Microscopy Microanalysis
Microsc. Microanal. 20, 124–132, 2014 doi:10.1017/S1431927613014049
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An Inexpensive Approach for Bright-Field and Dark-Field Imaging by Scanning Transmission Electron Microscopy in Scanning Electron Microscopy Binay Patel* and Masashi Watanabe Department of Materials Science and Engineering, Lehigh University, Bethlehem, PA 18015, USA
Abstract: Scanning transmission electron microscopy in scanning electron microscopy ~STEM-in-SEM! is a convenient technique for soft materials characterization. Various specimen-holder geometries and detector arrangements have been used for bright-field ~BF! STEM-in-SEM imaging. In this study, to further the characterization potential of STEM-IN-SEM, a new specimen holder has been developed to facilitate direct detection of BF signals and indirect detection of dark-field ~DF! signals without the need for substantial instrument modification. DF imaging is conducted with the use of a gold ~Au!-coated copper ~Cu! plate attached to the specimen holder which directs highly scattered transmitted electrons to an off-axis yttriumaluminum-garnet ~YAG! detector. A hole in the copper plate allows for BF imaging with a transmission electron ~TE! detector. The inclusion of an Au-coated Cu plate enhanced DF signal intensity. Experiments validating the acquisition of true DF signals revealed that atomic number ~Z! contrast may be achieved for materials with large lattice spacing. However, materials with small lattice spacing still exhibit diffraction contrast effects in this approach. The calculated theoretical fine probe size is 1.8 nm. At 30 kV, in this indirect approach, DF spatial resolution is limited to 3.2 nm as confirmed experimentally. Key words: holder development, soft materials, atomic number contrast ~Z contrast!, polymers, image resolution, SMART macro
I NTR ODUCTION Soft materials characterization ~e.g., of polymers and biological materials! by electron microscopy @e.g., scanning electron microscopy ~SEM!, transmission electron microscopy ~TEM!, and scanning transmission electron microscopy ~STEM!# has been limited due to the lack of long range order in soft materials and their susceptibility to high-voltage electron beams. These challenges present a unique opportunity for innovation in the field of electron microscopy and may require an unconventional, hybrid characterization technique. STEM-IN-SEM ~e.g., Joy & Maher, 1976! may be a convenient characterization approach for these materials. Utilizing the lower accelerating voltages, larger field of view, and exclusion of a post-specimen projection lens in a SEM instrument, STEM-IN-SEM has shown results similar to TEM observation of polymer morphology ~Guise et al., 2011!. Furthermore, comparable image quality has been obtained with bright-field ~BF! STEM-IN-SEM as compared with BF TEM observation of silver nanoparticles ~Vanderlinde & Ballarotto, 2004! and bacteria ~Bogner et al., 2007!. Various specimen-holder geometries and detector arrangements have been used for STEM-IN-SEM imaging but have primarily focused on BF imaging ~e.g., Crawford & Liley, 1970; Woolf et al., 1972; Oho et al., 1987a, 1987b; Vanderlinde & Ballarotto, 2004; Merli & Morandi, 2005; Bogner et al., 2007! while work on DF imaging ~e.g., Crawford & Liley, 1970; Merli & Morandi, 2005; Acevedo-Reyes Received September 18, 2013; accepted December 2, 2013 *Corresponding author. E-mail: [email protected]
et al., 2008; Brodusch et al., 2013! has been limited by technology ~until recently! or by the costly acquisition of new instrumentation ~e.g., detectors!. STEM-IN-SEM imaging has been applied to morphological observation of various polymer-based latex particles ~Geng et al., 2013!, mask metrology ~Klein et al., 2012!, X-ray elemental mapping ~Stokes & Baken, 2007!, X-ray spectral imaging of thin specimens prepared by a focused ion beam method ~Kotula, 2009!, particle-size distribution measurements both in BF ~Klein et al., 2011! and DF ~with a dedicated detector! ~Acevedo-Reyes et al., 2008!, as well as extreme high resolution SEM, in which a monochromator and a 12-segment STEM detector provide the potential for sub-nanometer spatial resolution ~Roussel et al., 2009!. BF images of electron transparent specimens mainly display mass-thickness contrast. While such images may be used for particle-size and distribution analysis, these images do not provide intuitive image interpretation especially when investigating novel or unknown specimens. Conversely, the detection of DF signals, which correspond to the atomic number of constituent elements, would be advantageous to form atomic-number contrast ~so called Z-contrast! images when electrons scattered with higher angles are collected. Therefore, the goal of this study is to advance the potential of soft materials characterization through STEMIN-SEM by developing a method for the acquisition of BF and DF STEM-IN-SEM images with fabrication of a newly designed specimen holder. The new holder will facilitate BF and DF STEM-IN-SEM imaging without requiring costly modifications to an existing SEM instrument.
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Configuration of Fine Probe Settings All BF and DF STEM-IN-SEM images were collected in a Hitachi 4300 SEM instrument ~Hitachi, Hitachinaka, Japan! equipped with a Schottky field-emission gun, at an accelerating voltage of 30 kV ~the maximum for this instrument!. In this study, two detectors were used for BF and DF STEM-IN-SEM imaging in the SEM instrument: one is a TE detector located below the specimen stage for BF imaging and the other is a YAG detector located off-axis for DF imaging ~which is usually used for backscatter electron imaging!. Both detectors came standard when this SEM was acquired by Lehigh University. Based on the instrument configurations, the convergence semi-angle to form the minimum probe size at 30 kV was estimated as 3.2 mrad resulting in an estimated minimum probe size as fine as 1.8 nm, following a procedure outlined in Goldstein et al. ~2003!. This convergence semi-angle is achieved at a working distance of 4.6 mm in the Hitachi 4300. The spatial resolution of DF STEM-IN-SEM imaging is evaluated through image analysis from a standard combined test specimen for TEM ~Ted Pella, Redding, CA, USA!, consisting of small Au nanoparticles with a light deposition of graphitized carbon on a perforated carbon film, using the scanning microscope assessment and resolution testing ~SMART! macro ~Joy, 2002! with the Scion Image ~Scion Corporation, Frederick, MD, USA! software package. Previously, the manual procedure for SMART macro and its limitations ~e.g., partial user dependence! for resolution testing of STEM-IN-SEM images was discussed ~Probst et al., 2007!. Although, user dependence is an issue with manual SMART macro analysis, overall this method is far superior to line profile analysis ~through the measurement of the minimum distance between peaks that correspond to actual points in an image! for image resolution analysis. The only deviation here from the SMART procedure previously outlined is the recording of image resolution measurements from 256 ⫻ 256 pixel areas from five DF images. The Au standard combined test specimen is commonly used for resolution evaluation for TEM and STEM imaging.
Holder Design The new specimen holder ~Fig. 1! is made of aluminum and includes ~i! an entrenched flat housing for the specimen to reside, ~ii! a long vertical column that acts as structural support for the specimen housing and ~iii! a base designed to fit into the stage locking mechanism of the SEM instrument. The holder features an Au-coated Cu plate with a center hole and a fixed geometry. The Cu plate is positioned below the specimen housing and facilitates the direct detection of BF signals and the indirect detection of DF signals. Conductive graphite paste is used on the exteriors of both the holder body and the Cu plate to minimize unnecessary electron emission from these parts. BF and DF STEM-IN-SEM signal acquisition is achieved as follows ~shown schematically in Fig. 2!: The incident
Figure 1. A new specimen holder for BF and DF STEM-IN-SEM imaging in the Hitachi 4300SE.
