Ultramicroscopy 123 (2012) 90–98

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Ultramicroscopy
journal homepage: www.elsevier.com/locate/ultramic

Scanning transmission electron microscopy:

Albert Crewe’s vision and beyond
Ondrej L. Krivanek a,n, Matthew F. Chisholm b, Matthew F. Murfitt a, Niklas Dellby a
a
Nion Company, 1102 8th Street, Kirkland, WA 98033, USA
b
Oak Ridge National Laboratory, Materials Science and Technology Division, Oak Ridge, TN 37831-6069, USA

a r t i c l e i n f o a b s t r a c t

Available online 17 May 2012 Some four decades were needed to catch up with the vision that Albert Crewe and his group had for the
Keywords: scanning transmission electron microscope (STEM) in the nineteen sixties and seventies: attaining 0.5 Å
STEM resolution, and identifying single atoms spectroscopically. With these goals now attained, STEM
ADF imaging developments are turning toward new directions, such as rapid atomic resolution imaging and
EELS exploring atomic bonding and electronic properties of samples at atomic resolution. The accomplish-
Aberration correction ments and the future challenges are reviewed and illustrated with practical examples.
Single atom imaging & 2012 Elsevier B.V. All rights reserved.
Single atom spectroscopy

1. Introduction
The invention and successful implementation of the modern
scanning transmission electron microscope (STEM) by Albert
Crewe and his coworkers and students in Chicago in the 1960s
and 1970s were nothing short of revolutionary. For the first time
in human history, it became possible to analyze solid matter with
a real-space probe of atomic dimensions [1–5]. The STEM pro-
duced the first images of single atoms not subject to intense
electric fields (which are inevitable in a field ion microscope), in
their native environment [6]. It also pointed the way to quanti-
tative microanalysis with single-atom sensitivity, in which the
inelastic interaction between the electrons in the atom-sized
probe and the sample is used to determine the chemical type of
the atoms in the sample [7]. Also interesting to note, the
investigations with the Chicago STEM were all done at primary
energies r45 keV, thus minimizing the knock-on damage in the
specimen, and allowing phenomena like thermally-activated
atomic hopping to be investigated [8,9].
Four decades later, we are thoroughly accustomed to the idea
of probing matter and manipulating it on an atomic scale,
typically via a single atom that protrudes from the tip of a
scanning tunneling microscope (STM), or of an atomic-force
microscope (AFM) [10,11]. We have also learned how to form
atom-size probes from ions, which have some important advan-
tages over electron probes [12]. Unfortunately, perhaps due to the
large number of such techniques now available and the wealth of
results that they have brought, it is not always remembered that
the first time matter was investigated with an atom-sized probe
that could be manipulated at will was in the scanning transmis-
sion electron microscope built by Albert Crewe’s group in
Chicago.
In the field of electron microscopy, the achievements and
dreams of the Chicago group are usually given due credit, and
they have been followed up energetically. There were many major
milestones on the journey, such as improving the attainable
resolution by about 2x by going to higher operating voltages,
improving the performance of the cold field emission electron
gun, improving the resolution by a further 2–3x through the
introduction of working aberration correctors, improving the
energy resolution and the collection efficiency of electron energy
loss spectroscopy (EELS), improving the efficiency of X-ray
energy-dispersive spectroscopy, monochromating the primary
beam for improved EELS energy resolution, and returning to
lower operating energies, in order to eliminate knock-on radiation
damage. Most of these developments have been thoroughly
reviewed, e.g. in Pennycook’s ‘‘Scan through the History of STEM’’
chapter that gives over 500 references [13].
In this article we review progress in two of the research areas
of aberration-corrected STEM we have been involved with espe-
cially closely: rapid imaging made possible by increasing the
electron current contained in an ultra-small probe, and low keV
imaging and analysis.

2. Rapid imaging of single atoms

n
Corresponding author.
E-mail addresses: krivanek@nion.com, The framework for the imaging of single atoms in the STEM
krivanek.ondrej@gmail.com (O.L. Krivanek). was set out by Crewe, Wall and Langmore in their landmark

0304-3991/$ - see front matter & 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.ultramic.2012.04.004
 O.L. Krivanek et al. / Ultramicroscopy 123 (2012) 90–98
Science paper [6]. The main differences between their work and
the present are:
(a) We now have electron probes of o1 Å in diameter, whereas
only a 5 Å probe was available to Crewe and coworkers when
they first imaged single atoms. (A 2.5 Å probe became avail-
able in their microscope a few years later.)
(b) Significant progress has been made in the available probe
current: whereas 30 pA was a typical probe current available
in the Chicago microscope, we can now operate with probe
currents of the order of 1 nA in probes of 1.4–2 Å [14].
(c) We now pay more attention to suppressing the coherent
component of annular dark field (ADF) images of materials
that arises when low scattering angles contribute to the
image [15–17], and which can produce strong maxima
(‘‘coherent speckle’’) in images of amorphous materials used
as substrates for heavy atoms. Most ADF images of heavy
atoms are therefore formed using ADF detector inner angles
450 mr, for which the coherent component is considerably
reduced. Interestingly, the use of such detectors was first
suggested by Crewe and coworkers [18], though for different
reasons.
(d) More accurate and more detailed cross-section data for the
atomic scattering are now available, e.g. [19].
To detect single heavy atoms reliably, we must make sure that
the weakest signal coming from each heavy atom is larger than
the strongest signal from the substrate away from the atoms. For
an isolated heavy atom X lying on an amorphous carbon substrate
and illuminated by a beam of cross-sectional area A, the signal SX,
i.e. the number of electrons scattered while the beam is over the
atom and detected by the ADF detector will be:
SX ¼ ntðsX =A þ rC t sC Þ:
The first term in the bracket is the contribution from the heavy
atom, the second term the contribution from the substrate. n is
the probe current expressed as the number of electrons
per second, t is the time the probe spends over the atom, sX is
the ADF cross-section for atom X, rC is the density of the carbon
film (in atoms/Å3), t is the film thickness, and sC is the cross-
section for the carbon atom. The cross sections depend on the
atomic type, the ADF lower and upper cut-off angles b1 and b2,
the illumination semi-angle a and the primary beam energy Eo.
