shown in Figure 14(a). The majority of the δ phase in the coating suffers noticeable damage. A few more imaging passes using a very small ion

occurring FIB-induced artifacts, and in many cases, after hard lessons learnt.

2. low melting point gallium phase formation on sample surface,

beam current (only 32 pA) worsens the damage, as shown in Figure 14(b).

FIB IC chip circuit modification of Cu-based interconnect is generally problematic (Malis et al., 2002; Herschbein et al., 1998; Casey et al., 2000) due to the significantly different sputter rate of Cu metal interconnects. The sputter rate variation is related to Cu grain crystallographic orientations. Measurements of FIB sputter rates on single crystal Cu specimens (Phaneuf et al., 2003) show a sputter rate variation of about four times between fast milling orientations, such as (111), and slow milling orientations, such as (110). This difference in sputter rate is not limited only to Cu, but has also been observed in FIB milling of Au and other alloys with cubic structures. The slow sputtering of the Cu (110) orientation is attributed to the of Ga ion beam channelling, and the formation of an anomalous metal– gallium (MxGay) phase during FIB milling under conditions in which the incident FIB beam hits the specimen at angles far from glancing and closer to normal incidence. During FIB milling of Cu, some grains become dark in color; these dark islands grow and spread as the

4. beam induced grain growth in nano-crystalline materials,

3. beam damage on FIB prepared TEM specimens,

5. beam damage to most of the HCP materials, 6. materials redeposition during milling,

Common FIB induced artifacts include:

parameter.

7. surface amphorization.

ion dose increases (Shuman et al., 2001; Phaneuf et al., 2002). The appearance of such Garich phase is also found to appear on low-carbon steel as shown in Figure 15. There have been little reports on how to reduce Ga phase formation during FIB milling. It is not known if changing milling parameters (reduce beam energy etc.) will have any positive effect. However, one should always be aware of these gallium phases in any FIB milling.

Fig. 13. FIB drills on silicon using a 670 pA beam. (a) a well-aligned beam, (b) a poorly aligned beam.

Fig. 14. FIB cross-section of a galvanealed Zn coating on IF steel. (a) polished with a wellaligned beam of 210 pA and imaged with a 32 pA beam, (b) a few more imaging passes using 32 pA causes more damage to the coating.

Advanced Techniques in TEM Specimen Preparation 83

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Fig. 15. FexGay phase formation on low carbon steel during FIB imaging. (a) first image pass, (b) multiple imaging passes under 1.5 nA beam current, (c) multiple passes under 6 nA beam.

## **3. Conclusion**

Advances in materials engineering depend heavily on high-level microstructure characterization using a combination of state-of-the-art instruments. New characterization techniques and methodologies are critical in understanding the fundamentals of materials properties. High-quality TEM work is heavily dependent on the preparation of TEM foil with minimum artifacts. FIB lift-out techniques are versatile, and can produce almost any kind of TEM specimen (provided they are both vacuum and beam stable). The operator should always try to optimize milling parameters in order to reduce potential artifacts.

## **4. Acknowledgement**

The author gratefully acknowledges the efforts of the following individuals for their contributions of research and consultations leading to the completion of this book chapter: Tom malis, Pei Liu, Catherine Bibby, Wenyue Zheng and Darren Bibby (Materials Technology Laboratory, Natural Resources Canada), Michael Phaneuf (Fibics Incorporated), Chris Herd (University of Alberta) and George Kiporous (Dalhousie University).

#### **5. References**

Fig. 15. FexGay phase formation on low carbon steel during FIB imaging. (a) first image pass, (b) multiple imaging passes under 1.5 nA beam current, (c) multiple passes under 6 nA

Advances in materials engineering depend heavily on high-level microstructure characterization using a combination of state-of-the-art instruments. New characterization techniques and methodologies are critical in understanding the fundamentals of materials properties. High-quality TEM work is heavily dependent on the preparation of TEM foil with minimum artifacts. FIB lift-out techniques are versatile, and can produce almost any kind of TEM specimen (provided they are both vacuum and beam stable). The operator should always try to optimize milling parameters in order to reduce potential artifacts.

The author gratefully acknowledges the efforts of the following individuals for their contributions of research and consultations leading to the completion of this book chapter: Tom malis, Pei Liu, Catherine Bibby, Wenyue Zheng and Darren Bibby (Materials Technology Laboratory, Natural Resources Canada), Michael Phaneuf (Fibics Incorporated),

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beam.