Figure 2. Schematic diagram showing the optimal microscope configuration for BF and DF STEM-IN-SEM imaging in the Hitachi 4300SE.
electron beam interacts with an electron-transparent specimen. Subsequently, transmitted electrons leaving the specimen are collected differently depending on their scattering angle. The scattering of transmitted electrons is completely dependent on the elements from the specimen of interest, their composition and crystallographic orientations ~if they are crystalline materials!. Transmitted electrons that are scattered at low-angles travel through the center hole in the Au-coated Cu plate and are collected by an on-axis TE detector. The resultant BF STEM-IN-SEM images mainly exhibit mass-thickness contrast and/or diffraction contrast ~when crystalline materials are observed!. Transmitted electrons that are scattered at high-angles reach the Au-coated Cu plate and are backscattered from it toward an off-axis YAG detector. Finally, these transmitted @and subsequently backscattered electrons ~BSE!# are collected by the YAG detector for DF image formation. The DF STEM-IN-SEM images exhibit either diffraction contrast or Z-contrast depending on the type of specimen under investigation ~as described later, the contrast formation is dependent on the characteristic crystallographic orientation and resultant scat-
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tering behavior of a specimen!. This hybrid detection design makes efficient use of the detector configurations available on the Hitachi 4300 at Lehigh University ~and many other conventional SEM instruments as well!.
Optimization of DF Imaging Prior to fabrication of the holder, Monte Carlo simulation using the CASINO simulation package ~Drouin et al., 2007! was conducted to optimize the indirect detection method for DF STEM-IN-SEM imaging by utilizing the fine probe settings discussed above. First, to ensure significant electron signal collection for DF image formation, the minimum Au-coating thickness required for efficient electron backscattering off of the Cu plate surface was determined to be 600 nm. This coating thickness corresponds to achieving the maximum BSE yield of Au, as compiled by Joy ~Joy’s database contained in Goldstein et al., 2003!, without penetrating the coating layer. Second, the influence of Cu plate orientation on electron backscattering was studied. Monte Carlo simulations were performed on a Cu plate with 600 nm of Au-coating. In the simulations, the Cu plate remained stationary and interacted with electron beams tilted at 0, 30, 60, and 808 relative to the surface of the plate, which is geometrically equivalent to changing the Cu plate orientation against a normal electron beam. As the electron beam tilt angle increases, the signal generation also increases but is oriented toward higher backscattered directions, which agrees with experimental data on the forwardscattering of 30 kV electrons from an Au surface ~Darlin´ski, 1981!. Based on the simulation, an exaggerated Au-coating thickness of 1.5 mm was used to ensure sufficient electron backscattering for the holder design in this study. In addition, a center hole with a diameter of 2 mm was created on the Au-coated Cu plate corresponding to 55 mrad in this geometry at 30 kV. The use of a center hole is essentially equivalent to an inner angle of an annular DF detector, i.e., the hole serves as the inner cut-off angle for DF imaging. The measured signal intensities from BF and DF STEMIN-SEM images were used to determine the optimal Cu plate orientation for DF signal generation. Flat-polished Cu plates were first coated with gold to a thickness of 1.5 mm and then bent to inclination angles of 0, 10, 20, 40, and 808. The Au-standard combined test specimen was used for imaging. Five BF and DF STEM-IN-SEM images were recorded at a magnification of 150,000⫻ using each flatpolished plate and with no plate present, to serve as a control. The measured signal intensity from each image was determined using Image J software ~Schneider et al., 2012!.
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Optimization of BF and DF Signal Collection For this holder configuration to achieve efficient BF and DF STEM-IN-SEM imaging, optimizing the inclination angle of the Cu plate is critical. Figure 3 shows a BF and DF STEM-IN-SEM image pair of the Au standard with a Cu
Figure 3. A pair of BF ~left! and DF ~right! STEM-IN-SEM images of Au standard combined test specimen using the 108 Aucoated Cu plate.
Figure 4. Measured BF STEM-IN-SEM signal intensity as a function of Cu plate inclination angle. Also included is a reference value obtained without the Cu plate.
plate angle of 108. The Au nanoparticles appear dark in the BF image ~left! and bright in the DF image ~right!. Five DF STEM-IN-SEM images of the Au nanoparticles, collected with a Cu plate angle of 108 ~selected as discussed below!, were used to calculate a DF spatial resolution of 3.2 nm by SMART macro analysis for this indirect approach at 30 kV. There was no deviation in the DF spatial resolution observed from measurements collected from the five DF STEMIN-SEM images because consistent thresholding, through user-control, was applied to each image during the SMART macro analysis. Furthermore, from such images obtained with different plate inclination angle settings, signal intensities were evaluated. Figure 4 shows the measured BF signal intensity in gray level ~GL! plotted against the inclination angle of the Cu plate. Since the BF signals were collected by the TE detector below the holder, BF images can be obtained without the Cu plate. For reference, the measured average BF intensity was 219 6 2.3 GL without the plate present ~shown in Fig. 4 as reference!. As shown in Figure 4, the inclusion of the plate slightly reduces the BF signals especially for inclination angles ranging from 0 to 408. Yet, no significant difference was observed in the BF signals which fluctuated from 202 6 5.1 to 220 6 3.1 GL within the range of inclination angles tested. In fact, the difference in the measured BF
Inexpensive BF and DF STEM-IN-SEM Imaging
Figure 5. Measured DF STEM-IN-SEM signal intensity as a function of Cu plate inclination angle. Also included is a reference value obtained without the Cu plate.
signals is below 20 GL, which is not visually noticeable. Thus the addition of the Cu plate with the hole of 55 mrad does not disturb BF signal collection through the TE detector within an inclination angle range of 0–808. Similar experiments were also performed for DF STEMIN-SEM imaging ~Fig. 5!. The measured DF signals were the lowest when using no plate and an 808 inclined Cu plate, producing 58 6 0.1 and 63 6 0.6 GL, respectively. When no plate is present ~shown as reference in Fig. 5!, the DF signal collection is drastically lowered because transmitted electrons are not backscattered on surfaces of the holder due to the low atomic number graphite coating ~i.e., no effective backscattering toward the YAG detector!. When the 808 inclined plate is used, the BSE on the plate for DF imaging are no longer optimally oriented toward the YAG detector in the Hitachi 4300SE. Conversely, the use of plates with low inclination angles leads to nearly double the measured DF signals compared with the absence of a plate ~averaging 105 6 1.2 GL!. In addition, the measured DF signal does not change significantly between inclination angles of 0–408. BSE from the plate have an unperturbed path to the YAG detector and small variations in inclination angle do not actually influence DF image formation if the plate angle is set between 0 and 408. In this study, the 108 inclined plate was selected for BF and DF STEM-IN-SEM imaging but any plate inclined ,408 may be used on the Hitachi 4300SE microscope to produce similar results. Figure 6 shows a BF and DF STEMIN-SEM image pair of unstained 10 vol% Nanosilica-filled Diglycidly Ether of Bisphenol A ~DGEBA! epoxy. The sample was cryo-microtomed to ;100 nm in thickness. While the DF image is noisy, it highlights the sensitivity of this approach for imaging low atomic number differences in soft materials. Indeed, DF signals in the discrete nanosilica particles ~effective Z ⫽ 10! are higher than those in the epoxy matrix ~effective Z ⫽ 3.7! in this uniformly thick specimen.