They can be worked out in many different ways, at least two of
which give nearly identical results [19].
The weakest signal collected from a heavy atom will be:
SX 2uN x ¼ nt½sX =A þ rC t su½ntðsX =A þ rC t sÞ0:5
where Nx is the mean statistical noise in the image intensity of the
heavy atoms (plus the underlying substrate), and we model the
peak deviation as being u times larger than the mean noise.
The strongest signal from an area of the substrate equivalent
to the image area of individual heavy atoms will be:
SC þvN C ¼ nt rC t sC þvðgnt rC t sC Þ0:5
where SC is the average signal from the substrate, NC is the mean
statistical noise in the image intensity of the substrate, the peak
deviation in the substrate image is modeled as being v times
larger than the mean noise, and g accounts for the fact that the
coherent contribution to the random speckle in a dark field image
of an amorphous carbon film will cause the speckle to be stronger
than the incoherent shot-noise variation in the number of
detected electrons. The magnitude of g depends on how coherent
the dark field image is, which in turn depends on the ADF
collection geometry. Our estimate is that for inhomogeneous
substrate films and small inner ADF cut-off wavevectors of the
91
order qo  b1/l o1 Å  1 the speckle is partially coherent and g can
reach 2 and more, but that for homogeneous films and
qo 42.5 Å  1, the substrate image is largely incoherent and g
decreases towards 1.
The visibility criterion for the single heavy atoms is therefore:
SX uN x 4SC þ vN c ð4aÞ
ntsX =Au½ntðsX =A þ rC t sÞ0:5 4vðgntrC t sC Þ0:5 ð4bÞ
The criterion will be attained if the electron dose Dx that is
delivered to each atom X is greater than:
DX ¼ nt 4ðA=sX Þ2 ½uðsX =A þ rC t sÞ0:5 þ vðgrc t sC Þ0:5 2 ð5Þ
which becomes possible when the time the probe spends over the
atom:
t 4ðA=sX Þ2 ½uðsX =A þ rC tsÞ0:5 þ vðgrc tsC Þ0:5 2 =n: ð6Þ
The choice of u determines the probability of false negatives
(missing actual heavy atoms); the choice of v determines the
probability of false positives (identification of heavy atoms where
none exist). Selecting u ¼3 gives a probable rate of false negatives
of 1.3  10  3, i.e. one out of about 700 heavy atoms being missed.
Selecting v ¼5 gives a probable rate of false positives of
o3  10  7, allowing large areas of the sample to be examined
without mistaking random maxima for heavy atoms.
The above expressions ignore inelastic scattering, whose con-
tribution to the ADF signal is considerably weaker than the elastic
one even for the carbon substrate, and much more so for the
heavy atom. They assume that there are no major probe tails, and
that the atoms stay still while under the beam. They also assume
that the ADF detector is 100% efficient, and they do not specifi-
cally account for the noise term that will arise if the carbon film
thickness varies randomly (although this can be included by
ð1Þ
increasing the speckle factor g).
Using Eq. (6) with u ¼3 and v ¼5, and the cross-section data
tabulated for various detector geometries by Treacy [19] for two
specific cases:
(A) single Au atoms being detected in an optimized microscope
with a bright CFEG at 200 kV, on a non-optimized
substrate, and
(B) single Au atoms being detected in an optimized microscope
with a bright CFEG at 60 kV, on an optimized substrate,
we arrive at the values listed in Table 1, in which the important
results are shown in bold. The table predicts that in both cases,
o40 scattered electrons detected on the ADF detector are
ð2Þ
sufficient to detect each Au atom, and that such a signal can be
collected in about 0.5 ms in the non-optimized case A, and about
0.1 ms in the optimized case B. The short times needed to detect
single heavy atoms have important implications for rapid sequen-
cing of DNA by STEM imaging, as discussed below.
For both cases, we kept the same illumination aperture and
ADF detector angles. The illumination angle is typically not varied
ð3Þ
greatly as the primary beam energy is changed in Cc-limited
systems such as the Nion UltraSTEM [14,20], in which the
increase in sensitivity to Cc at lower primary energy Eo mostly
offsets the fact that larger absolute deviations of the aberration
function (expressed in units of length) can be tolerated at lower
energies than at high ones. The detection angles b1 and b2 should
ideally be adjusted to keep the scattering wavevectors approxi-
mately constant at any primary energy. But in the present
example, in which the C substrate is thinner for the 60 kV case,
thus making the coherent speckle contribution less bothersome, it
is acceptable to keep the scattering angles roughly the same for
the two cases. Because of the shorter electron wavelength l in the
92 O.L. Krivanek et al. / Ultramicroscopy 123 (2012) 90–98
Table 1
Two scenarios for detecting a single Au atom on a carbon substrate. Estimated parameters are shown in italic script, the important results in bold script.
Case A: 200 keV, 200 Å thick C substrate
Primary energy Eo 200 keV
Probe semi-angle a 30 mr
Zero current probe size do 0.6 Å
Coherent probe current Ic 0.3 nA
Actual probe current Ip 1.2 nA
Actual probe size dp ¼do(1þIp/Ic)0.5 1.4 Å
ADF lower cut-off (b1) 60 mr
ADF upper cut-off (b2) 200 mr
Min. scat. wavevector qo 2.39 Å  1
sAu 0.01284 Å2
sC 0.00010 Å2
C film density r 0.12 atm./Å3
C support film thickness t 200 Å
Speckle factor g 1.2
s 5.2  10  7 s
Electrons incident on Au atom during time s 3926 electrons
Average number of ADF electrons detected per Au atom 34 electrons
200 keV case A, the inner cut-off (minimum) scattering wave-
vector qo is then higher than in case B: 2.39 Å  1 vs. 1.23 Å  1. This
plus the strong dependence of the cross-section on the primary
energy (cross-sections scale as l  2 for equivalent scattering
geometries) has made both the Au and the C cross-sections about
10x higher in the 60 keV case B than in the 200 keV case A.
