A Virtual Introduction
Each autumn for the last 16 years, WITec has hosted the Confocal Raman Imaging Symposium. This year, we
decided to postpone it due to the ongoing pandemic. It was a very difficult decision for us as the conference is
one of the most important events in our calendar, and one that we especially look forward to. It’s therefore agreat consolation that every speaker we’d invited has confirmed their participation in next year’s event, the 17th
Confocal Raman Imaging Symposium, which will be held in September 2021.
The primary aim of our Symposium is to bring the Raman community together and provide a platform forpresenting and discussing the latest developments and applications in Raman imaging microscopy. Scientificexchange is even more important in the current situation, when international travel involves new precautions
and complications, but research around the world continues to make rapid progress. With this year’s conference
postponed, we wanted to offer an alternative forum for the Raman community to engage and interact. Our
efforts resulted in the first Virtual Raman Imaging Poster Summit, a one-week online poster session that offeredresearchers the possibility to share and discuss results from the convenience of their home offices.
We were amazed at how well the format was received. There were about 250 participants and a total of 55
posters covering various fields of application. The enthusiastic response confirmed that scientists are eager toshare their recent results and find new modes of engagement with their colleagues. It was also apparent thatthe online format of the conference gave it a very international character, because it facilitated participationfrom every global region.
This spring, many researchers had only limited access to their labs, which of course made it challenging tocontinue performing measurements as usual. We saw that many scientists focused on data analysis in thesemonths and profited from the extensive functionality of our software suite and in particular from our special
Home Office License. However, experimental research in laboratories also continues even when the amount oftime spent in home office has increased. In line with these developments, WITec launched the new generationalpha300 apyron in April. This fully automated confocal Raman microscope can carry out self-alignment andself-calibration to optimize reproducibility and performance. Another advantage is that it can be controlled
completely remotely, with only the placement of the sample requiring physical interaction. Thus, even measurements from home offices become possible.
We thank everyone who contributed to the success of the first Virtual Raman Imaging Poster Summit and wehope to welcome many of you next year in Ulm at the 17th Confocal Raman Imaging Symposium.
Stay safe and healthy.

Olaf Hollricher and Joachim Koenen
Managing Directors at WITec GmbH

Poster Abstracts
Advanced Materials Analysis
p. 5
Correlative Imaging Applications
p. 29
Environmental and Geosciencep. 35
Life Sciences, Biomedical andPharma Research
p. 45

L
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UN
IT Y 31
BM AR
SU NU
JA

WITec Paper Award
The WITec PaperAward recognizes exceptional scientific publications in a peerreviewed journal that include results and/or images acquired with a WITec
microscope system. Scientists from all over the world are encouraged to submit
their papers published (print or online) in the current year.
The use of a WITec microscope system should be clearly documented either
in the „Materials and Methods“ section of the article or by other supporting
documentation.
Each submitted paper is valid for one giveaway and any author of a paper
can submit it.
A WITec jury will judge the submitted papers in terms of scientific relevance,data quality and the level of instrument feature utilization. The awards forthe three best papers will be given to the first authors.
Once a year, a jury will appoint the winners of the annual award.
Entry deadline is January 31st of each year.

How to contribute:

01
02
03

Send your paper (PDF format) to papers@witec.de
and include your full contact information.
Receive a WITec thank-you gift foreach new paper submitted.
Automatically participate in the WITec Paper
Award competition. The first authors of thethree best papers will receive a 500 e (GOLD),
300 e (SILVER) or 200 e (BRONZE) Amazon gift card.

Submit new scientific results acquired with a WITec
microscope system to be placed in consideration for the
WITec Paper Award. Each qualifying paper will automaticallycompete for the prize and WITec will send a small present
to the person that submitted a paper.
WITec Headquarters
WITec GmbH
Lise-Meitner-Straße 6 . D-89081 Ulm . Germany
Phone +49 (0) 731 140700 . Fax +49 (0) 731 14070200
info@WITec.de . www.WITec.deAdvanced Materials Analysis

Advanced Materials Analysis
P-110

Investigation of Properties of ZnO Nanostructures by First- Principals Calculations
S. OUDJERTLI
Research Center In Industrial Technologies. CRTI. P.O.BOX 64, Cheraga, Algeria, ALGERIA, Algeria

Structural and microstructural properties were investigated by first-principals computing. The ZnO powder asTransparent Ceramics exhibited a hexagonal crystal structure with space group p63mc of ZnO. We applied
the present first-principals approach to the electronic structure of the ZnO structures. Band structure anddensity of states of the phase of crystal ZnO computed using first principal methods, confirmed that pureZnO is a direct band gap semiconductor when obtained in the B4 type structure phase.