Validation of DF Signal Acquisition In order to determine if this DF STEM-IN-SEM approach produces images that may be interpreted in the same manner as high-angle annular DF ~HAADF! STEM images three critical factors must be addressed: ~i! the influence of the SEM instrument, particularly it is scanning system, on
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Figure 6. A pair of BF ~left! and DF ~right! STEM-IN-SEM images of unstained 10 vol% nanosilica-filled DGEBA epoxy using the 108 Au-coated Cu plate.
image interpretability, ~ii! the origin of DF signal based on electron-beam specimen interactions, and ~iii! the underlying contrast mechanism~s! present in DF images based on the new holder design. Influence of the Scanning Electron Beam on STEM-IN-SEM Imaging In dedicated STEM instruments, a scanning system consists of double deflection coils, which ensure that the electron beam remains parallel to the optic axis of the instrument ~i.e., normal to a nontilted specimen! during imaging. Conversely, only one deflection coil is used for scanning in conventional SEM instruments. This may cause abnormal interaction between the electron beam and a nontilted specimen and may also shift the electron beam off-axis, especially when a large field of view needs to be scanned, i.e. at low magnifications. The tilted incident beam may promote severe image distortion and uneven signal distribution, etc., making it nearly impossible to ascertain the underlying contrast mechanism~s! present in DF STEM-INSEM images. To evaluate the influence of SEM scanning on STEMIN-SEM imaging, a series of BF images of a polystyrene latex standard @particle size 0.3 mm ~6 0.01!# ~Ted Pella! were recorded at magnifications ranging from 10,000 to 300,000⫻. One droplet of polystyrene latex particles in solution was placed on a standard holey carbon TEM grid for use during imaging. The average diameter of the polystyrene latex particles was measured, using Image J software ~Schneider et al., 2012! from each image and compared with the known particle size distribution. Any significant deviation in the average particle size measurements would suggest that the deflection of electron beam to off-axis is no longer negligible in image formation. BF images of polystyrene latex particles ~such as the one shown in Fig. 7A! over a range of magnifications did not yield statistically significant image distortion ~Fig. 7B!. The average particle diameter at each magnification was within the nominal particle size distribution provided by the supplier. Therefore, the deflection present in the scanning electron beam is small enough that image distortion does not occur within the magnification range covered in
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Figure 7. Polystyrene Latex Particles. A: BF STEM-IN-SEM image showing average particle diameter. B: Variation in average particle diameter with imaging magnification.
Figure 8. A pair of BF ~left! and DF ~right! STEM-IN-SEM images of Au standard combined test specimen obtained at a working distance of 4.5 mm with signal intensity plot profile shown ~bottom! with particular emphasis on the copper grid and hole regions.
this study. The absence of image distortion suggests that the electron beam still remains close to parallel to the optic axis. Therefore, the resultant scattering of electrons transmitted through the specimen after electron beam–specimen interactions may be directly attributed to the specimen itself. Authentication of DF Signal In the imaging method outlined, DF STEM-IN-SEM imaging is conducted by an off-axis YAG detector, which is usually used for BSE imaging. BSE images are generated directly from the primary electron beam by interaction with a specimen at its surface ~or near sub-surface! ~Goldstein et al., 2003! whereas DF images are generated from highly scattered transmitted electrons after interaction with the specimen. To investigate the origin of the DF STEM-INSEM signals in the instrument/holder geometry proposed in this study two experiments were conducted. First, BF and DF STEM-IN-SEM images were recorded from the same field of view on the Au standard combined test specimen at two different working distances: A shorter working distance of 4.5 mm ~optimal for STEM-IN-SEM imaging, where the specimen is positioned above the YAG detector! and a longer working distance of 13.5 mm ~optimal for BSE imaging, where the specimen is positioned below the YAG detector!. Figures 8 and 9 show two pairs of
BF and DF STEM-IN-SEM images from the combined test specimen with the Cu grid bar, recorded at the optimum and longer working distances, respectively. The contrast mechanisms between each pair of images are compared based on the average measured signal intensity from sections of the Cu grid on which the Au standard specimen resides. Image analysis was conducted using Gatan Digital Micrograph ~Gatan Inc., Pleasanton, CA, USA!. The Cu grid regions on traditional TEM grids are too thick for electron transparency. When the shorter working distance, optimized for the STEM-IN-SEM approach, was chosen the measured DF signal intensity of Cu grid bar region is 24.1 6 1.4 GL in average. Conversely, the DF signal intensity is 84.7 6 8.2 GL at the same region with the longer working distance. Since the YAG detector is located above the specimen in this case, the higher signal intensity must include BSE signals from the grid bar. In the field of view shown in Figures 8 and 9, holes can also be found in the carbon support film. The average DF signal intensities from one of the holes is 24.0 6 1.2 GL for the optimum and 24.3 6 1.0 GL for the longer working distance, respectively. Ideally, holes in the specimen should display neither DF signals nor BSE signals since no scattering occurs from such regions. As shown in Figure 8, the DF signals exhibit exactly the same level from the Cu grid bars
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Figure 9. A pair of BF ~left! and DF ~right! STEM-IN-SEM images of an Au standard specimen obtained at a working distance of 13.5 mm with signal intensity plot profile shown ~bottom! with particular emphasis on the copper grid and hole regions.
