Near-Atomic Three-Dimensional Mapping for Site-Speciﬁc Chemistry
of ‘Superbugs’
Vahid R. Adineh,†
Ross K. W. Marceau,‡
Tony Velkov,§
Jian Li,∥
and Jing Fu*,†,⊥
†
Department of Mechanical and Aerospace Engineering, Monash University, Clayton, Victoria 3800, Australia
‡
Institute for Frontier Materials, Deakin University, Geelong, Victoria 3216, Australia
§
Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria 3052,
Australia
∥
Monash Biomedicine Discovery Institute, Department of Microbiology, Monash University, Clayton, Victoria 3800, Australia
⊥
ARC Centre of Excellence for Advanced Molecular Imaging, Monash University, Clayton, Victoria 3800, Australia
*S Supporting Information
ABSTRACT: Emergence of multidrug resistant Gram-negative bacteria has caused a global health crisis and last-line class of
antibiotics such as polymyxins are increasingly used. The chemical composition at the cell surface plays a key role in antibiotic
resistance. Unlike imaging the cellular ultrastructure with well-developed electron microscopy, the acquisition of a highresolution
chemical map of the bacterial surface still remains a technological challenge. In this study, we developed an atom probe
tomography (APT) analysis approach to acquire mass spectra in the pulsed-voltage mode and reconstructed the 3D chemical
distribution of atoms and molecules in the subcellular domain at the near-atomic scale. Using focused ion beam (FIB) milling
together with micromanipulation, site-speciﬁc samples were retrieved from a single cell of Acinetobacter baumannii prepared as
needle-shaped tips with end radii less than 60 nm, followed by a nanoscale coating of silver in the order of 10 nm. The
signiﬁcantly elevated conductivity provided by the metallic coating enabled successful and routine ﬁeld evaporation of the
biological material, with all the beneﬁts of pulsed-voltage APT. In parallel with conventional cryo-TEM imaging, our novel
approach was applied to investigate polymyxin-susceptible and -resistant strains of A. baumannii after treatment of polymyxin B.
Acquired atom probe mass spectra from the cell envelope revealed characteristic fragments of phosphocholine from the
polymyxin-susceptible strain, but limited signals from this molecule were detected in the polymyxin-resistant strain. This study
promises unprecedented capacity for 3D nanoscale imaging and chemical mapping of bacterial cells at the ultimate 3D spatial
resolution using APT.
KEYWORDS: Cell imaging, atom probe tomography, nanoscale chemical mapping, drug resistance, polymyxin
Infections caused by ‘superbugs’, extremely antibiotic
resistant bacteria, represent a signiﬁcant global health
problem, and it is estimated that antimicrobial resistant
infections will kill one person every three seconds by 2050.1
Based on the ongoing studies on antibiotic resistance, a wide
range of biochemical and physiological mechanisms have been
proposed and debated.2
The diﬀerences between the chemical
composition and structure of susceptible and resistant strains
are considered to carry the fundamental information on the
underlying resistance mechanisms. However, nanoscale or even
near-atomic scale chemical mapping of cellular targets still
remains a technological challenge, and advances could have
immediate impacts on both fundamental and clinical research
particularly for the ﬁght against antibiotic resistance.