6Advanced Materials Analysis
P-113

3D Stress Mapping and Texture Analysis of Nacre using Correlative Micro-indentation andPiezo-Raman
1

1, 2, 31, 2

1

H.-C. Loh , T. Divoux, F.-J. Ulm , A. Masic
2
MIT, Department of Civil and Environmental Engineering, Cambridge, MA, United States, CNRS–MIT, MultiScale
3
Material Science for Energy and Environment, Cambridge, MA, United States, Univ Lyon, Ens de Lyon, Univ Claude
Bernard, CNRS, Laboratoire de Physique, Lyon, France
1

Nacre (mother-of-pearl) has been extensively studied for its excellent combination of high stiffness, strength,
and toughness. These outstanding mechanical properties have been mostly attributed to the interplay
between aragonite platelets (~0.5 µm thick) and organic matrices in the typical brick-and-mortar structure.
Here, we report a new length scale of ~20 µm that contributes to nacre’s toughening mechanism,
investigated by correlating micro-indentation and piezo-Raman spectroscopy studies. A series of microindentation tests with different maximum force is performed on nacre samples, and the images of theindented area (SEM; Figure 1a) are correlated to the crystallographic texture (polarized Raman; Figure 1b)
and the stress distribution (piezo-Raman; Figure 1c-d). First, the crystallographic texture analysis shows thatstacks of aragonite tablets have the same crystal orientation forming co-oriented aragonite columns with acharacteristic dimension of around 20 µm. Then, by correlating piezo-Raman and micro-indentation results,
we quantify the residual strain energy associated with the strain hardening capacity of nacre samples.
Plotting the residual stress energy stored in the material against the effective indent size, we find that a strain
hardening mechanism is activated when the indent size is in the order of the characteristic aragonite column
height. Our results show that the co-oriented aragonite columns effectively store energy through cooperative
plastic deformation, defining a new length scale of nacre toughening. This work also highlights theeffectiveness of correlative SEM-Raman imaging tools in quantifying and mapping the plastic deformationand the 3D residual stress field, as well as analyzing the crystallographic texture with sub-micron spatial
resolution.

Fig 1 Characterization of an indented nacre sample (a) SEM image (b) Crystallographic texture map; Red and blue colors indicate
different crystal orientations (c) Raman peak position map (d) Residual stress map calculated with piezo-Raman calibration

7

Advanced Materials Analysis
P-114

Characterization of virgin and ion irradiated graphite surface by Raman spectroscopy
M. Wilczopolska, Ł. Kurpaska, J. Jagielski, M. Frelek-Kozak
National Centre for Nuclear Research, Otwock, Poland

Graphite due to its good properties such as good thermal conductivity, high melting point and high thermal
shock resistant is currently used as a core structural component for High Temperature Gas Cooled Reactor(HTGR). Virgin and ion irradiated graphite was investigated by Raman spectroscopy to characterizematerials used in nuclear technologies. Ion irradiation was performed using a 150 keV Ar+ beam in order tointroduce defects in the structure that could be similar to those created by neutrons in the nuclear reactor.
First-order Raman spectra were compared. The lattice damage of graphite is evaluated by comparison of the-1-1
disorder-induced peak (~1348 cm ) with respect to the Raman active E2g2 mode peak (~1581 cm ). Virgin
spectra are significantly different from irradiated graphite surface.

8Advanced Materials Analysis
P-118

Towards vibrational tomography of citrate on dynamically changing individual silver
nanoparticles
T. Ahuja, K. Chaudhari, G. Paramasivam, G. Ragupathy, J. S. Mohanty, T. Pradeep
IIT Madras, Chemistry, Chennai, India

