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6 Progress in Electron Microscopy

6 Progress in Electron Microscopy

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Progress in Electron Microscopy




Fig. 2.25 (a) Schematic of spherical aberration of a converging lens. The scattering power

increases with increasing angle (with respect to the optical axis) at which the electrons enter the

lens. As a result, the electrons passing the lens at its periphery are focused at a distance in front of

the image that is defined by low-angle beams. The image of a point P of the specimen is broadened into a “point-spread disk” of radius R (red). (b) Aberration correction. Spherical aberration is

compensated by combining the converging lens with a suitable diverging lens. In electron optics,

diverging lenses are realized by combinations of multipole lenses. (Reprinted with permission from

[2.12]. © 2008 AAAS)

in the exit plane wave function (EPWF). This can be derived by focus-variation

techniques (see [2.12]) where EPWF is calculated by fitting model functions to a

series of images taken under different defocus values. Then the EPWF is calculated

for a model structure of the sample which is iteratively improved to obtain the best

fit between the calculated and experimental EPWFs. The result is generically not an

image in the conventional sense but a computer model of the structure that gives the

atomic species and coordinates.

Theoretically, the optimum imaging contrast is achieved by combining a small

defocus with a small negative value of the spherical-aberration parameter Cs

(see [2.12]). Under these negative spherical-aberration imaging (NCSI) conditions,

atoms appear bright on a dark background. The strong contrast achieved by NCSI,

which makes few-electron atoms such as oxygen, nitrogen, or even boron visible, is

considered to be one of the major advances of spherical-aberration correction (see

[2.12]). In a

3 {111} twin boundary of BaTiO3 (Fig. 2.26), all the atom species

including oxygen (arrows) can be identified. By evaluating the local intensity signal,



Microscopy – Nanoscopy

Fig. 2.26

3 {111} twin boundary in BaTiO3 . All atomic species, including oxygen, can be

identified as confirmed by image simulation. Quantitative site-occupation measurements derived

from the local intensity values indicate that in the interfacial oxygen columns only about 40–70%

of the sites are occupied. This provides evidence for oxygen deficiency, which is presumed to have

a strong influence on the electronic properties of interfaces. (Reprinted with permission from [2.12,

2.73]. © 2008 AAAS)

the oxygen occupancies (see upper part of Fig. 2.26) in the boundary can be determined to 40–70% of the bulk value, providing direct evidence of oxygen deficiency

in the boundary.

The resolution of aberration-corrected 300 keV electron microscopes is about

0.08 nm. A resolution of 0.05 nm is achieved in the transmission electron aberrationcorrected microscope (TEAM; [2.12] and Fig. 5.43). The TEAM instrument offers

both TEM and STEM operation, a monochromator in the electron-beam forming system and a future additional corrector for chromatic aberration (see [2.12,

2.13]). The accuracy at which the separation of well-isolated atoms can be measured is with about 0.006 nm much better than the resolution (see [2.12]). This

accuracy was demonstrated in a study of polarization domain walls in ferroelectric

Pb(Zr0.2 Ti0.8 )O3 (PZT) (Fig. 2.27). The high-precision measurement of the shifts

of the individual atoms enables the derivation of the local polarization vector and

thereby the determination of the structure of the domain wall.

In STEM, the prevailing imaging mode is high-angle annular dark-field

(HAADF; see [2.12]), where the contrast arises from incoherent scattering intensity which can be understood directly in terms of the atomic number. The HAADF

mode is also referred to as Z-contrast imaging. In imaging a silicon crystal along a

[112] direction by aberration-corrected STEM the atom-pair separation of 0.078 nm

can be clearly resolved (Fig. 2.28a). The low-angle scattered electrons passing the


Progress in Electron Microscopy


Fig. 2.27 (a) Transversal inversion polarization domain wall in ferroelectric Pb(Zr0.2 Ti0.8 )O3