For case A, we have assumed a non-optimized carbon sub-
strate 200 Å thick, i.e. a substrate that is readily purchased
commercially, for case B, we have assumed an optimized carbon
substrate 20 Å thick, i.e. a substrate of the type that Crewe and
coworkers developed. The smaller inner ADF collection angle
(expressed in Å  1) of case B means that the substrate speckle
will be more important for the assumed 60 keV set-up than for
the 200 keV one, and we have taken account of this by setting the
speckle factor g to 1.2 and 2 for A and B, respectively.
Had identical support films been assumed for the two cases,
the minimum times t per heavy atom would have been within a
factor of 2 for the two cases considered. This shows that once the
sample and the microscope have been optimized, the variations
available when adjusting the primary energy, illumination and
signal collection geometries are not very strong.
Fig. 1 illustrates that performance similar to that documented
in Table 1 is now indeed possible. It shows ADF imaging at
200 keV of single Au atoms on a carbon substrate about 200 Å
thick, with probe formation and ADF signal collection conditions
essentially as given by case A. The probe current was 1.2 nA, and
the probe diameter was about 1.5 Å. Several 512  512 images
were acquired unusually rapidly for STEM: the dwell time was
just 0.167 ms per pixel, and the pixels were 0.8 Å  0.8 Å. Just
4 pixels therefore spanned each Au atom, and the dwell time per
atom was only about 0.7 ms. The frame repetition rate was about
23 frames/s, i.e. the imaging was done at close to TV rate.
Fig. 1(a)–(g) shows the same 45  170 pixel sub-area in
7 consecutive time-averaged frames, each one of which is a sum
of 4 consecutive frames. It shows the evolution of the sample over
about 1 s. Single Au atoms are clearly visible in each of the frames,
but they disappear in some frames and come back in other
frames. For instance, the three Au atoms indicated by vertical
arrows in (a) stay in the same place throughout the sequence,
whereas the two atoms indicated by horizontal arrows in
(g) move around. The bottom one of the two atoms is present
in frames (c) and (d), disappears in frame (e), and comes back in
frame (f), in a slightly different place. The disappearance is
probably due to the atom moving too much in the frame (e) to
Case B: 60 keV, 20 Å thick C substrate
Primary energy Eo 60 keV
Probe semi-angle a 30 mr
Zero current probe size do 1.0 Å
Coherent probe current Ic 0.3 nA
Actual probe current Ip 0.6 nA
Actual probe size dp ¼do(1þIp/Ic)0.5 1.7 Å
ADF lower cut-off (b1) 60 mr
ADF upper cut-off (b2) 200 mr
Min. scat. wavevector qo 1.23 Å  1
sAu 0.11747 Å2
sC 0.00120 Å2
C film density r 0.12 atm./Å3
C support film thickness t 20 Å
Speckle factor g 2
s 1.1  10  7 s
Electrons incident on Au atom during time s 347 electrons
Average number of ADF electrons detected per Au atom 21 electrons
be clearly visible. Along with the statistical variation in the
intensity of the atomic images, the atomic motion is also
responsible for the varying intensity of the same atoms in
different images: atoms that move about give less visible peaks
than atoms that stay still.
When individual frames of the original sequence are exam-
ined, individual atoms are visible too, but they disappear much
more frequently. When the successive frames are combined into a
movie and played back suitably slowed down [21], the impression
one gets is that the atoms are ‘‘swimming about’’ rather like fish
in an aquarium—randomly, slowly and steadily, with only rare
large sudden movements. This may seem surprising at first, as
there is no obvious direct mechanism that would displace the
heavy atoms continuously by small amounts. The explanation
probably lies in the substrate: 200 keV electrons can transfer up
to 43 eV energy to a carbon atom, and the carbon bonding energy
is only about 15 eV. This means that C atoms are continuously
displaced and even knocked away from the sample under 200 keV
irradiation. An amorphous carbon film that appears to ‘‘swim’’, i.e.
to change continuously under an electron beam, is a familiar sight
to most high resolution electron microscopists. The ‘‘swimming’’
is quite energetic at 200 keV. The Au atoms, riding mostly on top
of the C film, therefore simply join in the collective ‘‘swim’’, i.e.
they move about in response to the changes in the supporting
film. At 60 keV, the ‘‘swimming’’ motion of heavy atoms is largely
absent, but other, less frequent types of atomic motion can be
observed [22].
Because of the induced atomic motion, 200 keV is not a good
primary energy for localizing Au atoms with sub-nm precision.
However, the substrate atomic motion mostly stops at 60 keV,
and appears to stop completely at 40 keV.
The above discussion indicates that rapid imaging of Au and
other heavy atoms on 20 Å thick C substrates is now possible very
much as shown in Table 1, i.e. at o1 ms/atom. Being able to image
atomic motion at a fast rate is a welcome new capability for
atomic resolution STEM, which has typically used slower frame
refresh rates than shown here. The new capability may be useful
for studying rearrangements of small crystals and sample edges in
materials such as small catalytic particles and graphene.