This study explored changes in binding modes of the ubiquitous ligand, citrate on silver nanoparticles(AgNPs) using single-particle surface-enhanced Raman scattering (SP-SERS). Single AgNPs of 50±10 nm
anchored on clean glass slides, having plasmonic scattering in the regime of 620-670 nm, were monitored
for time-dependent SERS with a 632.8 nm excitation laser at 1.3 μW power per nanoparticle. We observed
distinct spectra of citrate during time-dependent SERS. A set of 1400 spectra was analyzed by cluster
analysis which showed the existence of two major sets of clusters, termed ‘SERS-Favorable (SERS-F)’ and‘SERS-Transient (SERS-T)’ spectra, based on their probability of appearance. These distinct spectra
corresponded to a multitude of structures, binding modes, and variants of photocatalyzed products of citrateon the surface of dynamically changing AgNPs. Density functional theory (DFT) simulations were performed
to model the structures and binding modes of citrate on an Ag(111) surface and corresponding Raman
spectra were computed. Experiments performed with deuterated citrate-capped AgNPs provided additional
evidence to understand the shifts in vibrational features obtained in SP-SERS of citrate-capped AgNPs. Our
strategy of systematic analysis of time-dependent SP-SERS spectra can probe structural variations, which
can be used for reconstruction and vibrational tomography of ligands at the single-particle level.

Towards vibrational tomography of citrate on single AgNP

A paper based on this work is in the process of publication.

9

Advanced Materials Analysis
P-119

Investigation of inherent stress using confocal Micro Raman in GaN Heterostructure for RF
HEMT Applications
1, 2

1

1

1

1

1

R. Kaneriya , C. Karmakar , G. Rastogi , R. B. Upadhyay , P. Kumar , A. N. Bhattacharya
2Space Application Centre, ISRO, Microelectronics Group, Ahmedabad, India, Indian Institute of Space Science andTechnology, Department of Physics, Thiruvananthapuram, India
1

Abstract
The influence of AlGaN barrier layer and 6H-SiC substrate on inherent stress in GaN heterostructure isinvestigated using confocal micro Raman measurement. Depth profile measurements were carried out toextract the inherent stress for Al0.3Ga0.7N/GaN heterostructure on 6H-SiC wafer. It has been reported a valley
type inherent stress profile in GaN heterostructure. As we move toward 2DEG from top of AlGaN barrier
layer, pronounced increase of inherent tensile stress is observed. Furthermore, transition from tensile tocompressive stress is found towards the 6H-SiC substrate.
MethodThe inherent stress in growth play a crucial role in the generation of carriers, especially for polarization
depended GaN heterostructure.The GaN heterostructure has been grown by MOCVD on c-plane 6H-SiC
substrate. Layer stakes, thickness and composition for heterostructure is depicted in inset of Fig. 1(a), which
include 60nm AlN nucleation layer, 2 micron GaN buffer layer and 20nm undoped Al 0.3Ga0.7N layer on 6HSiC. We evaluate heterostructure properties by room temperature micro Raman scattering experiments
using WiTec alpha 300 Raman confocal microscope equipped with the 532 nm laser as the excitation lightsource, 1200 lines/mm grating, and 100 X objective lens. The Raman spectra give the information about
lattice vibration and corresponding stress in the material. We adopted Fig. 1(a) measurement methodology
to extract the true nature of inherent stress in depth profiling.

Fig.1 (a) Measurement method (Inset:
Investigated GaN Heterostructure), (b)Depth profile of Inherent stress in GaNHeterostructure

Results
Room temperature Raman spectra of the GaN heterostructure are measured and variation in E 2 (high)phonon peak throughout 4 micron depth extracted. The E2 peak in GaN is very much sensitive to stress inthe films.
-1
The relation between biaxial stress σxx and Raman shift Δω (568 cm select GaN strain free E2-High phonon
peak [2]) is given by[1,2]Δω=Kσxx (1)where K is linear stress coefficient.
For GaN on 6H-SiC,-1
K=2.7 cm /GPa [1,2]The inherent stress calculated using equation (1) is depicted in Fig.1(b).
Conclusion:
The influence of AlGaN barrier layer and 6H-SiC substrate on inherent stress in GaN heterostructure has
been calculated through E2-high phonon peak shifting. This study can be used to improve GaN HEMT
devices performance for RF applications.
References[1] J.-M. Wagner, et al. Applied Physics Letters 77, 346 (2000)[2] V. Yu. Davydov, et al. Appl. Phys. 82, 5097 (1997)
Acknowledgment
We are thankful to Hitesh Mamgain, Application Scientist, WITec, GmbH for providing Raman facility

10Advanced Materials Analysis
P-120

Cascaded nanooptics to probe microsecond atomic-scale phenomena
M. Kamp
University of Cambridge, Physics, Cambridge, United Kingdom