**3. Conclusion**

**4. Acknowledgement**

**5. References**

**5**

*USA*

**Low-Dose Imaging Techniques for**

**Transmission Electron Microscopy**

*Department of Molecular and Cellular Biology, University of California at Davis,*

Transmission electron microscopes have the ability to visualize almost any specimen at the nanoscale. Unfortunately, modified imaging protocols are needed when samples are composed predominantly of low atomic number elements. In this chapter we review several methods that are currently employed to mitigate the adverse effects caused by electron irradiation to materials at the interface of biology and nanotechnology. We also highlight future technological advancements to the microscope platform that may further enhance the

Although material scientists are able to routinely image inorganic specimens at atomic resolution, the same is not true for organic and biological specimens. This divergence in attainable resolution is largely a result of varying tolerance to electron beam exposure. Organic and biological specimens exhibit increased sensitivity to high-energy electron irradiation as compared to inorganic materials. For example, metals and monocrystals of silicon can handle very large doses of radiation without showing significant signs of damage. However, radiation damage, in the form of bond breakage and mass loss, from the electron beam is both deleterious and unavoidable for organic samples made of low atomic number elements such as carbon, nitrogen, and oxygen (Thach & Thach, 1971). The damage response of biological specimens is dependent on the cumulative exposed dose and was estimated using spot fading diffraction experiments on two-dimensional (2D) crystals of the amino acid L-valine. In those experiments, a dose of 6 e-/Å2 at 80 keV resulted in the complete disappearance of diffraction spots and this value is typical, within an order of magnitude, of most biological specimens (Glaeser, 1971). Aliphatic polymers such as polyethylene are equally sensitive to electron irradiation and display similar critical doses to that of proteins (Revol & Manley, 1986). However, more aromatic polymers such as polystyrene, anthracence and poly-xylene can withstand higher irradiation doses due to the stability of the phenyl group (36, 42 and 120 e-/Å2 respectively) (Kumar & Adams, 1990; Williams & Carter, 2004). Importantly, it should be noted that although zeolites and ceramics are technically inorganic specimens consisting of high atomic number elements, they exhibit intermediate sensitivity to electron irradiation with critical doses of

**2. Need for low-dose imaging with transmission electron microscopy**

approximately 100 and 600 e-/Å2, respectively (Pan & Crozier, 1993).

**1. Introduction**

quality of imaging for beam sensitive samples.

David B. Carlson and James E. Evans

## **Low-Dose Imaging Techniques for Transmission Electron Microscopy**

David B. Carlson and James E. Evans *Department of Molecular and Cellular Biology, University of California at Davis, USA*

#### **1. Introduction**

84 The Transmission Electron Microscope

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Transmission electron microscopes have the ability to visualize almost any specimen at the nanoscale. Unfortunately, modified imaging protocols are needed when samples are composed predominantly of low atomic number elements. In this chapter we review several methods that are currently employed to mitigate the adverse effects caused by electron irradiation to materials at the interface of biology and nanotechnology. We also highlight future technological advancements to the microscope platform that may further enhance the quality of imaging for beam sensitive samples.

#### **2. Need for low-dose imaging with transmission electron microscopy**

Although material scientists are able to routinely image inorganic specimens at atomic resolution, the same is not true for organic and biological specimens. This divergence in attainable resolution is largely a result of varying tolerance to electron beam exposure. Organic and biological specimens exhibit increased sensitivity to high-energy electron irradiation as compared to inorganic materials. For example, metals and monocrystals of silicon can handle very large doses of radiation without showing significant signs of damage. However, radiation damage, in the form of bond breakage and mass loss, from the electron beam is both deleterious and unavoidable for organic samples made of low atomic number elements such as carbon, nitrogen, and oxygen (Thach & Thach, 1971). The damage response of biological specimens is dependent on the cumulative exposed dose and was estimated using spot fading diffraction experiments on two-dimensional (2D) crystals of the amino acid L-valine. In those experiments, a dose of 6 e- /Å2 at 80 keV resulted in the complete disappearance of diffraction spots and this value is typical, within an order of magnitude, of most biological specimens (Glaeser, 1971). Aliphatic polymers such as polyethylene are equally sensitive to electron irradiation and display similar critical doses to that of proteins (Revol & Manley, 1986). However, more aromatic polymers such as polystyrene, anthracence and poly-xylene can withstand higher irradiation doses due to the stability of the phenyl group (36, 42 and 120 e-/Å2 respectively) (Kumar & Adams, 1990; Williams & Carter, 2004). Importantly, it should be noted that although zeolites and ceramics are technically inorganic specimens consisting of high atomic number elements, they exhibit intermediate sensitivity to electron irradiation with critical doses of approximately 100 and 600 e-/Å2, respectively (Pan & Crozier, 1993).

Low-Dose Imaging Techniques for Transmission Electron Microscopy 87

In addition to the primary damage mechanisms of radiolysis and knock-on damage, one must also consider secondary and tertiary damage. Secondary damage results from inelastic collision events which form phonons, free radical species and secondary electrons that can each propagate through the sample and heat the sample or cause further damage via chemical reaction or secondary scattering (Williams & Carter, 2004). Tertiary damage effects arise from the evolution and buildup of gas (generally hydrogen and oxygen) following the

For specimens that are good thermal conductors, phonon induced heating is usually negligible (Williams & Carter, 2004). However, the majority of biological and organic specimens (as well as ceramics) are thermal insulators and therefore such heating can be considerable, sometimes to the point of melting the sample. Simulations of proteins frozen and imaged at liquid nitrogen temperature (77 K) suggest that this heating may be