and the hole, which definitely proves that no signal from these regions is detected from electrons transmitted through the thin specimen using the off-axis YAG detector, at least when the working distance is set at 4.5 mm. Possible reasons for the nonzero DF signals from the Cu grid bars and from the hole may be from either spurious electrons such as SE III ~Goldstein et al., 2003! or detector “dark current.” To confirm the origin of DF signals, a second set of experiments was performed. First, BF and DF STEM-IN-SEM images were collected from a large hole in the Au standard specimen with the optimum working distance of 4.5 mm at a low magnification of 3,000⫻. Next, the magnification was increased until both the BF and DF imaging scans were completely inside the hole of interest, which means that there is no electron scattering event with the specimen within the field of view. Then, BF and DF STEM-IN-SEM images were taken inside the hole of interest at a magnification of 20,000⫻ by blocking the incident electron beam with an objective aperture ~located above the specimen!. Figure 10 shows two pairs of BF and DF STEM-INSEM images ~a! with and ~b! without the electron beam. Measured DF signal from the hole with the electron beam is 55.2 6 2.1 GL, which is compatible with those without the electron beam ~54.7 6 2.4 GL!. Since the electron beam was not present in the latter image ~Fig. 10B! the minimal measured signal intensity observed in DF STEM-IN-SEM images is not attributed to electron beam–specimen interactions and to any spurious electrons in the column. Therefore, the source of the measured signal intensity is attributed to the “dark current” present in the YAG detector. The YAG detector used for DF STEM-IN-SEM imaging has no voltage bias applied to it. Thus, no electrons are attracted to the detector. Only electrons oriented toward the detector take part in image formation. Since electron signal acquisition may be low upon conversion of the electron signal to a photon signal for digital imaging, high amplification is required. The minimum measured signal intensity observed
is the result of electronic noise, or “dark current”, as a result of the amplification process. It should be noted that the dark-current levels are different between Figures 8 and 10. This is simply due to the difference in the acquisition time. In comparison, the TE detector used for BF STEM-INSEM imaging has a small positive voltage bias applied which attracts more electrons to participate in image formation. Thus, upon conversion of the electron signal to a photon signal for digital imaging, amplification of the electron signal is not as intense as in the YAG detector. As a result, electronic noise from the TE detector ~0.00 6 0.0 GL, Fig. 10B! is significantly lower than in the YAG detector. Determination of DF Signal Contrast Mechanism DF images may exhibit either diffraction contrast or Z-contrast, depending on the collection semi-angle selected for imaging. Diffraction contrast involves coherent electron scattering, where the phase relationships between atomic lattices lead to contrast variations which make image interpretation difficult if crystalline materials are being characterized. However, Z-contrast involves incoherent electron scattering, where only highly scattered electrons that have no phase relationship between atomic lattices are detected. Thus, the contrast variation in Z-contrast images is directly related to the atomic number of atoms in the specimen, which makes image interpretation much more straightforward ~Nellist & Pennycook, 2000!. To determine the contrast mechanism for DF STEM-IN-SEM images the minimum collection semi-angle for Z-contrast is calculated using the following relation as described by Volkenandt et al. ~2010!: bmin ⫽ 2 arcsin~bM l/d!,
~1!
where b ⫽ 0.61, l is the wavelength of the electrons, and d is the atomic spacing along the lattice. The minimum collection semi-angle is evaluated for 30 kV electrons. The lattice spacings for four materials @Au, silica ~SiO2 !, polyethylene ~PE!, and PS# are chosen to illustrate the role of the mini-
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Figure 10. A pair of BF ~left! and DF ~right! STEM-IN-SEM images of a hole in the Au standard specimen. ~A! at 3,000⫻ magnification and with an unblocked electron beam and ~B! at 20,000⫻ magnification with a blocked electron beam. Signal intensity plots profiles ~bottom! in both images emphasis the hole regions.
mum collection semi-angle on image contrast. The lattice spacings for gold in the @111# direction and SiO2 in the @100# direction were obtained from Web Electron Microscopy Applications Software ~WebEMAPS! ~Zuo & Mabon, 2004!. Owing to the noncubic crystal structures of PE ~orthorhombic! and PS ~trigonal! in their nearly crystalline states, average lattice spacings are used, as obtained from Young and Lovell ~1991!. An analysis of the scattering angles of transmitted electrons at 30 kV revealed that harder materials ~e.g., Au, SiO2 !, with characteristically shorter lattice spacing, still exhibit diffraction contrast effects during DF STEM-INSEM imaging under the current geometrical set up ~Fig. 11!. In fact, any material with lattice spacings smaller than 8.8 Å, corresponding to the 55 mrad cut-off angle on the Cu plate, will exhibit some diffraction contrast effects in DF STEMIN-SEM imaging. Under the current configuration of the Cu-plate, the simplest crystallizable polymer structure, PE ~ bmin ⫽ 73 mrad in the orthorhombic structure!, does not exhibit pure Z-contrast during DF imaging. However, many soft materi-
als, specifically nearly crystalline polymer systems ~e.g., PS, bmin ⫽ 40 mrad in the trigonal structure!, with characteristically larger lattice spacing, may exhibit Z-contrast through DF STEM-IN-SEM imaging under this approach. These larger lattice spacings of polymers are a consequence of the increased functionality of side groups as these polymers become more complex ~Young & Lovell, 1991!. As polymers are generally amorphous, lacking long range order, their effective lattice spacings are further enlarged and diffraction contrast effects are further reduced ~i.e., bmin decreases!. Thus, such polymer systems ~e.g., PS latex particles and 10 vol% nanosilica-filled DGEBA epoxy! do exhibit Z-contrast through DF STEM-IN-SEM imaging under this approach.
Applications and Limitations of BF and DF STEM-IN-SEM Imaging This technique may be used both in industry and academia. The breadth of information available from BF and DF STEM-IN-SEM images may prove as a suitable alternative to TEM characterization of materials. Specifically for soft materials, this technique may be a cost-efficient method for
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Massachusetts Medical School ~Worchester, MA! is acknowledged for ultramicrotoming the 10 vol% nanosilica-filled DGEBA epoxy sample. Finally, the authors thank Mr. William Mushock and Dr. Robert Keyes from Lehigh University ~Bethlehem, PA! for value discussion during this study.
R EFER ENCES
Figure 11. Minimum collection semi-angle ~ bmin ! for achieving Z-contrast during DF imaging ~dashed line! with reference to the cut-off angle on Cu plate ~full line!, plotted against lattice spacing. Lattice spacings and corresponding collection semi-angles for select materials are included.
full characterization of specimen morphology. Additional Cu plates with appropriate hole sizes may be developed to accommodate specimen-specific electron scattering behavior. Characterization of hard materials ~e.g., metals and ceramics! is limited by weak electron penetration power at 30 kV ~typically the highest accelerating voltage used for SEM!. Nonetheless, this technique may serve as a valuable precursor to subsequent TEM and STEM characterization @e.g., high resolution TEM ~HRTEM!, X-ray analysis, and diffraction studies# for these materials.
C ONCLUSION A new specimen holder has been developed to facilitate BF and DF STEM-IN-SEM imaging. The specimen holder includes a low-angle inclined plate which facilitates DF image formation. DF STEM-IN-SEM images are solely attributed to the electron beam’s interactions with the specimen. No image distortion was observed from pre-specimen electron beam deflection effects. Post-specimen scattering behavior at 30 kV was used to evaluate the underlying contrast mechanism~s! present in DF STEM-IN-SEM images. In general, harder materials display diffraction contrast using 30 kV STEM-IN-SEM due to their smaller lattice spacings whereas soft materials exhibit Z-contrast due to their characteristically larger lattice spacings. The underlying contrast mechanism of electron microscopy images is often of greater importance than the resolution obtained. In this regard, BF and DF STEM-IN-SEM imaging enables intuitive and informative interpretation of specimen morphology.
A CKNOWLEDGMENTS The authors thank Dr. Raymond Pearson from Lehigh University ~Bethlehem, PA! for providing the 10 vol% nanosilicafilled DGEBA epoxy sample and for valuable discussion. In addition, Dr. Gregory Hendricks from the University of
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Vanderlinde, W.E. & Ballarotto, V.W. ~2004!. Microscopy at the nanoscale. In ISTFA 2004: 30th International Symposium for Testing and Failure Analysis, pp. 1–8. Materials Park, OH: ASM International. Volkenandt, T., Müller, E., Hu, D.Z., Schaadt, D.M. & Gerthsen, D. ~2010!. Quantification of sample thickness and inconcentration of InGaAs quantum wells by transmission measurements in a scanning electron microscope. Microsc Microanal 16, 604–613. Woolf, R.J., Joy, D.C. & Tansley, D.W. ~1972!. A transmission stage for the scanning electron microscope. J Phys E Sci Instrum 5, 230–233. Young, R.J. & Lovell, P.A. ~1991!. Introduction to Polymers. London: Chapman & Hall. Zuo, J.M. & Mabon, J.C. ~2004!. Web-based electron microscopy application software: Web-EMAPS. Microsc Microanal 10, 1000– 1001. Available at http://emaps.mrl.uiuc.edu/.