So far, several advanced microscopy and mass spectroscopy
techniques have been applied to subcellular imaging. Transmission
electron microscopy (TEM) has been a principal tool
for investigating the ultrastructures of cells, and incorporation
of a cryogenic environment allows high-resolution imaging of
the cellular architectures in their near-native state.3
The
introduction of ﬂuorescence probes and improved resolution
Received: August 13, 2016
Revised: September 15, 2016
Published: September 21, 2016
Letter
pubs.acs.org/NanoLett
© 2016 American Chemical Society 7113 DOI: 10.1021/acs.nanolett.6b03409
Nano Lett. 2016, 16, 7113−7120
in light microscopy also provide an eﬀective yet indirect
approach to examine the spatiotemporal pattern of certain
proteins in cells.4,5
A recent cutting-edge biological imaging
tool is atom probe tomography (APT), which has demonstrated
a signiﬁcant potential in achieving three-dimensional
(3D), near-atomic resolution of biomaterials.6,7
The principle
of APT is well-established:8,9
atoms are progressively removed
from the surface of a cryogenically cooled, needle-shaped
specimen under ultrahigh vacuum conditions by the process of
ﬁeld evaporation. Upon application of a high voltage to the
specimen, a very intense electrostatic ﬁeld is produced at the tip
apex having a radius of curvature less than 100 nm, and by
using either voltage or laser pulsing (notably, the mechanism of
ﬁeld evaporation is diﬀerent between the two pulsing modes:
voltage pulsing increases the applied electric ﬁeld, whereas laser
pulsing increases the thermal energy to overcome the barrier to
ﬁeld ionization, and thereby trigger ﬁeld evaporation) surface
atoms are subsequently ﬁeld ionized and evaporated toward a
position-sensitive detector in a controlled manner. The
chemical identity of each ion is determined by time-of-ﬂight
mass spectrometry (ﬂight time from the tip apex to the
detector), from which the mass-to-charge-state ratio is
determined.8,9
The mass-resolving power of the APT technique
allows the detection of all of the atoms in the periodic table
Figure 1. Schematic of the proposed approach for site-speciﬁc 3D atomic-scale analysis of bacterial cells. (a) A specimen from a single bacterial cells
was retrieved using FIB-lift-out technique, and (b) a site-speciﬁc ﬁnal needle-shaped specimen tip was achieved by precise annular FIB milling and
contained a speciﬁc region of the original cell (either cell envelope or intracellular domain). (c−d) Selected site speciﬁc needle-shaped specimens
from the cell envelope and intracellular domains were observed with conventional TEM. (e) Prior to APT, suﬃcient electrical conductivity is
achieved with a nanoscale layer of metallic coating, which allows ﬁeld evaporation of the specimen tip via pulsed-voltage APT and leads to (f) a massto-charge-state
ratio spectrum of the ionic species followed by (g) an actual 3D reconstruction of the tomographic map at near-atomic resolution.
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with equal sensitivity, including their individual isotopes, and in
various ionic charge states. Following the atom probe
experiment, to produce a tomographic data set, the evaporated
volume is reconstructed in 3D using an inverse projection
reconstruction algorithm and the sequence of detected events.
The tomographic data set typically spans tens to hundreds of
nanometers in depth and contains the spatial coordinates and
elemental identities of tens to hundreds of millions of atoms
with near atomic resolution (∼0.1−0.3 nm in depth and 0.3−
0.5 nm laterally).10,11
The nature of conventional pulsed-voltage APT requires the
specimen to be analyzed to satisfy two criteria: (1) to conform
to a needle-shaped tip with an end radius <100 nm and (2) to
possess suﬃciently high electrical conductivity. For biological
samples, the ﬁrst challenge has been addressed with FIB-based
“lift-out” approaches that were originally developed for TEM
sample preparation12
and have recently been utilized for
specimen preparation of atomic force microscopy (AFM) to
probe interior sections of biological materials.13
After retrieval
of the micron-sized “lift-out” sample from the bulk material,
annular milling with low-current FIB is performed to gradually
reduce the tip radius to the required size.14
The conductivity
requirement has been circumvented by the introduction of
pulsed-laser APT to assist ﬁeld evaporation at the tip surface.
Using both FIB lift-out and laser-assisted APT, it has been
demonstrated that 3D atomic imaging is feasible from a single
Figure 2. Mass-to-charge-state ratio spectra acquired from the cell envelope of A. baumannii, revealing distinct information from both a susceptible
strain (ATCC 19606 S) highlighted in blue and a resistant strain (ATCC 19606 R) highlighted in red.
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mammalian cell (e.g., HeLa cell).15
However, there exists a
well-known challenge of optimizing the laser parameters,
namely, laser duration, wavelength, and energy, which
collectively can contribute to spatial and compositional
errors.16,17
Furthermore, since organic molecules tend to
undergo structural phase transitions at much lower temperatures
relative to inorganic materials, thermally induced ﬁeld
evaporation of organic materials using a pulsed laser is much
more sensitive to these operational parameters and can result in
poorer spatial resolution.18
As an alternative to laser pulsing of an electrically insulated
target, it has been hypothesized that suﬃcient ﬁeld evaporation
can be achieved in pulsed-voltage APT in the presence of a
thin, conductive coating layer on the specimen.18
In previous
studies, numerical simulations of voltage distribution in both
uncoated and coated bacterial cell APT specimens have been
performed, using physical data relating to bacterial cells found
in the literature. The simulation results suggested that, given a
conductive shell of thickness 10 nm or more, a remarkable
uniform voltage distribution throughout the specimen can be
achieved, which is desirable for pulsed-voltage APT study of
biological specimens.19
In contrast, a signiﬁcantly nonuniform
voltage distribution was found on an equivalent uncoated cell
specimen.
