Application Note

Characterization of Nanoparticles
ZEISS ORION NanoFab SIMS

He
SE
Se-
Author Christelle Guillermier, Ph.D
Carl Zeiss SMT, Inc.

The development of nanotechnology has radically transformed diverse research fields such as physics, chemistry,
material science, and biology. Continued progress relies on the further advancement of characterization techniques
at the nanoscale level. ZEISS ORION NanoFab enables researchers to visualize their samples using a helium beam that
can be focused to a probe size less than 1 nm. And most recently, NanoFab has been equipped with a focused neon
beam and a Secondary Ion Mass Spectrometer (SIMS) to provide elemental imaging with spatial resolution smaller
than 15 nm. The combination of high resolution He+ imaging with Ne+ SIMS elemental mapping on the same platform
yields a direct correlative technique particularly attractive for exploring nanoparticles (NPs) and, in general, nanofeatures
of sizes smaller than 100 nm.

Over the past several years, nanotechnology has played a crucial
role in the field of nanomedicine, diagnostics, drug delivery,
and therapeutics with the development of nanoparticle-based
strategies. The characterization of these nanoparticles (NP) prior
to administration is of the upmost importance. For instance,
their size, morphology, and composition are key parameters,
the control of which will lead to the desired performance.
Conversely, NPs and nanomaterials in our environment are an
increasing threat to human health, both directly and indirectly.
Nanoparticles in the environment will potentially affect biodiversity,
having unpredictable effects on complex and often poorly under-
stood ecosystems (both aquatic and terrestrial). Investigation of
their nano-toxicity will allow the formulation of strategies to limit
these risks. Transmission electron microscopy (TEM) or scanning
electron microscopy (SEM) coupled with energy dispersive X-ray
spectroscopy (EDX) are the conventional analytical techniques to
determine the nanoparticles’ morphology, size, and elemental
composition. However, although sensitive enough to detect trace
elements, SEM and EDX cannot provide elemental information
for the smallest features on a bulk sample or for the lightest
elements; nor can they provide isotopic analysis.
ZEISS ORION NanoFab is a multi-ion beam microscope that
allows for high resolution secondary electron (SE) imaging with
a He+ focused ion beam capable of achieving a sub-nanometer
probe size. The recent addition of a custom-designed magnetic
sector secondary ion mass spectrometer (SIMS) allows for the
detection of all periodic table elements including molecular
fragments and small clusters[1]. Importantly, SIMS with Ne+
provides elemental imaging with spatial resolution smaller
than 15 nm. The combination of high resolution He+ imaging
(<1 nm) with Ne+ SIMS elemental mapping on the same platform
yields a direct correlative technique particularly attractive for
exploring nanoparticles [2] and, in general, features with sizes
smaller than 100 nm.
Characterization of Biogenic NPs
Over the past several years, the use of both nanoparticles and
nanostructured surfaces have emerged as an alternative solution
to antibiotic-resistant bacteria as they effectively decrease bacterial
survival without being highly toxic to mammalian cells. These
nanoparticles, whose sizes span 10 nm to several hundred nm,
are composed of a variety of materials such as pure metals,
metal oxides, and metalloids. Their synthesis relies on physical,
chemical, and recently on an environmentally-friendly approach
involving plants and bacteria[3,4]. The characterization of these
synthesized nanoparticles is of the upmost importance.
Case 1: Tellurium nanoparticles produced via aloe vera extract
Tellurium nanoparticles (TeNPs) were synthesized via a plant-based
synthesis approach. In this approach, aloe vera extract is used as
the reducing and stabilizing agent to prepare TeNPs using Na2TeO3
salt as precursor. The average size of the resulting TeNPs is 20 nm,
as determined by TEM.
Figure 1a and b show the mass spectra obtained via Ne+ SIMS of
a concentrated aloe vera extract and Na2TeO3 salt solution on a
silicon substrate. All the elements detected with SIMS are consistent
with the results of an EDX analysis performed on a similar sample;
however, Secondary Ion Mass Spectrometry provides additional
valuable information that EDX cannot deliver. For example, the
negative mass spectrum reveals the presence of 12C14N-, an
organic fragment often detected with SIMS, as well as 130Te16O-
and 130Te16O - cluster ions not detectable via EDX. Cluster
2
formation is especially interesting as it can only be a result of
the material’s atoms being co-locally present. TeO - in this case,
2
could be an indicator of the NPs chemical composition or a
signature of residual Na2TeO3 salts in the solution.