Microsc. Microanal. 20, 124–132, 2014 doi:10.1017/S1431927613014049
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An Inexpensive Approach for Bright-Field and Dark-Field Imaging by Scanning Transmission Electron Microscopy in Scanning Electron Microscopy Binay Patel* and Masashi Watanabe Department of Materials Science and Engineering, Lehigh University, Bethlehem, PA 18015, USA
Abstract: Scanning transmission electron microscopy in scanning electron microscopy ~STEM-in-SEM! is a convenient technique for soft materials characterization. Various specimen-holder geometries and detector arrangements have been used for bright-field ~BF! STEM-in-SEM imaging. In this study, to further the characterization potential of STEM-IN-SEM, a new specimen holder has been developed to facilitate direct detection of BF signals and indirect detection of dark-field ~DF! signals without the need for substantial instrument modification. DF imaging is conducted with the use of a gold ~Au!-coated copper ~Cu! plate attached to the specimen holder which directs highly scattered transmitted electrons to an off-axis yttriumaluminum-garnet ~YAG! detector. A hole in the copper plate allows for BF imaging with a transmission electron ~TE! detector. The inclusion of an Au-coated Cu plate enhanced DF signal intensity. Experiments validating the acquisition of true DF signals revealed that atomic number ~Z! contrast may be achieved for materials with large lattice spacing. However, materials with small lattice spacing still exhibit diffraction contrast effects in this approach. The calculated theoretical fine probe size is 1.8 nm. At 30 kV, in this indirect approach, DF spatial resolution is limited to 3.2 nm as confirmed experimentally. Key words: holder development, soft materials, atomic number contrast ~Z contrast!, polymers, image resolution, SMART macro
I NTR ODUCTION Soft materials characterization ~e.g., of polymers and biological materials! by electron microscopy @e.g., scanning electron microscopy ~SEM!, transmission electron microscopy ~TEM!, and scanning transmission electron microscopy ~STEM!# has been limited due to the lack of long range order in soft materials and their susceptibility to high-voltage electron beams. These challenges present a unique opportunity for innovation in the field of electron microscopy and may require an unconventional, hybrid characterization technique. STEM-IN-SEM ~e.g., Joy & Maher, 1976! may be a convenient characterization approach for these materials. Utilizing the lower accelerating voltages, larger field of view, and exclusion of a post-specimen projection lens in a SEM instrument, STEM-IN-SEM has shown results similar to TEM observation of polymer morphology ~Guise et al., 2011!. Furthermore, comparable image quality has been obtained with bright-field ~BF! STEM-IN-SEM as compared with BF TEM observation of silver nanoparticles ~Vanderlinde & Ballarotto, 2004! and bacteria ~Bogner et al., 2007!. Various specimen-holder geometries and detector arrangements have been used for STEM-IN-SEM imaging but have primarily focused on BF imaging ~e.g., Crawford & Liley, 1970; Woolf et al., 1972; Oho et al., 1987a, 1987b; Vanderlinde & Ballarotto, 2004; Merli & Morandi, 2005; Bogner et al., 2007! while work on DF imaging ~e.g., Crawford & Liley, 1970; Merli & Morandi, 2005; Acevedo-Reyes Received September 18, 2013; accepted December 2, 2013 *Corresponding author. E-mail: [email protected]
et al., 2008; Brodusch et al., 2013! has been limited by technology ~until recently! or by the costly acquisition of new instrumentation ~e.g., detectors!. STEM-IN-SEM imaging has been applied to morphological observation of various polymer-based latex particles ~Geng et al., 2013!, mask metrology ~Klein et al., 2012!, X-ray elemental mapping ~Stokes & Baken, 2007!, X-ray spectral imaging of thin specimens prepared by a focused ion beam method ~Kotula, 2009!, particle-size distribution measurements both in BF ~Klein et al., 2011! and DF ~with a dedicated detector! ~Acevedo-Reyes et al., 2008!, as well as extreme high resolution SEM, in which a monochromator and a 12-segment STEM detector provide the potential for sub-nanometer spatial resolution ~Roussel et al., 2009!. BF images of electron transparent specimens mainly display mass-thickness contrast. While such images may be used for particle-size and distribution analysis, these images do not provide intuitive image interpretation especially when investigating novel or unknown specimens. Conversely, the detection of DF signals, which correspond to the atomic number of constituent elements, would be advantageous to form atomic-number contrast ~so called Z-contrast! images when electrons scattered with higher angles are collected. Therefore, the goal of this study is to advance the potential of soft materials characterization through STEMIN-SEM by developing a method for the acquisition of BF and DF STEM-IN-SEM images with fabrication of a newly designed specimen holder. The new holder will facilitate BF and DF STEM-IN-SEM imaging without requiring costly modifications to an existing SEM instrument.
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Configuration of Fine Probe Settings All BF and DF STEM-IN-SEM images were collected in a Hitachi 4300 SEM instrument ~Hitachi, Hitachinaka, Japan! equipped with a Schottky field-emission gun, at an accelerating voltage of 30 kV ~the maximum for this instrument!. In this study, two detectors were used for BF and DF STEM-IN-SEM imaging in the SEM instrument: one is a TE detector located below the specimen stage for BF imaging and the other is a YAG detector located off-axis for DF imaging ~which is usually used for backscatter electron imaging!. Both detectors came standard when this SEM was acquired by Lehigh University. Based on the instrument configurations, the convergence semi-angle to form the minimum probe size at 30 kV was estimated as 3.2 mrad resulting in an estimated minimum probe size as fine as 1.8 nm, following a procedure outlined in Goldstein et al. ~2003!. This convergence semi-angle is achieved at a working distance of 4.6 mm in the Hitachi 4300. The spatial resolution of DF STEM-IN-SEM imaging is evaluated through image analysis from a standard combined test specimen for TEM ~Ted Pella, Redding, CA, USA!, consisting of small Au nanoparticles with a light deposition of graphitized carbon on a perforated carbon film, using the scanning microscope assessment and resolution testing ~SMART! macro ~Joy, 2002! with the Scion Image ~Scion Corporation, Frederick, MD, USA! software package. Previously, the manual procedure for SMART macro and its limitations ~e.g., partial user dependence! for resolution testing of STEM-IN-SEM images was discussed ~Probst et al., 2007!. Although, user dependence is an issue with manual SMART macro analysis, overall this method is far superior to line profile analysis ~through the measurement of the minimum distance between peaks that correspond to actual points in an image! for image resolution analysis. The only deviation here from the SMART procedure previously outlined is the recording of image resolution measurements from 256 ⫻ 256 pixel areas from five DF images. The Au standard combined test specimen is commonly used for resolution evaluation for TEM and STEM imaging.