The single most exciting potential application of the rapid
imaging of single heavy atoms is however to be found in biology
rather than materials science: sequencing DNA. In this approach,
labels containing single (or multiple) heavy atoms would be
attached to a specific base type, preferably using a single uncoiled
 O.L. Krivanek et al. / Ultramicroscopy 123 (2012) 90–98
Fig. 1. Rapid STEM ADF imaging of single Au atoms. The images in the sequence
were collected 0.17 s apart. Atoms marked by vertical arrows stayed approxi-
mately stationary, atoms marked by horizontal arrows moved about. Ultra-
STEMTM200, Eo ¼200 keV, probe current¼ 1.2 nA.
strand of the DNA, the DNA with the labels would be laid down on
a thin substrate such as a very thin amorphous carbon film, and
the heavy atoms constituting the labels would be imaged. This
should reveal where the bases of the labeled type are located.
Repeating the experiment with at least one other type of bases
labeled (and using the fact that in the double strands, adenine is
always paired with thymine, and guanine with cytosine) should
then allow the complete base sequence to be ‘‘read out’’.
Perhaps not surprisingly, Crewe’s group originated this tech-
nique [23,24]. It is now being pursued again [25–27], with better
chemical labeling methods, better specimen preparation techni-
ques, and more sophisticated electron microscopes. A unique
advantage of the EM-based approach should be longer read
93
lengths [28] than possible when using the so-called shot-gun
sequencing approach, in which the DNA is cut up into segments at
most a few tens of bases long. But even with this advantage, the
EM-based approach will need to be accurate and fast, if it is to be
competitive with the many different DNA sequencing methods
that are now available or being developed [28].
The theoretical and experimental results presented above
show that single heavy atoms should be detectable if the electron
probe spends just 0.1–1 ms over them. Assuming that ways can be
found that allow the probe to progress along each DNA strand and
not wonder pointlessly in the ‘‘wilderness’’ between the strands,
e.g. using a strategy whereby the positions of the strands are
mapped at lower magnification and the probe is then made to
advance only over the strands at the higher magnification, it
therefore appears that around 106 bases/s could potentially be
read using the ADF STEM approach. The human genome has about
3  109 base pairs, which means that the above approach may be
able to cover the whole genome in about 1 h per one type of label.
The ‘‘read’’ would then need to be repeated using heavy atom
labels attached to one of the non-complementary bases, i.e. either
adenine or thymine would need to be read out plus either guanine
or cytosine. Further, ‘‘overcoverage’’ would be needed to build up
enough statistics to allow correcting various mistakes that are
likely to occur in the chemical labeling, the laying out of the DNA,
and the imaging. Picking a 20x overcoverage as a likely possibility
indicates that the above approach may be able to sequence the
human genome in about 40 h, and perhaps as little as 10 h with
further optimization.
3. Single atom imaging and spectroscopy below the knock-on
damage threshold
For primary energies less than about 100 keV, the maximum
energy Emax that can be transferred by a primary beam electron to
an individual atom in the sample is approximately proportional to
Eo/A, where Eo is the primary energy and A is the atomic mass
number [29,30]. More specifically, a maximum energy transfer of
15 eV is possible for hydrogen atoms (1H) at a primary energy
Eo ¼6.9 keV, boron (10B) at Eo ¼65 keV, carbon (12C) at Eo ¼78 keV,
silicon (28Si) at Eo ¼167 keV, and gold (197Au) at Eo ¼774 keV.
Binding energies of the order of 15 eV are common in many
different types of crystals. The short list just given therefore
shows that hydrogen displacement cannot be avoided in a typical
transmission electron microscope with a lowest primary energy
setting of 20 keV, even in the absence of ionization damage, that
imaging carbon without knock-on damage requires Eo smaller
than 100 keV, that imaging silicon without knock-on requires Eo
smaller than 200 keV, and that strongly bonded heavy atoms can
be imaged without knock-on damage at 300 keV and even higher.
In a previous article [22], we have called STEM imaging free of
knock-on damage ‘‘gentle STEM’’. For light atoms such as B, C, N
and O, gentle STEM can typically be done at 60 keV primary
energy [31], except at the sample’s edge or in disordered materi-
als, where the atoms are bonded less strongly.
Atomic resolution is more difficult to attain with gentle STEM
than when operating at higher energies. Prior to aberration
correction, STEM spatial resolution was typically not good enough
to resolve atomic columns in regular crystals when the operating
energy was less than 100 keV. Interestingly, the microscope that
came closest to giving atomic resolution at low primary energies
was the Chicago high resolution STEM [32], operated at up to
45 keV. It had an objective lens with low spherical and chromatic
aberration coefficients (Cs ¼0.46 mm, Cc  0.8 mm,), and it was
eventually able to produce probe sizes of about 2.5 Å at 40 keV.
94 O.L. Krivanek et al. / Ultramicroscopy 123 (2012) 90–98
Present-day aberration-corrected STEMs are able to improve
on such performance by 2–2.5x [14,33]. Their probe size at
energies lower than about 200 keV is typically limited by chro-
matic aberration rather than geometric aberrations. For a negli-
gibly small source size (i.e., very small beam current), the smallest
attainable probe size is given by [34]:
dchrom ¼ 0:5ðlC c dE=Eo Þ0:5
where l is the electron wavelength, Cc is the coefficient of
chromatic aberration, dE is the energy spread and Eo is the
primary energy.
Since the electron wavelength l is proportional to Eo 0.5 at low
primary energies, Eq. (7) shows that dchrom is proportional to
Eo 0.75. This means that the resolution grows worse quite quickly
as Eo is lowered. For 60 keV electrons, Cc ¼ 1.2 mm and dE¼ 0.4 eV
(as is readily attainable with a cold field emission gun (CFEG)) and
negligibly small source size, the equation predicts dchrom ¼1.0 Å.