Plasmonic nanostructures can focus light far below the diffraction limit, and the nearly thousandfold fieldenhancements obtained routinely enable few- and single-molecule detection. However, for processes
happening on the molecular scale to be tracked with any relevant time resolution, the emission strengths
need to be well beyond what current plasmonic devices provide. Here [1], we develop hybrid nanostructures
incorporating both refractive and plasmonic optics, by creating SiO 2 nanospheres fused to plasmonic
nanojunctions. Drastic improvements in Raman efficiencies are consistently achieved, with (single7
−1 −1
5
−1 −1
−1
wavelength) emissions reaching 10 counts⋅mW ⋅s and 5 × 10 counts∙mW ∙s ∙molecule , for11enhancement factors >10 . We demonstrate that such high efficiencies indeed enable tracking of single gold
atoms and molecules with 17-µs time resolution, more than a thousandfold improvement over conventional
high-performance plasmonic devices. Moreover, the obtained (integrated) megahertz count rates rival (evenexceed) those of luminescent sources such as single-dye molecules and quantum dots, without bleaching or
blinking.
[1] M. Kamp et al. PNAS 117, p. 14819-14826 (2020).

11Advanced Materials Analysis
P-124

On using statistical Raman Spectroscopy for lithium battery materials characterization
1

1

2

D. Pelegov , B. Slautin , A. Koshkina
2
Ural federal university, Institute of Natural Sciences and Mathematics, Ekaterinburg, Russian Federation, Institute ofSolid State Chemistry UB RAS, Ekaterinburg, Russian Federation
1

Lithium battery (LB) is a cornerstone element of the today’s global economy. About two decades ago, LB
enabled the genesis of portable electronics industry. Today, it plays the crucial role in the global transition
toward electromobility and renewable energy generation. And the Nobel prize in Chemistry 2019 awarded
jointly to John B. Goodenough, M. Stanley Whittingham, and Akira Yoshino "for the development of lithiumion batteries" features its importance both for science and industry.
Raman Spectroscopy is a rather common tool for structural characterization of LB electrode materials [1], but
it is usually considered as an auxiliary one. Generally speaking, there are three most popular applications ofRaman Spectroscopy for LB electrode materials: 1) target phase validation, 2) structural characterization ofcarbon additive, and 3) in-situ studies during lithium intercalation and deintercalation. We believe that there
is some room to grow and Raman spectroscopy usage for electrode material characterization can be more
intensive.
In this presentation, the alternative big-data or statistical approach is discussed and its application for defects
and local heterogeneities probing is proposed. For the first time, the idea of using the statistical analysis ofmultiple Raman spectra for quality control of LB electrodes was proposed in 2001 in paper [2]. Unfortunately,
this idea was not adopted by LB community, but later the similar approach was used for carbonaceous
materials studies [3,4]. Recently, we have revived this approach and demonstrated its advantages forheterogeneity characterization of LB electrodes. In order to discuss all the peculiarities of Raman
Spectroscopy studies in electrode materials, we will focus on two case studies. First, this statistical approach
will be used for the advanced structural defect characterisation in pristine lithium titanate (Li 4Ti5O12) withspinel structure [5]. Second, Raman spectroscopy will be used for structural and morphologicalcharacterization of carbon coverage efficiency in LTO-C composites [6].
References[1] Baddour-Hadjean R., Pereira-Ramos J.-P. Chem. Rev. (2010) 110, 1278
[2] Panitz J. C., Novák P., J. Power Sources (2001) 97–98, 174.
[3] Beyssac O. et al., Spectrochim. Acta Part A Mol. Biomol. Spectrosc. (2003) 59, 2267.
[4] Englert J. M. et al., ACS Nano (2013) 7, 5472.
[5] Pelegov D.V. et al., J. Power Sources (2017) 346, 143.
[6] Pelegov D.V. et al., J. Raman Spectrosc. (2019) 50, 1015.

12

Advanced Materials Analysis
P-125

Chemical Vapor Deposition and Characterization of Transition Metal Chalcogenide
Heterostructures
1, 2

1, 2

1, 2

3

1, 2, 31, 2

L. Peters , C. Ó Coileáin , C. Cullen , R. Siris , G. S. Duesberg, N. McEvoy
2
3
Trinity College Dublin, School of Chemistry, Dublin, Ireland, CRANN, Dublin, Ireland, Universität der Bundeswehr
München, Institute of Physics, München, Germany