(PZT). Arrows give the direction of the spontaneous polarization, which can be deduced from

the local atomic displacements. The shifts of the oxygen atoms (blue circles) out of the Ti/Zr atom

rows (red circles) can be seen. (b) Atomic resolution measurements of the shifts of oxygen (δ O ),

and titanium/zirconium (δTi/Zr ) atoms in a longitudinal inversion domain wale and the value of

the local polarization P that can be calculated from theses data [2.12, 2.74, 2.75]. (Reprinted with

permission from [2.74]. © 2009 Nature Publishing Group)

annular HAADF detector can be used for electron-energy-loss spectroscopy (EELS)

providing an analysis of the electronic structure with atomic-scale resolution (see

Fig. 2.29 and [2.79, 2.80]). In Fig. 2.28b the atomic resolution EELS spectrum of

a La0.7 Sr0.3 MnO3 /SrTiO3 multilayer is imaged. A spectral electron-energy resolution of 55 meV has been achieved at the sub-electron volt sub-Ångström microscope

(SESAM) which can be exploited to study changes in the width of the local

electron-energy band gap near interfaces in semiconductor nanodevices (see [2.12]).



Microscopy – Nanoscopy

Fig. 2.28 (a) High-angle annular dark-field (HAADF) image in scanning transmission electron microscopy (STEM), of Si along a [112] direction. The atom-pair separation of 0.078 nm

is clearly resolved in the 300 keV instrument [2.12, 2.76]. (b) Spectroscopic imaging of a

La0.7 Sr0.3 MnO3 /SrTiO3 multilayer showing the different chemical sub-layers in a 64 ×-64 pixel

spectrum extracted from 650 eV wide electron-energy-loss spectroscopy (EELS) data recorded at

each pixel. Red (Mn-L edge)-green (La–M edge)-blue (Ti-L edge) false-color image showing the

Mn, La, and Ti sub-lattices as derived from the EELS data. The purple color at the interface indicates Mn–Ti intermixing at the Ti sub-lattice [2.12, 2.77]. (Reprinted with permission from [2.12].

© 2008 AAAS)

Fig. 2.29 Spectroscopic imaging of a single La atom inside CaTiO3 by an aberration-corrected

scanning transmission electron microscope with electron energy loss spectroscopy (EELS). (a)

Z-contrast image with (b) EELS traces showing spectroscopic identification of a single La atom

of atomic resolution with the same beam used for imaging [2.78]. The characteristic M4,5 lines

of La are seen strongly in spectrum 3. (Reprinted with permission from [2.78]. © 2004 American

Physical Society)


Progress in Electron Microscopy


2.6.2 TEM Nanotomography and Holography

For high-resolution (∼13 nm3 ) 3D tomographic imaging in TEM, images are

recorded every one or two degrees about a tilt axis. Typically, the ensemble of

images is then “back-projected” to form a 3D reconstruction. The STEM HAADF

signal is most appropriate for tomographic applications and is also sensitive to

changes in composition (see [2.14, 2.81, 2.82]) because it is approximately proportional to Z1.8 , where Z is the atomic number. STEM tomography can be used to

relate the distribution of ruthenium-platinum catalyst nanoparticles to the underlying surface curvature (see Fig. 2.30a), showing in this case the strong preference of

the particles (red) for saddle-shaped anchor points. 3D compositional information

can be extracted from energy-filtered transmission electron micrograph (EF-TEM)

series. Electron tomography can also be used for studying dislocation networks

and EF-TEM tomography is useful for the investigation of precipitates in alloys

(see [2.14]). True atomic resolution tomography appears to be possible by using

aberration-corrected TEM or STEM instruments (see [2.14]).

Electron holography [2.83], which is based on the formation of an interference

pattern in the TEM, overcomes the TEM limitations of using only intensities in

imaging. By contrast, holography allows the phase shift of the electron wave to be

recovered (see [2.14]). As the phase shift is sensitive to local variations in magnetic and electrostatic potential, the technique can be used to obtain quantitative

information about magnetic and electric fields in materials and devices with spatial resolution that can approach the nanometer scale. In the TEM off-axis electron

holography mode (Fig. 2.30b), a field-emission electron gun is used to provide

a highly coherent source of electrons. By electron holography, magnetic fields

in fine-particle recording media, flux vortices in superconductors, the Aharonov–

Bohm effect (see [2.14]), or spontaneous polarization [2.84] have been observed.