Holder Design The new specimen holder ~Fig. 1! is made of aluminum and includes ~i! an entrenched flat housing for the specimen to reside, ~ii! a long vertical column that acts as structural support for the specimen housing and ~iii! a base designed to fit into the stage locking mechanism of the SEM instrument. The holder features an Au-coated Cu plate with a center hole and a fixed geometry. The Cu plate is positioned below the specimen housing and facilitates the direct detection of BF signals and the indirect detection of DF signals. Conductive graphite paste is used on the exteriors of both the holder body and the Cu plate to minimize unnecessary electron emission from these parts. BF and DF STEM-IN-SEM signal acquisition is achieved as follows ~shown schematically in Fig. 2!: The incident
Figure 1. A new specimen holder for BF and DF STEM-IN-SEM imaging in the Hitachi 4300SE.
Figure 2. Schematic diagram showing the optimal microscope configuration for BF and DF STEM-IN-SEM imaging in the Hitachi 4300SE.
electron beam interacts with an electron-transparent specimen. Subsequently, transmitted electrons leaving the specimen are collected differently depending on their scattering angle. The scattering of transmitted electrons is completely dependent on the elements from the specimen of interest, their composition and crystallographic orientations ~if they are crystalline materials!. Transmitted electrons that are scattered at low-angles travel through the center hole in the Au-coated Cu plate and are collected by an on-axis TE detector. The resultant BF STEM-IN-SEM images mainly exhibit mass-thickness contrast and/or diffraction contrast ~when crystalline materials are observed!. Transmitted electrons that are scattered at high-angles reach the Au-coated Cu plate and are backscattered from it toward an off-axis YAG detector. Finally, these transmitted @and subsequently backscattered electrons ~BSE!# are collected by the YAG detector for DF image formation. The DF STEM-IN-SEM images exhibit either diffraction contrast or Z-contrast depending on the type of specimen under investigation ~as described later, the contrast formation is dependent on the characteristic crystallographic orientation and resultant scat-
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tering behavior of a specimen!. This hybrid detection design makes efficient use of the detector configurations available on the Hitachi 4300 at Lehigh University ~and many other conventional SEM instruments as well!.
Optimization of DF Imaging Prior to fabrication of the holder, Monte Carlo simulation using the CASINO simulation package ~Drouin et al., 2007! was conducted to optimize the indirect detection method for DF STEM-IN-SEM imaging by utilizing the fine probe settings discussed above. First, to ensure significant electron signal collection for DF image formation, the minimum Au-coating thickness required for efficient electron backscattering off of the Cu plate surface was determined to be 600 nm. This coating thickness corresponds to achieving the maximum BSE yield of Au, as compiled by Joy ~Joy’s database contained in Goldstein et al., 2003!, without penetrating the coating layer. Second, the influence of Cu plate orientation on electron backscattering was studied. Monte Carlo simulations were performed on a Cu plate with 600 nm of Au-coating. In the simulations, the Cu plate remained stationary and interacted with electron beams tilted at 0, 30, 60, and 808 relative to the surface of the plate, which is geometrically equivalent to changing the Cu plate orientation against a normal electron beam. As the electron beam tilt angle increases, the signal generation also increases but is oriented toward higher backscattered directions, which agrees with experimental data on the forwardscattering of 30 kV electrons from an Au surface ~Darlin´ski, 1981!. Based on the simulation, an exaggerated Au-coating thickness of 1.5 mm was used to ensure sufficient electron backscattering for the holder design in this study. In addition, a center hole with a diameter of 2 mm was created on the Au-coated Cu plate corresponding to 55 mrad in this geometry at 30 kV. The use of a center hole is essentially equivalent to an inner angle of an annular DF detector, i.e., the hole serves as the inner cut-off angle for DF imaging. The measured signal intensities from BF and DF STEMIN-SEM images were used to determine the optimal Cu plate orientation for DF signal generation. Flat-polished Cu plates were first coated with gold to a thickness of 1.5 mm and then bent to inclination angles of 0, 10, 20, 40, and 808. The Au-standard combined test specimen was used for imaging. Five BF and DF STEM-IN-SEM images were recorded at a magnification of 150,000⫻ using each flatpolished plate and with no plate present, to serve as a control. The measured signal intensity from each image was determined using Image J software ~Schneider et al., 2012!.
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Optimization of BF and DF Signal Collection For this holder configuration to achieve efficient BF and DF STEM-IN-SEM imaging, optimizing the inclination angle of the Cu plate is critical. Figure 3 shows a BF and DF STEM-IN-SEM image pair of the Au standard with a Cu
Figure 3. A pair of BF ~left! and DF ~right! STEM-IN-SEM images of Au standard combined test specimen using the 108 Aucoated Cu plate.
Figure 4. Measured BF STEM-IN-SEM signal intensity as a function of Cu plate inclination angle. Also included is a reference value obtained without the Cu plate.
plate angle of 108. The Au nanoparticles appear dark in the BF image ~left! and bright in the DF image ~right!. Five DF STEM-IN-SEM images of the Au nanoparticles, collected with a Cu plate angle of 108 ~selected as discussed below!, were used to calculate a DF spatial resolution of 3.2 nm by SMART macro analysis for this indirect approach at 30 kV. There was no deviation in the DF spatial resolution observed from measurements collected from the five DF STEMIN-SEM images because consistent thresholding, through user-control, was applied to each image during the SMART macro analysis. Furthermore, from such images obtained with different plate inclination angle settings, signal intensities were evaluated. Figure 4 shows the measured BF signal intensity in gray level ~GL! plotted against the inclination angle of the Cu plate. Since the BF signals were collected by the TE detector below the holder, BF images can be obtained without the Cu plate. For reference, the measured average BF intensity was 219 6 2.3 GL without the plate present ~shown in Fig. 4 as reference!. As shown in Figure 4, the inclusion of the plate slightly reduces the BF signals especially for inclination angles ranging from 0 to 408. Yet, no significant difference was observed in the BF signals which fluctuated from 202 6 5.1 to 220 6 3.1 GL within the range of inclination angles tested. In fact, the difference in the measured BF
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Figure 5. Measured DF STEM-IN-SEM signal intensity as a function of Cu plate inclination angle. Also included is a reference value obtained without the Cu plate.
signals is below 20 GL, which is not visually noticeable. Thus the addition of the Cu plate with the hole of 55 mrad does not disturb BF signal collection through the TE detector within an inclination angle range of 0–808. Similar experiments were also performed for DF STEMIN-SEM imaging ~Fig. 5!. The measured DF signals were the lowest when using no plate and an 808 inclined Cu plate, producing 58 6 0.1 and 63 6 0.6 GL, respectively. When no plate is present ~shown as reference in Fig. 5!, the DF signal collection is drastically lowered because transmitted electrons are not backscattered on surfaces of the holder due to the low atomic number graphite coating ~i.e., no effective backscattering toward the YAG detector!. When the 808 inclined plate is used, the BSE on the plate for DF imaging are no longer optimally oriented toward the YAG detector in the Hitachi 4300SE. Conversely, the use of plates with low inclination angles leads to nearly double the measured DF signals compared with the absence of a plate ~averaging 105 6 1.2 GL!. In addition, the measured DF signal does not change significantly between inclination angles of 0–408. BSE from the plate have an unperturbed path to the YAG detector and small variations in inclination angle do not actually influence DF image formation if the plate angle is set between 0 and 408. In this study, the 108 inclined plate was selected for BF and DF STEM-IN-SEM imaging but any plate inclined ,408 may be used on the Hitachi 4300SE microscope to produce similar results. Figure 6 shows a BF and DF STEMIN-SEM image pair of unstained 10 vol% Nanosilica-filled Diglycidly Ether of Bisphenol A ~DGEBA! epoxy. The sample was cryo-microtomed to ;100 nm in thickness. While the DF image is noisy, it highlights the sensitivity of this approach for imaging low atomic number differences in soft materials. Indeed, DF signals in the discrete nanosilica particles ~effective Z ⫽ 10! are higher than those in the epoxy matrix ~effective Z ⫽ 3.7! in this uniformly thick specimen.