With a 60 pA probe current coming from a source giving a
coherent current Ic of 150 pA, the finite size of the electron source
increases the probe size to 1.2 Å [34]. For 30 keV electrons,
Cc ¼0.8 mm and dE ¼0.4 eV, dchrom ¼1.4 Å with a negligible beam
current, and 1.6 Å for a 60 pA probe. (Note that probe size is a
more stringent performance criterion than the transfer of a given
spatial frequency into the image: for instance, a probe of 1.2 Å
full-width at half-maximum (FWHM) can easily transfer spatial
frequencies higher than (1 Å)  1.)
In practice, there are several other influences, besides the
chromatic aberration and the finite source size, which can poten-
tially become resolution-limiting. These include poorly tuned
geometric aberrations, finite size of the atoms, statistical noise in
the images, and instrumental instabilities. They are discussed in
[34–36]. Even with these limitations, present-day aberration-
corrected STEMs are readily able to resolve all the individual atoms
in graphene and single sheet BN at 60 keV, including nearest
neighbors that are separated by o1.5 Å in these two materials.
It is useful to remember that due to the hexagonal nature of
graphene and monolayer BN sheets, the reflections that must be
transferred strongly in order to clearly resolve the near-neighbor
atomic pairs in these materials, the (11–20)-type ones, have a
smaller spacing than the nearest-neighbor distance (d(11–20) ¼
1.23 Å in graphene vs. 1.42 Å nearest-neighbor (nn) distance,
and 1.26 Å in hexagonal BN vs. 1.45 Å nn distance). In other
words, in order to achieve atomic resolution in graphene and
BN, a probe size that is 15% smaller than the nearest neighbor
separation is very useful. A strong transfer of the (11–20) reflec-
tions was first achieved with a STEM for graphene in 2009, and
Fig. 2. MAADF images of monolayer graphene containing Si substitutional atoms and other defects. (a) Topologically perfect, but distorted graphene lattice containing a
single Si atom, (b) Si atom bonded in a 9-fold ring, and (c) Si atoms at graphene edge. UltraSTEMTM100, Eo ¼60 keV.
clearly resolved STEM images of nearest neighbor pairs (as well as
various topological defects such as 5-fold rings and a single
carbon atom and a single impurity atom hanging off the edge of
graphene) were published in early 2010 [22].
As an interesting aside, a subsequent paper by Jinschek et al.
[37], which used through-focus reconstruction in a conventional
TEM (CTEM) to resolve C atoms in a single and double graphene
ð7Þ
sheet (but not at the graphene’s edge), made no mention of the
STEM work. A News and Views article in Nature Materials then
wrongly attributed the first atomic-resolution electron micro-
scope images of graphene to the CTEM work [38]. The episode
shows that some parts of the electron microscopy community
unfortunately have yet to acknowledge the contribution of the
STEM to our knowledge of materials, even 40 years after Albert
Crewe’s pioneering results.
Fig. 2 shows medium-angle annular dark field (MAADF)
images of graphene acquired at 60 keV, with a beam current of
50 pA and a dwell time of 64 ms per each pixel, 0.06  0.06 Å large.
The probe semi-angle was 30 mr, and the MAADF detector
extended from 55 mrad semi-angle up to about 200 mrad. The
images were filtered by a rotationally symmetric two-Gaussian
filter that nulled the probe tail at 1.42 Å from the probe center
(i.e., at the nearest-neighbor distance), and also removed image
shot noise with spacing smaller than about 1 Å (i.e., noise at
higher spatial frequencies than those meaningfully transferred to
the image), as explained in Ref. [22]. Here we simply note that
whenever the probe tail contribution at the near neighbor
distance (and beyond) is not zero, images of individual atoms
contain significant contributions from the atom’s neighbors even
in monolayer samples. Accurate analysis of the image intensity of
different types of atoms that may be present as substitutional
impurities is then not possible.
There was another image defect present: the graphene lattice
was imaged slightly distorted, probably because the graphene
sheet was not precisely perpendicular to the incoming electron
beam. This is especially visible in Fig. 2(b). It does not affect the
interpretation of the images greatly, and it was not corrected.
The imaging conditions were nearly identical to those we have
used in the past for imaging BN monolayers with substitutional
impurities [31]. The same power law relating the intensity Iz of
the atom’s image to its atomic type Z in tail-subtracted images
was found once more:
Iz ¼ Icarbon ðZ=6Þ1:64 ð8Þ
For Si, Isilicon ¼4.0  Icarbon. The medium-intensity impurity
atoms seen in several places in Fig. 2 were found to have
 O.L. Krivanek et al. / Ultramicroscopy 123 (2012) 90–98
3.8–4.2x carbon atom intensity, demonstrating that the impuri-
ties were Si. The presence of Si in the sample was further
confirmed by EELS analysis of large aggregates of the impurity
atoms, which produced the Si L2,3 edge signature.
The preparation of monolayer graphene often involves close
contact with materials such as SiO2 or SiC, and silicon is a
common contaminant of graphene. In the present sample, Si
atoms were detected in several types of lattice sites, and Fig. 2
shows a selection of them. Some Si atoms were incorporated into
the perfect graphene lattice, as demonstrated by the Si atom in
Fig. 2(a), and the right atom in Fig. 2(b). The preferred length of
the single C–Si bond is 1.85 Å [39], i.e. considerably more than the
1.42 Å C–C distance in graphene. The result is that the silicon’s
near neighbor carbon atoms are pushed away, and this can be
seen directly in the image. Density functional theory (DFT; [40])
calculations also show that the graphene surface around the Si
atom becomes buckled and the Si atom stick outs a small distance
above (or below) the graphene plane [41], i.e. that it forms the
apex of a very small triangular pyramid.
The buckling is hinted at in the image of the right Si atom in
Fig. 2(b). The foreshortening of the average graphene hexagon in
the image indicates that the graphene sheet was tilted along an
axis running approximately from bottom left to top right, which
means that the projected faces of a pyramid sticking out of the
sheet should have different sizes. The three hexagons that include
the Si atom are indeed unequal in size: the top left hexagon is
smaller than the other two, and this indicates that the Si atom
was either above or below the plane of the graphene sheet.