2
 a b
1500

600

1000
Counts/s

Counts/s
400

500
200

0 0
0 20 40 60 80 100 120 140 160 10 20 30 40 50 60 70
m/z (amu) m/z (amu)

Figure 1 Mass spectra obtained with Neon-SIMS on a concentrated aloe vera and Tellurium salt solution: a) negative secondary ions; b) positive secondary ions.

ORION NanoFab also allows the generation of mass filtered
images or “elemental maps”, with up to four designated masses
detected simultaneously. In this study, we first used the helium
beam to acquire a high resolution secondary electron (SE) image
of an area containing aggregated nanoparticles. The same region
was then imaged with the neon beam using the SIMS spectro-
meter to determine the distribution of 16O-, 12C14N-, 130Te-
and 130Te16O2- (Figure 2a). Interestingly, and although easily
detectable (Figure 1b), the TeO - ion signal is on average five
2
times smaller than that for Te- in these images. CN- and O-
signals also fully correlate with Te- and TeO -. The co-registration
2
of the SE and Te- SIMS images (Figure 2b) confirms that the NPs
are highly enriched in Tellurium and the low TeO - signal may
2
provide additional evidence, at the nanometer scale, that the
salt was effectively reduced to elemental TeNPs. In addition,
the detection of CN- indicates the presence of molecules in
or at the periphery of the NPs.

a b

O- CN- Te- TeO2-

Figure 2 4 μm x 4 μm field of view of aggregated aloe vera nanoparticles:

a) SIMS images of O-, CN-, Te- and TeO2- negative secondary ions acquired simultaneously with
a 4 pA current neon beam at 20 keV of energy; b) overlay of the co-registered, in situ SE (gray)
He
and Te- ions (red) images. SE
Te-

3
a b
CN- Se-
Figure 3 8 μm x 8 μm field of view of S. Aureus and nanoparticles:
SIMS images (a, b) of CN- and Se- negative secondary ions acquired simultaneously with a 4 pA current
neon beam at 20 keV of energy; c) CN and Se ions SIMS images overlay; d) overlay of the co-registered,
in situ SE (gray) and Se- ions (red) images.
Case 2: Nanoparticles produced from S. Aureus
Bacteria, such as Staphylococcus aureus, can reduce selenium-
base compounds within the cultured medium to elemental
selenium. The bacteria that possess selenium detoxification
processes can convert Na2SeO4 salts into elemental selenium
nanoparticles. The selenium nanoparticles diameters span a
range of 80 to 200 nm.
Figures 3a and 3b show the CN- and Se- SIMS images of a
Na2SeO4 salt and media-containing bacteria solution prepared
as a drop on a silicon chip. The CN- image may indicate the
location of bacteria on the substrate, and the hot spots observed
in the Se- image may be attributed to SeNPs. The overlay of Se-
and CN- images (figure 3c) shows the concomitance of organic
materials with selenium particles along with some isolated. The
in situ secondary electron image (Figure 3d) of the exact same
field acquired prior to the SIMS analysis, shows unambiguous
identification of both the S. aureus bacteria and NPs (white
spheres). The overlay of the Se- SIMS image with the directly
correlated SE image demonstrate the selenium composition
of the NPs. This overlay also uniquely reveals the presence of
NPs inside the bacteria, an observation not accessible without
direct correlation of SIMS and SE imaging modalities.
c d
CN- Se-
He
SE
1 µm
Se-
Characterization of Nanoparticles Potential Toxicity
Silver nanomaterials (AgNMs) are one of the most widely
used Engineered Nano Materials (ENMs), with more than
300 commercially available products in Europe. Silver nano-
particles are well established as an antibacterial agent while
silver nanowires (AgNWs) are used in the production of liquid
crystal displays, touch screens and solar cells. However, the
release of AgNMs to the ambient air during manufacturing
processes is of increasing concern for health as, when inhaled,
ENMs efficiently deposit in the respiratory tract [5].
In this study the authors evaluated the potential toxicity of
spherical silver particles and PVP-coated silver nanowires on
an alveolar barrier (lung) 3D representative model. The silver
nanoparticle sizes were 20 nm (Ag20) and 200 nm (Ag200)
respectively. The diameter and length of the AgNW were
50 nm and up to 50 μm respectively. Here, NanoFab SIMS
was used to evaluate the uniformity of the deposit, the
NPs shapes, their agglomeration and chemical composition
after nebulization and settling processes, an important
characterization step for this specific toxicology study.
4
The secondary electron images (Figure 4a) shows that Ag20 a
and Ag200 NPs tend to agglomerate after gravitational settling
on a silicon substrate while the sprayed AgNWs remained
dispersed and maintained their needle-like shape, with the
same initial ratio between length and diameter. The SIMS images
1 µm He 1 µm He 1 µm He
(Figure 4b) for Ag200 reveal that in addition to silver, sodium SE SE SE
and potassium are also present. The 23Na+, 39K+, and 107Ag+
overlay image demonstrates an inhomogeneous repartition
of these elements within the agglomerate. b