Validation of DF Signal Acquisition In order to determine if this DF STEM-IN-SEM approach produces images that may be interpreted in the same manner as high-angle annular DF ~HAADF! STEM images three critical factors must be addressed: ~i! the influence of the SEM instrument, particularly it is scanning system, on
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Figure 6. A pair of BF ~left! and DF ~right! STEM-IN-SEM images of unstained 10 vol% nanosilica-filled DGEBA epoxy using the 108 Au-coated Cu plate.
image interpretability, ~ii! the origin of DF signal based on electron-beam specimen interactions, and ~iii! the underlying contrast mechanism~s! present in DF images based on the new holder design. Influence of the Scanning Electron Beam on STEM-IN-SEM Imaging In dedicated STEM instruments, a scanning system consists of double deflection coils, which ensure that the electron beam remains parallel to the optic axis of the instrument ~i.e., normal to a nontilted specimen! during imaging. Conversely, only one deflection coil is used for scanning in conventional SEM instruments. This may cause abnormal interaction between the electron beam and a nontilted specimen and may also shift the electron beam off-axis, especially when a large field of view needs to be scanned, i.e. at low magnifications. The tilted incident beam may promote severe image distortion and uneven signal distribution, etc., making it nearly impossible to ascertain the underlying contrast mechanism~s! present in DF STEM-INSEM images. To evaluate the influence of SEM scanning on STEMIN-SEM imaging, a series of BF images of a polystyrene latex standard @particle size 0.3 mm ~6 0.01!# ~Ted Pella! were recorded at magnifications ranging from 10,000 to 300,000⫻. One droplet of polystyrene latex particles in solution was placed on a standard holey carbon TEM grid for use during imaging. The average diameter of the polystyrene latex particles was measured, using Image J software ~Schneider et al., 2012! from each image and compared with the known particle size distribution. Any significant deviation in the average particle size measurements would suggest that the deflection of electron beam to off-axis is no longer negligible in image formation. BF images of polystyrene latex particles ~such as the one shown in Fig. 7A! over a range of magnifications did not yield statistically significant image distortion ~Fig. 7B!. The average particle diameter at each magnification was within the nominal particle size distribution provided by the supplier. Therefore, the deflection present in the scanning electron beam is small enough that image distortion does not occur within the magnification range covered in
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Figure 7. Polystyrene Latex Particles. A: BF STEM-IN-SEM image showing average particle diameter. B: Variation in average particle diameter with imaging magnification.
Figure 8. A pair of BF ~left! and DF ~right! STEM-IN-SEM images of Au standard combined test specimen obtained at a working distance of 4.5 mm with signal intensity plot profile shown ~bottom! with particular emphasis on the copper grid and hole regions.
this study. The absence of image distortion suggests that the electron beam still remains close to parallel to the optic axis. Therefore, the resultant scattering of electrons transmitted through the specimen after electron beam–specimen interactions may be directly attributed to the specimen itself. Authentication of DF Signal In the imaging method outlined, DF STEM-IN-SEM imaging is conducted by an off-axis YAG detector, which is usually used for BSE imaging. BSE images are generated directly from the primary electron beam by interaction with a specimen at its surface ~or near sub-surface! ~Goldstein et al., 2003! whereas DF images are generated from highly scattered transmitted electrons after interaction with the specimen. To investigate the origin of the DF STEM-INSEM signals in the instrument/holder geometry proposed in this study two experiments were conducted. First, BF and DF STEM-IN-SEM images were recorded from the same field of view on the Au standard combined test specimen at two different working distances: A shorter working distance of 4.5 mm ~optimal for STEM-IN-SEM imaging, where the specimen is positioned above the YAG detector! and a longer working distance of 13.5 mm ~optimal for BSE imaging, where the specimen is positioned below the YAG detector!. Figures 8 and 9 show two pairs of
BF and DF STEM-IN-SEM images from the combined test specimen with the Cu grid bar, recorded at the optimum and longer working distances, respectively. The contrast mechanisms between each pair of images are compared based on the average measured signal intensity from sections of the Cu grid on which the Au standard specimen resides. Image analysis was conducted using Gatan Digital Micrograph ~Gatan Inc., Pleasanton, CA, USA!. The Cu grid regions on traditional TEM grids are too thick for electron transparency. When the shorter working distance, optimized for the STEM-IN-SEM approach, was chosen the measured DF signal intensity of Cu grid bar region is 24.1 6 1.4 GL in average. Conversely, the DF signal intensity is 84.7 6 8.2 GL at the same region with the longer working distance. Since the YAG detector is located above the specimen in this case, the higher signal intensity must include BSE signals from the grid bar. In the field of view shown in Figures 8 and 9, holes can also be found in the carbon support film. The average DF signal intensities from one of the holes is 24.0 6 1.2 GL for the optimum and 24.3 6 1.0 GL for the longer working distance, respectively. Ideally, holes in the specimen should display neither DF signals nor BSE signals since no scattering occurs from such regions. As shown in Figure 8, the DF signals exhibit exactly the same level from the Cu grid bars
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Figure 9. A pair of BF ~left! and DF ~right! STEM-IN-SEM images of an Au standard specimen obtained at a working distance of 13.5 mm with signal intensity plot profile shown ~bottom! with particular emphasis on the copper grid and hole regions.