In Fig. 2.30c the holographic imaging of the magnetic interaction of closely spaced

cobalt nanoparticles is shown. The images indicate an approximately 50:50 mixture of clockwise and anticlockwise ground-state configurations to which the rings

relax after exposure to a magnetic field (see [2.14]). Electron holography can also

provide quantitative information about electrostatic potentials in doped semiconductors and in ferroelectrics (see [2.14]). In a silicon p-n junction (Fig. 2.30d) the

p-type and n-type regions are delineated clearly as darker and lighter holographic

contrast, respectively.

An exciting prospect is the combination of electron holography with electron

tomography to characterize electrostatic and magnetic fields inside nanostructured

materials with nanometer spatial resolution in three dimensions (see [2.14]).

2.6.3 Cryoelectron Microscopy and Tomography

Cryoelectron microscopy is a technique in nanobiology for imaging single

organelles (∼5 nm) in cells such as the protein-packaging Golgi apparatus or

energy producing mitochondria [2.85, 2.86] for a better understanding of their



Microscopy – Nanoscopy

Fig. 2.30 (a) Electron tomography for reconstruction of a heterogeneous catalyst. Ruthenium–

platinum nanoparticles on disordered mesoporous silica. The surface has been color-coded

according to the curvature of the surface. The nanoparticles (red) appear to prefer to anchor

themselves at the (blue) saddle points (width 20 nm) [2.14]. (b) Schematic of electron holography microscopy. A voltage is applied to an electron biprism located close to a conjugate image

plane in a field-emission electron gun (FEG) TEM, to overlap a vacuum reference electron wave

that has passed through a region of the specimen to form an off-axis electron hologram. Variations

in the spacing and direction of the holographic interference fringes contain information about the

projected magnetic flux density inside and outside the crystals. (c) Magnetic phase contours formed

by the magnetic contributions to the electron phase shift measured on a cobalt nanoparticle ring

in magnetic-field-free conditions. The magnetization of the nanoparticles is indicated by arrows

[2.14]. (d) Representative electrostatic phase image reconstructed from an off-axis electron hologram of a silicon p–n junction sample. (Reprinted with permission from [2.14]. © 2009 Nature

Publishing Group)


Progress in Electron Microscopy


functionalities. For this purpose the cells are flash-frozen (105 K/s) to, e.g., 4.2 K.

They then do not need to be fixed or have their membranes disrupted and additionally can withstand a ten times higher electron dose for imaging (6500 e- nm2 [2.87])

than at ambient temperatures. By the electron beam, 2D projection slices of the

particle are imaged with subsequent processing to a 3D image by computer back

projection routines. A few examples of cell components imaged by cryoelectron

microscopy are shown in the following.

In Fig. 2.31a stereoview of the human SF3b splicing factor complex imaged by

cryoelectron tomography is given at a resolution of less than 1 nm [2.88]. The splicing factor SF3b with a mass of ∼450 kDa consists of seven proteins and is essential

for the accurate excision of introns (segments of a gene that do not function in coding for protein synthesis) from pre-messenger RNA. The SF3b factor is involved in

the recognition of the pre-messenger RNA’s branch site and is required for the premessenger RNA splicing [2.88]. 3D reconstruction of SF3b reveals the architecture

of an envelope (wall thickness ∼2.5 nm) enclosing a large inner cavity containing

a single density element (yellow) like a pearl in an oyster (see Fig. 2.31a) and the

protrusions (see Fig. 2.31b) might be identified as possible RNA-recognition motif

(RRM) domains.

Microtubule doublets (see Fig. 2.32) are structural components of axonemes

which form the core of eukaryotic flagella and cilia, e.g., in sea urchin sperm.