In summary, these studies demonstrate the capabilities of

ORION NanoFab SIMS for the characterization of metalloids and
metal nanoparticles, <100 nm in size, synthetized via biogenic
approaches. More specifically, the combination of high resolution
secondary electrons He+ imaging (0.5 nm) with Ne+ SIMS elemental
mapping on the same platform allowed to uniquely reveal the
presence of NPs inside bacteria, information not easily accessible Ag+ K+
with other techniques, if at all.

Additionally, the SIMS analytical technique’s intrinsic property at

generating clusters (TeO2) yielded unique information on TeNPs
compositions and confirmed at the nanoscale – the conclusion
drawn by the author from macroscopic observation. Finally, the Ag+
simultaneous imaging of several elements possible with NanoFab
K+
SIMS allowed highlighting the existence of sub-100 nm elemental
heterogeneities in agglomerated 200 nm silver NPs. Na+ Na+

Figure 4 a) Secondary electron images of the Ag20, Ag200 and AgNWs

distribution, respectively, after nebulization and gravitational setup on a silicon
chip; b) Ag+, K+, Na+ individual SIMS images recorded simultaneously and
overlay of the Na+, K+, Ag+ mass filtered images.

References
[1] Wirtz T, Philipp P, Audinot JN, Dowsett D, Eswara S. High-resolution high sensitivity elemental imaging by secondary ion mass spectrometry:
from traditional 2D and 3D imaging to correlative microscopy. Nanotechnology. 2015;26(43):434001.
[2] Dowsett D, Wirtz T. Co-registered in situ secondary electron and mass spectral imaging on the helium ion microscope demonstrated using
lithium titanate and magnesium oxide nanoparticles. Anal Chem. 2017; 89(17):8957–65.
[3] Medina Cruz, D. et al., 2019. Submitted manuscript
[4] Medina Cruz, D., Mi, G. and Webster, T.J., 2018. Synthesis and characterization of biogenic selenium nanoparticles with antimicrobial
properties made by Staphylococcus aureus, methicillin‐resistant Staphylococcus aureus (MRSA), Escherichia coli, and Pseudomonas
aeruginosa. Journal of Biomedical Materials Research Part A, 106(5), pp.1400-1412.
[5] Fizeșan, I., Cambier, S., Moschini, E., Chary, A., Nelissen, I., Ziebel, J., Audinot, J.N., Wirtz, T., Kruszewski, M., Pop, A. and Kiss, B., 2019.
In vitro exposure of a 3D-tetraculture representative for the alveolar barrier at the air-liquid interface to silver particles and nanowires.
Particle and fibre toxicology, 16(1), p.14.

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