1

2D materials are promising materials for numerous electronic applications. Especially transition metal
dichalcogenides are of interest as these have a range of properties varying from metallic to semi-conducting
depending on layer number. This suggests the amazing prospect of creating electronic devices solely from
2D materials through the use of heterostructures. Heterostructures of two dissimilar TMDs result into devices
with atomically sharp and clean interfaces in which the properties of the material can be tuned and the band
1,2
gap can be aligned .
In this work a single-step chemical vapor deposition (CVD) growth method is developed in which a micro3
reactor is used to synthesize MoSe2/WSe2 and MoS2/WS2 heterostructures . This growth method allows theformation of both lateral and vertical heterostructures offering a large range of possible device architectures.
In case of MoSe2/WSe2 automatically-thin pn-junctions can be created without further processing steps dueto the combination of n-type MoSe2 and ambipolar WSe2.
The quality and properties of the synthesized materials were intensely studied by various characterization
techniques such as (low-frequency) Raman spectroscopy, photoluminescence, spectroscopy, x-ray
photoelectron spectroscopy and atomic force microscopy (AFM). Advanced AFM modes such as conductive
AFM and Kelvin probe force microscopy offered deeper insight into the properties of these materials.
1 A. K. Geim and I. V Grigorieva, Nature, 2013, 499, 419–25.
2 A. V. Kretinin, Y. Cao, J. S. Tu, G. L. Yu, R. Jalil, K. S. Novoselov, S. J. Haigh, A. Gholinia, A. Mishchenko,
M. Lozada, T. Georgiou, C. R. Woods, F. Withers, P. Blake, G. Eda, A. Wirsig, C. Hucho, K. Watanabe, T.
Taniguchi, A. K. Geim and R. V. Gorbachev, Nano Lett., 2014, 14, 3270–3276.
3 M. O’Brien, N. McEvoy, T. Hallam, H.-Y. Kim, N. C. Berner, D. Hanlon, K. Lee, J. N. Coleman and G. S.
Duesberg, Sci. Rep., 2014, 4, 7374.

13

Advanced Materials Analysis
P-127

Laser-induced LiFePO4 decomposition
A. Ryabin, D. Pelegov
Ural Federal University, Institute of natural sciences and mathematics, Ekaterinburg, Russian Federation

During the last decade, the lithium battery industry is thriving due to steady demand from automobile andenergy industries. This maturing phase strongly needs production scale growth and cell price decreasing.
Both trends increase the role of quality control (QC) tools, especially ones, suitable for industrial applications.
Raman spectroscopy (RS) is one of the most promising candidates for industrial QC tools due to relatively
low acquisition times, inexpensive equipment and ability to be built-in production lines. So, we are promoting
the idea of using Raman spectroscopy for popular electrode material lithium iron phosphate (LiFePO 4 or
LFP). But there is a significant problem of LFP characterization by Raman spectroscopy – the laser-induced
decomposition.
To understand processes resulting in LFP decay under the action of laser irradiation, we used the
combination of statistical and local approaches to Raman spectroscopy. First, we carried out 26
measurements on powder samples with different values of laser power. Each measurement represents itself
as a scan (mapping) of the 10´10 µm region and consisted of 100 individual Raman spectra. It was expectedthat the probability of LFP decay will rise with increasing laser power. However, the results present anirregular dependence. The observed peculiarity of LFP decay is discussed in the terms of several stages ofthe decomposition kinetics [1]. Second, the decomposition of single particles was studied. We measured
Raman spectra using 1.59 mW power for 75 single particles sedimented on Si substrate. Such an approachallowed to reveal different scenarios of laser-induced phase transitions and estimate the stability of major
band parameters to the continuous action of laser irradiation.

Photo of decomposed single particle and typical spectra of Raman image scan for it

References:[1] A. A. Ryabin, B. N. Slautin, D. V. Pelegov, Three-stage kinetics of laser-induced LiFePO4 decomposition,
J. Raman Spectrosc. (2019). doi: 10.1002/jrs.5803

14

Advanced Materials Analysis
P-131

Non-contact evaluation of carrier concentration for power electronics
A. Myalitsin, O. Milikofu
Nissan ARC, Yokosuka, Japan