Some Si atoms preferred to segregate to topological defects, at
which the longer Si–C bond is more easily accommodated, as
shown by the left Si atom in Fig. 2(b). Finally, the edge of the
graphene sheet was often decorated by dense one-dimensional
arrays of Si atoms. Some of these atoms were incorporated into
5-fold rings located at the graphene edge, where they substituted
for two carbon atoms that complete similar rings in the usual
‘‘armchair’’ graphene edge termination. This was the case for the
2nd and 3rd Si atoms (counting from the top) in Fig. 2(c). Other Si
atoms simply dangled off the graphene edge, attached to just one
C atom in the graphene (atoms 4 and 5 in Fig. 2(c)). The average
distance of the Si atoms from their nearest neighbor carbon atoms
was about 1.9 Å for both the doubly bonded and the singly
bonded Si atoms, confirming that the preferred C–Si bond length
is indeed much larger than the C–C one.
There was also a single nitrogen atom, whose intensity was
1.3x the average carbon atom intensity. It is shown in Fig. 2(b),
located at a 7-fold ring. N along with B were intentionally
incorporated in the graphene sample, which was prepared by
reducing doped graphene oxide [42], but they were observed less
frequently than the Si atoms.
Fig. 2 makes it clear that a large amount of information can be
obtained by STEM ADF imaging on monolayer materials consist-
ing of light atoms. The STEM is able to image all the atoms, clearly
resolved, in a single exposure, and it identifies the atomic number
Z of the atoms simply by the image intensity of the individual
atoms. The resultant information is similar to the information
about a BN monolayer sheet that incorporated C and O impurities,
which we were able to acquire by ADF STEM earlier [31], but the
detailed types of the observed structures are very different. For a
thorough understanding, modeling the observed structures, e.g.
with density-functional theory, is of course essential. STEM work
focused on understanding the materials science behind the
addition and incorporation of foreign atoms on and in graphene
sheets can also be found in Refs. [43,44].
Determining the atomic type from the atom’s MAADF image
intensity is straightforward if there is no major contribution to
the image of each atom from the other atoms present in the
95
sample. This applies to tail-subtracted images of flat monolayers,
of atoms dangling off the sample’s edge, and of linear chains of
atoms. The main attraction of the technique is that it can provide
an unambiguous identification of single atoms at smaller electron
doses than what is required for spectroscopic identification.
Electron doses of about 5  106 electrons/Å2 are sufficient for
the MAADF images to be sufficiently noise-free so that atoms
differing by DZ¼ 7 1 can be reliably distinguished from each
other [31], whereas single atom identification by EELS requires
doses of the order of 108 electrons/Å2 and higher [22]. For more
complicated samples, e.g. double and thicker layers, monolayers
covered by adatoms or contaminants, nanotubes and nanopods,
etc., some degree of overlap of the atomic images cannot be
avoided. The simple relationship between the observed intensity
and the atomic type is then no longer available. 3D reconstruction
from multiple projections may be possible, but it will not be easy
in the presence of possible radiation damage and other changes in
the sample from exposure to exposure. The reconstruction will
also need to account for the fact that because of the partially
coherent nature of MAADF imaging (in the beam direction), the
intensity of an MAADF image maximum due to atoms that
precisely overlap each other in projection is typically slightly
more than the simple sum of the individual images of the two
overlapping atoms [36]. In all such cases, element-specific spec-
troscopic data will be invaluable.
4. Electron energy loss spectroscopy (EELS) of single atoms
Single atoms of uranium (Z¼92) were tentatively identified by
EELS in a non-corrected STEM already in 1991 [45], and single
atoms of Gd (Z ¼64) were identified much more unambiguously
in 2000 [46]. Subsequent demonstrations of single atom spectro-
scopy, obtained in aberration-corrected electron microscopes,
using nanopod-embedded single atoms of Er (Z ¼68) at 60 keV
[22], and of La (Z ¼57) and Ce (Z ¼58) at 30 keV [47] were even
clearer. N4,5 and O4,5 edges, with relatively large cross sections,
were used in the above experiments. A single La atom present in a
CaTiO3 crystal was also identified in an aberration-corrected
STEM, using the La M4,5 edge that was highly visible due to its
prominent threshold peaks [48].
Improvements in the beam current available in an aberration-
corrected STEM [14], in probe stability relative to the sample [34],
and in the efficiency of the coupling of the EELS signal into an
energy loss spectrometer [20] have now made it possible to
extend single-atom spectroscopy studies to single light atoms,
using K-edges with much smaller cross-sections.
Fig. 3 shows the results of a spectrum-imaging (SI) experiment
performed on B and N substitutional impurities in a graphene
sheet, using 60 keV electrons, in which the EELS signal from
individual light atoms is unmistakable. Fig. 3(a) shows an MAADF
STEM image of a different area of the same type of sample as was
used for Fig. 2. The area contained single N and B impurities: the
image shows one more intense and one less intense atom.
Fig. 3(b)–(d) show boron, nitrogen and carbon elemental maps
computed from a spectrum-image (SI) of this area acquired with a
Gatan UHV Enfina spectrometer. The SI acquisition parameters
were: map size 7  11 pixels, pixel size 0.66 Å, time per pixel 1 s,
1340 energy channels, each energy channel 0.3 eV wide. The
spectrometer acceptance half-angle was about 50 mrad, i.e. it was
significantly larger than the radius of the bright field disk. The
elemental maps were computed using power law background
extrapolation and subtraction.