and the hole, which definitely proves that no signal from these regions is detected from electrons transmitted through the thin specimen using the off-axis YAG detector, at least when the working distance is set at 4.5 mm. Possible reasons for the nonzero DF signals from the Cu grid bars and from the hole may be from either spurious electrons such as SE III ~Goldstein et al., 2003! or detector “dark current.” To confirm the origin of DF signals, a second set of experiments was performed. First, BF and DF STEM-IN-SEM images were collected from a large hole in the Au standard specimen with the optimum working distance of 4.5 mm at a low magnification of 3,000⫻. Next, the magnification was increased until both the BF and DF imaging scans were completely inside the hole of interest, which means that there is no electron scattering event with the specimen within the field of view. Then, BF and DF STEM-IN-SEM images were taken inside the hole of interest at a magnification of 20,000⫻ by blocking the incident electron beam with an objective aperture ~located above the specimen!. Figure 10 shows two pairs of BF and DF STEM-INSEM images ~a! with and ~b! without the electron beam. Measured DF signal from the hole with the electron beam is 55.2 6 2.1 GL, which is compatible with those without the electron beam ~54.7 6 2.4 GL!. Since the electron beam was not present in the latter image ~Fig. 10B! the minimal measured signal intensity observed in DF STEM-IN-SEM images is not attributed to electron beam–specimen interactions and to any spurious electrons in the column. Therefore, the source of the measured signal intensity is attributed to the “dark current” present in the YAG detector. The YAG detector used for DF STEM-IN-SEM imaging has no voltage bias applied to it. Thus, no electrons are attracted to the detector. Only electrons oriented toward the detector take part in image formation. Since electron signal acquisition may be low upon conversion of the electron signal to a photon signal for digital imaging, high amplification is required. The minimum measured signal intensity observed
is the result of electronic noise, or “dark current”, as a result of the amplification process. It should be noted that the dark-current levels are different between Figures 8 and 10. This is simply due to the difference in the acquisition time. In comparison, the TE detector used for BF STEM-INSEM imaging has a small positive voltage bias applied which attracts more electrons to participate in image formation. Thus, upon conversion of the electron signal to a photon signal for digital imaging, amplification of the electron signal is not as intense as in the YAG detector. As a result, electronic noise from the TE detector ~0.00 6 0.0 GL, Fig. 10B! is significantly lower than in the YAG detector. Determination of DF Signal Contrast Mechanism DF images may exhibit either diffraction contrast or Z-contrast, depending on the collection semi-angle selected for imaging. Diffraction contrast involves coherent electron scattering, where the phase relationships between atomic lattices lead to contrast variations which make image interpretation difficult if crystalline materials are being characterized. However, Z-contrast involves incoherent electron scattering, where only highly scattered electrons that have no phase relationship between atomic lattices are detected. Thus, the contrast variation in Z-contrast images is directly related to the atomic number of atoms in the specimen, which makes image interpretation much more straightforward ~Nellist & Pennycook, 2000!. To determine the contrast mechanism for DF STEM-IN-SEM images the minimum collection semi-angle for Z-contrast is calculated using the following relation as described by Volkenandt et al. ~2010!: bmin ⫽ 2 arcsin~bM l/d!,
~1!
where b ⫽ 0.61, l is the wavelength of the electrons, and d is the atomic spacing along the lattice. The minimum collection semi-angle is evaluated for 30 kV electrons. The lattice spacings for four materials @Au, silica ~SiO2 !, polyethylene ~PE!, and PS# are chosen to illustrate the role of the mini-
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Figure 10. A pair of BF ~left! and DF ~right! STEM-IN-SEM images of a hole in the Au standard specimen. ~A! at 3,000⫻ magnification and with an unblocked electron beam and ~B! at 20,000⫻ magnification with a blocked electron beam. Signal intensity plots profiles ~bottom! in both images emphasis the hole regions.
mum collection semi-angle on image contrast. The lattice spacings for gold in the @111# direction and SiO2 in the @100# direction were obtained from Web Electron Microscopy Applications Software ~WebEMAPS! ~Zuo & Mabon, 2004!. Owing to the noncubic crystal structures of PE ~orthorhombic! and PS ~trigonal! in their nearly crystalline states, average lattice spacings are used, as obtained from Young and Lovell ~1991!. An analysis of the scattering angles of transmitted electrons at 30 kV revealed that harder materials ~e.g., Au, SiO2 !, with characteristically shorter lattice spacing, still exhibit diffraction contrast effects during DF STEM-INSEM imaging under the current geometrical set up ~Fig. 11!. In fact, any material with lattice spacings smaller than 8.8 Å, corresponding to the 55 mrad cut-off angle on the Cu plate, will exhibit some diffraction contrast effects in DF STEMIN-SEM imaging. Under the current configuration of the Cu-plate, the simplest crystallizable polymer structure, PE ~ bmin ⫽ 73 mrad in the orthorhombic structure!, does not exhibit pure Z-contrast during DF imaging. However, many soft materi-
als, specifically nearly crystalline polymer systems ~e.g., PS, bmin ⫽ 40 mrad in the trigonal structure!, with characteristically larger lattice spacing, may exhibit Z-contrast through DF STEM-IN-SEM imaging under this approach. These larger lattice spacings of polymers are a consequence of the increased functionality of side groups as these polymers become more complex ~Young & Lovell, 1991!. As polymers are generally amorphous, lacking long range order, their effective lattice spacings are further enlarged and diffraction contrast effects are further reduced ~i.e., bmin decreases!. Thus, such polymer systems ~e.g., PS latex particles and 10 vol% nanosilica-filled DGEBA epoxy! do exhibit Z-contrast through DF STEM-IN-SEM imaging under this approach.
Applications and Limitations of BF and DF STEM-IN-SEM Imaging This technique may be used both in industry and academia. The breadth of information available from BF and DF STEM-IN-SEM images may prove as a suitable alternative to TEM characterization of materials. Specifically for soft materials, this technique may be a cost-efficient method for
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Massachusetts Medical School ~Worchester, MA! is acknowledged for ultramicrotoming the 10 vol% nanosilica-filled DGEBA epoxy sample. Finally, the authors thank Mr. William Mushock and Dr. Robert Keyes from Lehigh University ~Bethlehem, PA! for value discussion during this study.
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Figure 11. Minimum collection semi-angle ~ bmin ! for achieving Z-contrast during DF imaging ~dashed line! with reference to the cut-off angle on Cu plate ~full line!, plotted against lattice spacing. Lattice spacings and corresponding collection semi-angles for select materials are included.
full characterization of specimen morphology. Additional Cu plates with appropriate hole sizes may be developed to accommodate specimen-specific electron scattering behavior. Characterization of hard materials ~e.g., metals and ceramics! is limited by weak electron penetration power at 30 kV ~typically the highest accelerating voltage used for SEM!. Nonetheless, this technique may serve as a valuable precursor to subsequent TEM and STEM characterization @e.g., high resolution TEM ~HRTEM!, X-ray analysis, and diffraction studies# for these materials.
C ONCLUSION A new specimen holder has been developed to facilitate BF and DF STEM-IN-SEM imaging. The specimen holder includes a low-angle inclined plate which facilitates DF image formation. DF STEM-IN-SEM images are solely attributed to the electron beam’s interactions with the specimen. No image distortion was observed from pre-specimen electron beam deflection effects. Post-specimen scattering behavior at 30 kV was used to evaluate the underlying contrast mechanism~s! present in DF STEM-IN-SEM images. In general, harder materials display diffraction contrast using 30 kV STEM-IN-SEM due to their smaller lattice spacings whereas soft materials exhibit Z-contrast due to their characteristically larger lattice spacings. The underlying contrast mechanism of electron microscopy images is often of greater importance than the resolution obtained. In this regard, BF and DF STEM-IN-SEM imaging enables intuitive and informative interpretation of specimen morphology.
A CKNOWLEDGMENTS The authors thank Dr. Raymond Pearson from Lehigh University ~Bethlehem, PA! for providing the 10 vol% nanosilicafilled DGEBA epoxy sample and for valuable discussion. In addition, Dr. Gregory Hendricks from the University of
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