A schematic diagram of the proposed approach adopted in
the present study is presented in Figure 1. The intracellular
domain and cell envelope samples retrieved from the target
bacterial cell were fabricated into needle-shape specimens
suitable for APT using well-established FIB lift-out techni-
que,20,21
followed by application of a nanoscale conductive
metallic coating prior to the APT experiments. Key steps of
sample preparation are presented in the Figure 1a−d, and the
complete protocol is provided in Supporting Information
(Supporting Figure 1). In parallel, aliquots of the bacterial
strains were also imaged by cryo-TEM, which allowed the
ultrastructure to be investigated in the near-native state and
provides invaluable insights for interpreting the APT data. Prior
to the atom probe experiments, selected specimens were also
imaged by SEM and TEM (Figure 1c,d) to verify site-speciﬁc
sample preparation from the cell envelope and intracellular
domains of target bacterial cells.
In this APT study, the areas of interest include the
intracellular and cell envelope regions of polymyxin-susceptible
and resistant strains of A. baumannii. Figure 2 demonstrates the
acquired mass-to-charge-state ratio spectra for the cell envelope
region. Also, the mass spectra for the intracellular domain of
polymyxin-susceptible and -resistant strains of A. baumannii are
presented in the Supporting Information (Supporting Figure
2). Assignment of the peaks to ionic species is nontrivial, as is
the case for APT spectra from most organic material,6,15,24
given the number of peaks and also the propensity for detection
of both atomic and molecular ions in various charge states, such
that peak overlap between ionic species can easily occur.6,15,22
Considering the regions of interest and their possible
compositions from the available literature, the peak assignments
presented in the Figure 2 represent the best possible matches.
Figure 3. 3D ion species maps from the APT reconstructions of (a) intracellular domain of a susceptible strain and (b) intracellular domain of a
resistant strain.
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Peaks for Ga+
and Ga2+
(69 and 34.5 Da, respectively), a
typical artifact caused by implantation during FIB sample
preparation, were present, but the amount of Ga implantation is
limited in all spectra (average concentration from 0.9 to
2.24%). Also, peaks corresponding to Ag+
, the conductive
coating material used in the sputter coating process, were
detected at 107 and 109 Da, where the source could either be
the coating layer itself or Ag atoms that have migrated into the
cellular specimens during coating. 1D average atomic
concentration (%) proﬁle of Ag along the length of the
tomographic reconstructions (z-axis) showed that Ag concentration
at the tip apex was relatively higher. After the cellular
region was exposed to the ﬁeld after the removal of the Ag
coating layer, the concentration of Ag signiﬁcantly decreased as
expected (Supporting Figure 3). These two atom types (Ga and
Ag), however, represented only a minor fraction of the
collected ions from the total composition of the intracellular
and cell envelope data (in the cell envelope, 1.12% Ga and
2.73% Ag detected in polymyxin-susceptible strain (ATCC
19606 S); and 1.3% Ga and 1.41% Ag in the resistant strain
(ATCC 19606 R); for specimens from intracellular domain,
2.24% Ga and 0.76% Ag from ATCC 19606 S and 0.9% Ga and
0.29% Ag from ATCC 19606 R). It is rational to exclude Ag
and Ga from further analysis as neither element is considered to
be present in native biological cells. Similarly, it should be
noted that, while hydrogen was detected in the current APT
experiments and could be expected to occur in molecular
species such as hydroxyl and carbonyl groups, some fraction is
likely due to remnant H atoms in the vacuum chamber of the
atom probe instrument. It is well-known that this small atom is
notoriously diﬃcult to remove, even from ultrahigh vacuum
systems.23
This serves to make the exact peak assignment
within the mass spectrum more challenging. However, a
comparison of the mass spectra retrieved from the intracellular
domain of mammalian cells using pulsed-laser APT15
with the
current intracellular bacterial APT data shows that there are
peaks in common, including 12
C+
(12 Da), 14
N+
(14 Da), 16
O+
(16 Da), and H2O+
(18 Da). Notably, there is also a peak at 28
Da, with potential candidates including the carbonyl group
(CO+
) and N2
+
. This particular ion was found in all cellular
specimens but was more pronounced in the mammalian cell15
and bacterial cell envelope. In the present study, the peak at 28
Da was designated as CO, although the chance of partial
involvement of N2 could not be excluded.