The boron map shows a strong peak where the less intense
atomic image is in the MAADF image; the nitrogen map shows a
strong peak where the more intense atomic image is. This
96 O.L. Krivanek et al. / Ultramicroscopy 123 (2012) 90–98

Fig. 3. MAADF image of graphene containing impurities and elemental maps. (a) MAADF image (acquired just before the spectrum-image used for the elemental maps),
(b) boron K-edge elemental map, (c) nitrogen K-edge elemental map, and (d) carbon K-edge elemental map. UltraSTEMTM100, Eo ¼ 60 keV.

confirms that the atoms were indeed boron and nitrogen, respec-
tively. The carbon map shows a decrease in the carbon signal
where the B and N atoms were located, which confirms that the
atoms were substitutional.
The carbon lattice is not resolved in the carbon-K EELS
map—not even the ‘‘holes’’ in the graphene sheet are visible. This
means that the spatial frequency of the (10–10) graphene reflec-
tion at (2.13 Å)  1 was not transferred into the carbon map. Since
the probe was about 1.2 Å in diameter, it means that the
delocalization of the carbon K-edge image was 41.7 Å (worked
out by adding the delocalization and the probe contributions to
the resolution in quadrature).
Fig. 4(a) shows spectra obtained by summing SI data from
9 pixels over the nitrogen atom (upper spectrum) and 9 pixels
over the boron atom (lower spectrum). The EELS edges due to the
single atoms are clearly identifiable. Fig. 4(b) shows an energy
loss spectrum from a single-layer BN with minor hydrocarbon
contamination obtained in a separate experiment but under
similar acquisition conditions. There is a clear difference between
the boron edge from the single B atom and from BN: the sharp pn
and sn threshold peaks that are strongly present in the BN
spectrum are largely absent for the single boron atom. The
difference between the two nitrogen K-edges is more subtle, with
the pre-edge peak appearing weaker for the single-atom spec-
trum than for BN. The carbon K edge in the BN spectrum is due to
amorphous carbon/hydrocarbon contamination. It has less pro-
nounced white lines than the graphene spectrum.
It is fascinating to note that EELS data recorded from single
Fig. 4. Comparison of EELS data from the B and N-containing graphene with a
atoms shown here and in [47] demonstrates that the Chicago spectrum from monolayer BN. (a) (top) EEL spectrum extracted from 9 pixels over
group’s 1975 prediction that a single atom of fluorine should be the boron atom in the EELS spectrum-image used for the maps shown in Fig. 3,
detectable by EELS in a well-designed STEM–EELS instrument [7] and (bottom) spectrum extracted from 9 pixels over the nitrogen atom, and
(b) spectrum from BN monolayer acquired in a separate experiment. Ultra-
has now been met, and extended to the point where the fine
STEMTM100, Eo ¼ 60 keV.
structure of EELS edges coming from single atoms can be studied
for many types of atoms, heavy and light. The fine structure
studies promise to provide a sensitive probe of the local atomic Experimental results indicating that delocalization may indeed
environment of individual atoms, and hence a versatile tool for improve at 30 keV have been published [47], but a systematic
achieving a detailed understanding of materials’ properties at the study of delocalization as a function of primary energy remains
atomic scale. to be done. Concentrating on K-edge imaging of the graphene
Powerful as the EELS approach is, it does have its drawbacks. (10–10) reflection, which should be just possible with a small
The main ones are the increased dose necessitated by the small probe and a primary energy of 30 keV, may prove fruitful for such
EELS cross-section, and the fact that the EELS interaction is experiments.
slightly delocalized, even with an optimized detection geometry
that accepts high scattering angles. The delocalization is expected
to decrease at lower primary energies as [30,34] 5. Discussion
3=4
ddeloc: ¼ 0:7  l=ðDE=Eo Þ ð9Þ
When optimizing the imaging of heavy atoms attached to DNA
E1/4
i.e., aso .This is not a strong dependence—lowering Eo from 100 strands, it may be useful to explore signals other than electrons
to 20 keV should improve the delocalization by 31%, and lowering scattered elastically with high momentum transfers, which was
it from 60 keV to 30 keV should give just a 15% improvement. the only signal used here.
 O.L. Krivanek et al. / Ultramicroscopy 123 (2012) 90–98
Crewe and collaborators were once more the pioneers in
combining the different signals available to maximum advantage.
They typically enhanced the visibility of single atoms by dividing
the ADF signal by the low-angle inelastic scattering signal Iin. The
Z dependence of the ADF image signal was modeled by them as
IADF,ZpZ1.33, the dependence of the inelastic signal as Iin,ZpZ0.33,
giving the intensity of the ratio image Ir as
Ir ¼ IADF,Z =Iin,Z pZ ð10Þ
It is worth noting that even though the Z exponent used by
Crewe et al. was too low to correctly model incoherent ADF
imaging that only uses electrons scattered to high angles, which
gives an exponent 41.5, the exponent of their model of inelastic
scattering was probably too low as well, and the relationship
IADF,Z/Iin,ZpZ may thus actually be approximately valid [19].
The ratio method was able to suppress the variation in the
signal arising from variations in the thickness of the supporting
film. With a 5 Å probe, such variations can look similar to the
signal from the single atoms. They modify the ADF and the
inelastic images in much the same way, and dividing the two
signals therefore does a good job of suppressing the variations.
With a 1–2 Å probe, however, the ADF signal due to a single atom
is typically much sharper than the variations in the inelastic
image of the support film, which are broadened by the delocaliza-
tion of the inelastic signal, and the danger of confusing the two is
much less. Another consideration then becomes dominant,
namely that the inelastic signal is an important source of
statistical noise, and that using the division method typically
leads to an increase in the overall noise with no other clear
benefit.
The ratio method may nevertheless be useful for suppressing
the background variation in single atom images taken from
samples of widely varying thickness. But the method is not often
used nowadays, almost certainly because of a practical problem:
most present-day electron energy loss spectrometers use CCD
detectors to record the inelastic scattering data, and do not have
the fast silicon surface barrier detector of the inelastic signal that
Crewe et al.’s STEM had [32] (or a scintillatorþPMT detector [20]
which is typically even faster). This means that they cannot
record the data needed for the ratio method with a sufficient
speed. It may therefore be useful in the future to provide fast
single-channel detectors of inelastic electrons once more.