The axoneme is one of the largest macromolecular machines. The structure of the

microtubule doublet derived from cryoelectron tomography provides insight into

tubulin–protein interaction and into the functions doublets perform in axoneme

machines, and may be used as basis for quantitative modeling of the mechanical

properties [2.87].


Fig. 2.31 (a) Stereoview of the SF3b complex by cryoelectron tomography. The SF3b155 proteins

are shown in rainbow colors, the two RNA recognition motifs (RRM) of SF3b49 protein in green

color, and the p14 RRM in the center of the complex in yellow. The potential hinge region in the

SF3b155 protein is indicated by asterisks. Scale bar, 2 nm. (Reprinted with permission from [2.88].

© 2003 AAAS)



Microscopy – Nanoscopy



Fig. 2.32 Electron tomography of microtubule doublets. (a) Protofilaments are well resolved in

the reconstruction, roughly parallel to the doublets. (b) A projection of a 26 nm thick cross-section

along the white line in (a). The field includes five complete and two broken doublets (black arrows

at missing proto filaments). A partially disassembled singlet microtubule is designated by a white

arrow. (Reprinted with permission from [2.87]. © 2006 Nature Publishing Group)

Fig. 2.33 A voxel (volume

pixel) projection of ribosomes

in the nematode C. elegans

from EF-TEM tomography

reconstructed from maps of

the characteristic electron

energy losses of phosphorus;

the ribosomes show no

contrast in a bright field (BF)

TEM image [2.89, 2.90].

(Reprinted with permission

from [2.90]. © 2004 Elsevier)

Tomography of energy-filtered transmission electron microscopy (EF-TEM) was

employed to reconstruct the ribosomes of the Caenorhabditis elegans nematode

worm by specifically detecting the P atoms in the ribosomes (Fig. 2.33) which are

particles composed of RNA and protein found in the cytoplasma of living cells and

which are active in the synthesis of proteins.

2.7 X-Ray Microscopy

X-ray microscopy in some sense fills the “resolution gap” between light microscopy

and electron microscopy [2.91]. Fresnel zone plate lenses, which focus light

by diffraction, or mirrors are used as optical elements for imaging (see [2.92,

2.93]). This type of microscopes successfully employs near-edge x-ray absorption


X-Ray Microscopy


fine-structure (NEXAFS) for the analysis of organic and magnetic materials [2.15].

X-ray nanotomography is applied in materials science [2.94] and biology [2.95,

2.96]. Spatial resolution down to 5 nm has been demonstrated by lens-less coherent x-ray diffraction imaging [2.91, 2.97, 2.98] with prospects of atomic resolution

imaging [2.97]. The intense, brief pulses of x-rays from upcoming free-electron

lasers (see [2.16]) will greatly extend x-ray microscopy to the femto-second time

domain and to interatomic length scales [2.17, 2.99] enabling the analysis of free

single molecules.

2.7.1 Lens-Based X-Ray Microscopy

Here, commonly Fresnel zone plates (FZPs; Fig. 2.34a) are used to condense the

beam unto the sample and then to use a micro FZP as a high-resolution objective

to produce a magnified full-field image of the object. Zernike phase contrast (see

Fig. 2.34a) is based on partially coherent illumination and uses a phase shifting

plate inserted in the back focal plane of the imaging FZP to phase shift the illumination beam by π/2 (positive phase contrast) or 3π/2 (negative phase contrast) with

respect to the light scattered by the sample. The combination of the phase shifted

and scattered light from the sample produces an image in the detector plane that has

the appearance of an absorption contrast image, but varies with the phase shift of the

sample structures (see [2.93]). The diffraction-limited spatial resolution achievable

with FZPs having dr as the outer most zone width is given by (see [2.93])

δ ∼ 1.22 dr /m

where m is the diffraction order. Resolutions to 15 nm have been achieved in a soft

x-ray microscope making use of an overlaid objective zone plate with 80 nm thick

gold plated opaque zones and a 15 nm outer zone width (Fig. 2.34b, c). Zone-platebased microscopes are of two main types (see [2.15]): In the scanning transmission

x-ray microscopes (STXMs) the sample is mechanically raster-scanned through the

focal spot provided by a zone plate. In the transmission x-ray microscope (TXM), a

zone plate magnifies the sample onto a 2D detector.