Runs containing 1−2 million ions were routinely recorded in
the present study, and long structural features, 5−10 nm wide
running through the tomographic reconstructions of the
intracellular domains of both polymyxin-susceptible and
resistant strains, have been observed (Figure 3a−b). Although
only indirectly supported by complementary cryo-TEM images
(Figure 4a−d), these long structural features were likely the
membrane of the intracellular vesicles. A one-dimensional (1D)
composition proﬁle across these band/interface-like features
suggests that they are rich in C, O, H, and COH and could
therefore be part of an energy storage region.
The atom probe data acquired from cell envelope, on the
other hand, displayed more complicated 3D structural forms
that resemble network/ﬁbril features (Figure 5a-b). A distinct
peak at 31 Da, relating to phosphorus (P), was detected in the
cell envelope but not pronounced in the intracellular specimen
(Figure 2 and Supporting Figure 2). 1D concentration proﬁles
of the network arms observed in the cell envelope specimen
revealed band regions in the order of 5−10 nm with
signiﬁcantly elevated concentrations of C and O. Moreover, a
peak at 58 Da was detected in the cell envelope of the
polymyxin-susceptible strain. The 58 Da species was not
present in the intracellular specimens of the both bacterial
strains in the current work, nor in the mammalian cell study by
Narayan et al.15
In addition, 58 Da was also below the detection
limit in the polymyxin-resistant strain. In the time-of-ﬂight
secondary ion mass spectrometry (TOF-SIMS) imaging of
Xenopus laevis oocytes using C60
+
(Buckminsterfullerene)
primary ions, it has been reported that a peak at m/z value
of 58 was attributed to a fragment of phosphocholine
(C5H15NO4P).24
Considering the region where the data were
collected (cell envelope) and given the fact that the elements C,
H, N, O and P have also been observed in the mass spectrum,
the peak at 58 Da in the APT mass spectrum from the
polymyxin-susceptible bacterial cell envelope likely represents a
phosphocholine fragment (C3H8N+
).24,25
As such, the band
region in the bottom-left area of the tomogram in Figure 5a (58
Da) detected in this study is considered as a candidate for the
phospholipid bilayer.24,25
On the basis of the literature,26,27
it has been hypothesized
that in A. baumannii polymyxin, resistance occurs due to the
loss of the initial binding target, the lipid A component of
lipopolysaccharide (LPS). A comparison of the data obtained
from the cell envelope of polymyxin-susceptible and resistant
strains reveals the presence of phosphocholine fragments only
in the cell envelope of polymyxin-susceptible strain, which
suggests loss or damage of the lipid layers of the polymyxinresistant
cells. Also, colocalization of species (59, 135, 137, 163,
and 165 Da) was observed within the cell envelope of the
Figure 4. Cryo-TEM images of the cell envelope from (a) control and
(b) polymyxin B treated A. baumannii susceptible strain (ATCC19606
S) and from the control and polymyxin B treated resistant strain
(ATCC19606 R) in (c) and (d), respectively.
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polymyxin-susceptible strain (Figure 5a), which implies that
these peaks represent fragments of the same molecule possibly
related to lipids.28,29
It should be highlighted that previous knowledge regarding
changes of the bacterial envelope due to drug resistance was
primarily hypothesized based on bulk analysis data, while our
current data provide the ﬁrst direct imaging of the
corresponding molecules in situ. Repeat mass-to-charge-state
ratio spectra (Supporting Figures 4−7) were also collected to
conﬁrm the consistency of the above-mentioned results and
reinforce the interpretations made. These data also conﬁrm the
repeatability of the pulsed-voltage APT method for analysis of
nonconducting biological material, and agreement between the
replicates for all four cases greatly strengthens the validity of the
work that was performed.