Detailed considerations of exactly how to optimize the ima-
ging parameters to maximize the speed of single atom imaging is
outside the scope of this paper. However, it may be useful to
speculate briefly about what approaches might lead to even faster
DNA sequencing than the performance discussed above. A speed-
ing up might for instance be possible if the probe was made
intentionally elongated, in such a manner that it would be narrow
along the direction of the DNA strands, and more extended
perpendicular to them. Such a probe shape can in principle be
obtained by using quadrupoles in the condenser part of the STEM
to give different source demagnifications in the two perpendicu-
lar directions. The convergence angle and the ADF detection
angles would be kept cylindrically symmetric—only the source
projected into the sample plane would be made ‘‘elliptical’’.
Additionally, the probe itself could then be made deliberately
astigmatic, so that it would be precisely focused in the direction
along the DNA strand (the ‘‘narrow’’ direction of the probe), but
slightly defocused in the perpendicular, ‘‘long’’ direction. The
Ronchigram pattern, possibly energy-filtered to show only inelas-
tically scattered electrons, would then contain real space infor-
mation about how the probe was centered on the DNA strand, and
this information could most likely be used to steer the probe so
that it remained centered on the strand as it was scanned along it.
97
A quadrupole gun lens is in fact built into every Nion Ultra-
STEM200 [14], and it may be interesting to experiment with using
the lens for this kind of imaging.
The jury is still out on whether DNA sequencing by imaging
labeled strands in the STEM will prove practical or not. However,
there is no doubt that the single atom imaging and spectroscopy
at reduced primary energies shown in Figs. 2–4 will be very useful
in untangling the various morphologies formed by dopants and
adatoms in and on graphene. Using this approach plus also
simultaneous bright field (BF) STEM imaging, Zan et al. have been
able to show several highly relevant results, such as that Cr
attaches to graphene more strongly than Au [43,44], a fact that is
used when fabricating electrical contacts to graphene. In future,
with further improvements in low keV imaging and analysis,
single atom spectroscopy should become nearly as routine as
column-by-column spectrum-imaging has become for crystalline
materials in the last few years [49–51].
It is also increasingly clear that STEM imaging and analysis
offer many advantages over phase-contrast imaging in a conven-
tional TEM (CTEM). Instead of needing to combine many images
of a through-focus series in order to compute an exit wave
reconstruction, as was done for instance in [37], the STEM is able
to show the exact atomic structure of the sample in a single
image, recorded at a specific point in time. It is also able to
identify the type of atoms present in the sample, both by the
recorded image intensities, and by the spectroscopic signature of
the individual atoms. It is even able to explore issues such as
bonding types and charge transfer, by revealing the fine structure
and the exact energy of the single-atom inner shell loss spectra.
This is usefully contrasted with a recent exploration of graphene
impurities [52] (using the most advanced CTEM instrument
presently available), which was unable to identify the impurities
unambiguously.
6. Conclusion
STEM imaging and analysis have come a long way since they
were introduced as practical electron microscopy techniques by
Crewe and co-workers. Thanks to aberration correctors, bright
cold field emission electron sources, and ultra-stable microscopes,
single atoms as light as boron can now be readily imaged, and
almost all single atoms can give highly informative electron
energy-loss spectra, provided of course that they sit still while
being illuminated. Single atoms have recently also been identified
using energy-dispersive X-ray spectroscopy [53,54]. It therefore
seems safe to state that most of Crewe’s goals for STEM instru-
mental performance have now been attained.
To fulfill Crewe’s vision completely, however, more may need
to be done in the area of biological application. Albert had high
ambitions for the STEM in biology, and it seems that the last few
years have brought many more advances in the applications of
STEM in inorganic materials, and that biology may have some
catching up to do. If the sequencing of DNA by atomic resolution
imaging in the STEM actually works out and goes on to find
important applications because of its long read lengths, it will be
especially satisfying to those of us interested in Albert’s legacy.
In the area of instrumentation, incremental advances in areas
such as corrector and electron source design will no doubt
continue to improve the resolution available, particularly at low
primary energies, where better resolution, say of the order of 1 Å
at 10–20 keV, would be very useful. The largest advances in this
direction are likely to come from chromatic aberration correctors,
an area that Albert probably thought too far in the distance to
worry about personally. But they are not likely to produce as
fundamental improvement of microscope performance as Cs and
98 O.L. Krivanek et al. / Ultramicroscopy 123 (2012) 90–98
C5 correctors did, and hence will not propel us far into the ‘‘post-
Crewe’’ electron microscopy era.
One inviting path that was not explored by Albert and his
coworkers is to improve the energy resolution of electron energy
loss spectroscopy. This path is especially promising because it is
very open-ended. Present day monochromators and spectro-
meters allow energy resolution of about 0.1 eV (on sample spectra
rather than just on the zero loss peak), typically at the cost of a
broadened electron probe. Much new information about the
sample would become available if the EELS data could be acquired
at 30, 10, 3 and ultimately even 1 meV energy resolution,
preferably with an atom-sized electron probe. We are now
pursuing this direction [55], and we look forward to being able
to report on what the post-Crewe landscape looks like along
this path.
Acknowledgments
We are grateful to our colleagues at Nion for their part in the
design and construction of the microscopes used here, to Profes-
sor H. Dai of Stanford U. for kindly supplying the graphene
sample, and to Prof. S.J. Pennycook for provision of facilities.
Research at Oak Ridge National Laboratory (MFC) was sponsored
by the Division of Materials Sciences and Engineering, Basic
Energy Sciences, of the US Department of Energy.
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