Compound lenses (Fig. 2.34d) can be made by chaining individual parabolic

lenses made from Al or Be to form the optic element of a hard x-ray microscope


Near-edge x-ray absorption fine-structure (NEXAFS) microscopy provides excellent compositional contrast in organic materials and is much less damaging than

electron energy loss spectroscopy in electron transmission (see [2.15]). Similarly,

the interaction of x-rays with magnetic materials provides element-specific contrast

that allows the determination of magnetic properties in multielement antiferromagnetic and ferromagnetic materials by making use of x-ray magnetic circular

dichroism (XMCD; see [2.15]), which also is used for studying dynamic magnetic

phenomena (see [2.15]). In Fig. 2.35 the magnetic domains of a bilayer of 1.0 nm



Microscopy – Nanoscopy

Fig. 2.34 Lens-based x-ray microscopy systems. (a) Fresnel zone plate (FZP) system (see [2.93]).

(b) Scanning electron micrograph of a zone plate with 15 nm outermost zone showing a detailed

view in the inset. The zonal period as indicated by the two black lines in the inset is 30 nm with

a zone overlay placement accuracy of 1.7 nm. (c) Soft x-ray image 15.1 nm half-period Cr/Si

multilayer image with the 15 nm zone plate shown in (b) at a photon energy of 815 eV and a

pixel size of 1.6 nm [2.92]. (d) Compound refractive imaging system (see [2.93]). (Reprinted with

permission from [2.93] (a), (d) and [2.92] (b), (c). © 2007 Elsevier (a), (d) and 2005 Nature

Publishing Group (b), (c))

of platinum on 1.2 nm cobalt on an antiferromagnetic LaFeO3 (100) substrate are

shown as imaged by soft x-ray XMCD microscopy. The contributions of the various

elements to the magnetic properties can be clearly distinguished because each element has its absorption edge at a different photon energy and the antiferromagnetic

domains of the substrate could be clearly correlated with the cobalt-specific ferromagnetic domains above it. The imaging of magnetic nanofeatures is reviewed in

Sect. 8.1.


X-Ray Microscopy


Fig. 2.35 Domain structure of a platinum–cobalt bilayer on an antiferromagnetic LaFeO3 substrate observed by photoelectron emission microscopy (PEEM). X-ray magnetic circular dichroism

(XMCD) can be used as a contrast mechanism in PEEM. The antiferromagnetic domains (a) and

the cobalt domains (b) are visualized. Comparison of the images shows that the ferromagnetic

cobalt domains align with the antiferromagnetic domains (light and dark regions inside outlined

areas) [2.15, 2.101]. (Reprinted with permission from [2.15]. Figure courtesy of J. Stöhr)

2.7.2 X-Ray Nanotomography

For imaging tungsten plugs at 60 nm resolution (Fig. 2.36), 890 nm-thick Au FZPs

have been used. These plugs interconnect the different layers of an integrated circuit.

When a plug contains a defect in its center (a “keyhole” defect) formed during the

electroplating process, it can cause the breakdown of the circuit.

X-ray microscopy can favorably image small biological objects containing elements of lower atomic numbers by using photon energies within the “water window”

between the absorption edge energies of carbon (284 eV or 4.4 nm wavelength)

and oxygen (543 eV or 2.3 nm) for probing whole hydrated biological cells. In

this energy range water is transparent to a 10 μm thickness whereas the more

carbon-rich organelles and other sub-cellular structures are more absorptive providing a natural contrast. 3D x-ray maps with 60 nm resolution of cryogenically fixed,

fully hydrated and unstained yeast [2.95] and bacterium cells [2.96] are shown in

Fig. 2.37.

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6 Progress in Electron Microscopy

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