Although the work presented here is an important step
toward adopting APT for biological analysis, there are indeed
some limitations that should be discussed. Notwithstanding the
fact that APT has extraordinary capabilities in terms of mass
and spatial resolution as demonstrated in this study, the ﬁeldof-view
or volume of analysis is extremely small which
inherently imposes restrictions on the translation of the results
into statistical meaningful information. From a sample
preparation point of view, while the in situ sample preparation
challenge has been addressed with the FIB lift-out approach,
precise retrieval and positioning of nanoscale biological targets
at the tip apex are very time-consuming. Moreover, for a
successful APT study of biological cells, dehydration is
recommended in order to avoid the possibility of cell
destruction.15
Although it has been previously shown that
proper protocols can be utilized for cell sample preparation to
limit the impact on the structure and chemical composition of
the cells,15,30,31
it should also be noted that perturbations in
chemical mapping of dehydrated specimens cannot be
completely avoided.15
Nevertheless, the proposed approach in
this study will remain as a promising solution until the
successful development of cryo-transfer of cryo-ﬁxed samples
into the APT instrument. Further challenges include understanding
the complexities in the probing/imaging process, with
respect to ﬁeld evaporation of biological molecules on the tip
apex in the pulsed-voltage mode. Similarly, laser−matter
interaction of biological material under thermal ﬁeld evaporation
in the pulsed-laser mode requires further investiga-
tions.18,22,32,33
Finally, since APT is a relatively new technique
for biological materials, establishing protocols for conﬁdent
peak identiﬁcation in the mass spectrum is essential for accurate
elemental composition analysis of the acquired data.
In conclusion, successful APT experiments have been
performed on bacterial cell specimens prepared by a FIB liftout
approach and subsequently coated with a nanoscale layer of
Ag. These results show that, even without pulsed-laser
acquisition, 106
−107
ions can be collected from coated bacterial
cell specimens compared to only 103
−104
ions from uncoated
control specimens (Supporting Figure 8). Mass spectra and
Figure 5. 3D ion species maps from the APT reconstructions of (a) cell envelope of susceptible strain, and (b) cell envelope of resistant strain.
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reconstructed 3D data volumes were obtained from both the
intracellular domain and cell envelope regions, while damage
from sample preparation, namely, Ga+
ion implantation, was
minimal. The application to explore the drug resistant
pathogens highlighted the capabilities of the proposed APT
method. The distinct mass spectra from polymyxin-susceptible
and resistant strains, primarily at the cell envelope, shed light
on the compositional changes involved in the development of
polymyxin-resistant mechanisms, which were not possible to be
imaged in situ in previous studies. We expect the experimental
approach demonstrated in this work will signiﬁcantly contribute
into the future as a tool to investigate the architecture and
chemistry of biological cells at the atomic-scale resolution.
Materials and Methods. Bacterial Cell Sample Preparation.
Colonies of A. baumannii, both susceptible (ATCC 19606
S) and resistant (ATCC 19606 R) to polymyxin B (from
ATCC), were inoculated into cation-adjusted Mueller-Hinton
broth (CAMHB). Overnight cultures were incubated in a
shaking water bath for 18 h. Log-phase cultures were prepared
by inoculating 1:100 of overnight culture into CAMHB and
incubated for 3.5 h. Also, sterile 1 mg/mL solution of
polymyxin B sulfate was prepared using Milli-Q water and a
0.22 μm syringe ﬁlter. Samples were washed 3 times in
phosphate buﬀered saline (PBS), then ﬁxed in 4% paraformaldehyde
in PBS for 20 min. For each sample, 10 μL of the
suspension was added onto silicon nitride membranes, and the
samples were air-dried in a biosafety cabinet.
The bacterial cells were subsequently transferred to a FIBSEM
instrument (Quanta 3D FEG, FEI Company) for initial
scanning electron microscopy (SEM) inspection at low voltage
(∼1 kV). After selection of a single cell, APT specimen
preparation was performed using the “lift-out” approach and a
chamber-installed micromanipulator (MM3A-EM, Kleindiek
Nanotechnik GmbH), with detailed steps presented in the
Supporting Information (Supporting Figure 1). After successful
lift-out, FIB annular milling was performed with decreasing
inner and outer diameters associated with decreasing current−
voltage parameters in order to achieve the tip radius required
for APT. The radii of the ﬁnal needle-shaped specimens
containing cellular material ranged from 60 to 110 nm.
Sputter Coating Experiments. Sputter coatings were carried
out using a high-resolution sputter coater (Cressington 208 HR
Sputter Coater, Cressington Scientiﬁc Instruments Inc.)
equipped with a Ag target. Imaging of coated tips was
performed using a SEM instrument (Nova NanoSEM 450
FEG SEM, FEI Company). Secondary electron (SE) imaging
was carried out in immersion mode with a 2 kV voltage and
nominal probe current of 14 pA, and imaging of the end tips
was performed with tilt angle of 52°.
Transmission Electron Microscopy. High-resolution imaging
of the biological tips prepared for APT was performed using
TEM (Tecnai G2 F20, FEI Company) with accelerating voltage
at 200 kV. For TEM sample preparation of coated APT tips, a
FIB lift-out approach with a micromanipulator (MM3A-EM,
Kleindiek Nanotechnik GmbH) was performed to attach the
coated APT specimens on the TEM grid with Pt gas
deposition. The conventional plunge freezing method was
also utilized for cryo-TEM imaging of the bacterial strains of
interest. The cells (suspended in the PBS medium) were
transferred onto a TEM grid followed by blotting using a
specialized paper (Waterman, 3M). These samples were then
rapidly immobilized in liquid nitrogen cooled by liquid ethane,
followed by transfer into the TEM with a cryo-sample holder.
Atom Probe Tomography. APT measurements were
conducted using a local electrode atom probe (LEAP 4000
HR, Cameca Instruments) in pulsed-voltage mode. Experiments
were performed with a voltage pulse repetition rate of
200 kHz, pulse fraction (ratio of the pulse voltage to the DC
standing voltage) of 20%, detection rate of 0.5% (0.005 ions/
pulse), and specimen temperature of ∼60 K. Reconstruction
and visualization of the APT data was performed using IVAS
3.6.12 software (Cameca Instruments). To consider the volume
of undetected atoms, a detector eﬃciency of ∼42% as quoted
by the manufacturer was considered in the tomographic
reconstruction steps. Further data visualization was performed
using the commercial software, Avizo 8.1.0 Fire (FEI
Company).
■ ASSOCIATED CONTENT
*S Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.nano-
lett.6b03409.
Details regarding FIB lift-out APT sample preparation
from bacterial cells (Supporting Figure 1), mass-tocharge-state
spectra acquired from intracellular domains
of susceptible and resistant strains of A. baumannii
(Supporting Figure 2), 1D atomic concentration (%)
proﬁle of Ag along the z-axis of the tomographic
reconstruction (Supporting Figure 3), repeats of massto-charge-state
ratio spectra collected from APT experiments
along with corresponding images of the specimen
tips before and after probing (Supporting Figures 4−7),
and application of metallic coating for APT specimen
preparation from bacterial cells (Supporting Figure 8)
(PDF)
■ AUTHOR INFORMATION
Corresponding Author
*E-mail: jing.fu@monash.edu. Fax: 61-3-99051825.
Notes
The authors declare no competing ﬁnancial interest.
■ ACKNOWLEDGMENTS
This study was funded by the Australian National Health &
Medical Research Council (NHMRC, APP1046561), Monash
University Interdisciplinary Research (IDR) Seed Fund, and
the Monash Centre for Atomically Thin Materials (MCATM)
for his PhD top-up scholarship. This work was performed in
part at the Melbourne Centre for Nanofabrication (MCN),
Victorian Node of the Australian National Fabrication Facility
(ANFF). The authors acknowledge use of facilities within the
Monash Centre for Electron Microscopy (MCEM), and the
Advanced Characterisation Facility within the Institute for
Frontier Materials (IFM) at Deakin University. This research
used equipment funded by the Australian Research Council
(LE0882821, LE110100223, and LE120100034). J.L. and T.V.
are supported by research grants from the National Institute of
Allergy and Infectious Diseases of the National Institutes of
Health (R01 AI098771 and AI111965). The content is solely
the responsibility of the authors and does not necessarily
represent the oﬃcial views of the National Institute of Allergy
and Infectious Diseases or the National Institutes of Health. J.L.
is an Australian NHMRC Senior Research Fellow. T.V. is an
Australian NHMRC Industry Career Development Level 2
Nano Letters Letter
DOI: 10.1021/acs.nanolett.6b03409
Nano Lett. 2016, 16, 7113−7120
7119
Research Fellow. The authors would like to thank Drs. Eric
Hanssen and Yu Chen for their assistance in TEM and cryoTEM
imaging.
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