With rapid growth of wide-band-gap semiconductors for efficient use of energy everywhere from homeelectronics to high speed railways and heavy machinery for mining and construction, the need for a fast andnon-destructive method to evaluate the raw materials, as well as device quality, also increases. Whilecurrently SIMS (secondary ion mass spectroscopy) is the most sensitive method to evaluate the dopant
concentration in semiconductor surfaces, it is destructive and therefore unfavorable to use between device
manufacturing steps. On the contrary, Raman spectroscopy is a fast, contact-free and non-destructive
method.
We used the confocal microscope “Alpha 300R” in back-scattering geometry to map the Raman signal of ann-type doped 4H-SiC epitaxial layer on a 4H-SiC wafer as a function of depth (figure 1a). It is well-known that-1
the A1 longitudinal optical (LO) phonon around 980 cm shows a significant peak shift and asymmetricbroadening as a function of dopant concentration, due to phonon-plasmon coupling. [1] As the concentrationof free electrons increases, the plasmon frequency shifts to higher wavenumbers and the LO plasmoncoupled mode (LOPC) becomes assymetrically broadened, while shifting to higher wavenumbers. This
broadening has been observed and modelled for single crystal 4H-SiC. [2-4] However, in the case of
epitaxially grown films, the sample cannot be considered uniform. Indeed, with increasing depth we see anincrease in free electron concentration at the interface (figure 1b). Therefore, special care should be taken
when applying the LOPC model to epitaxial wafers. Here we show a method for routine estimation of free
carrier concentration based on confocal Raman imaging of a sample surface/depth, and fitting of theexperimental Raman LO phonon line shape to theory.
[1] G. Irmer, V. V. Toporov, B. H. Bairamov and J. Monecke, Physica Status Solidi (b), 1983, 119, 595–603.
[2] S. Nakashima, T. Kitamura, T. Kato, K. Kojima, R. Kosugi, H. Okumura, H. Tsuchida and M. Ito, Applied
Physics Letters, 2008, 93, 121913.
[3] H. Harima, S. Nakashima and T. Uemura, Journal of Applied Physics, 1995, 78, 1996–2005.
[4] J. C. Burton, L. Sun, M. Pophristic, S. J. Lukacs, F. H. Long, Z. C. Feng and I. T. Ferguson, Journal ofApplied Physics, 1998, 84, 6268–6273.

Figure 1: (a) Depth profile of the n-doped 4H-SiC wafer (red), with the undoped epitaxial layer on top (green). (b) Spectra of the LOPC
mode as a function of depth.

15Advanced Materials Analysis
P-132

Raman and Photoluminiscence study in order to determined the aluminum concentration of
AlxGa1-xN
1

2

1

3

L. MARTINEZ-ARA , P. MALDONADO-ALTAMIRANO , J. AGUILAR-HERNANDEZ , M. HERNANDEZ-PEREZ , G.
1
CONTRERAS-PUENTE
1
2
NATIONAL POLYTECHNIC INSTITUTE, PHYSICS, MEXICO, Mexico, CINVESTAV-NATIONAL POLYTECHNIC
3
INSTITUTE, MEXICO, Mexico, NATIONAL POLYTECHNIC INSTITUTE, CHEMESTRY, MEXICO, Mexico

AlxGa1-xN films on different substrates, Si (111), Al2O3 (0001) and amorphous quartz were grown by Pulsed
Laser Deposition (PLD). A Nd-YAG laser tuned at 1064 nm wavelength, 12 ns pulse duration and arepetition rate of 50 Hz, was used for the ablation process. The energy of the laser beam focused at thetarget surface was 50 mJ/pulse. Aluminum concentration was determined by photoluminescence (PL) and
Raman spectroscopy. Photoluminescence (PL) measurements were carried out with a He-Cd laser as
exciting light, tuned at 325 nm, with a power of 40 mW. The PL at 10 K shows that the yellow band (YL) hasa shift of 170 meV at higher energy in the ternary in comparation with the gallium nitride (GaN). Such shift of
the PL maximun together with the widening of the YL band is related to the formation of the ternarycompound. Raman spectra were mesured at room temperature by using a He-Ne laser, 633 nm, it with an
output power of 30 mW, in a LabRam HR Evolution system. The Raman spectra show peaks at 569 and 655-1-1
cm modes associated to GaN and AlN respectively, and a peak at 772 cm , this frequency corresponds tothe vibrational mode A1(LO), this increases continuously from the mode of GaN to the corresponding A 1(LO)mode of the AlN when the aluminum concentration increases. Beginning with displacement of the vibrationalmode the aluminum concentration was determined, it turned out to be 15 %.

16Advanced Materials Analysis
P-133

Confocal Raman imaging of ferroelectric domain structure in KTA single crystals in the bulk
A. Akhmatkhanov, M. Chuvakova, E. Greshnyakov, V. Shur
Ural Federal University, School of Natural Sciences and Mathematics, Ekaterinburg, Russian Federation

The single crystals of potassium titanyl phosphate (KTiOPO 4, KTP) family [1] have long been recognized as
one of the main materials of nonlinear optics. These uniaxial ferroelectrics are commonly used for creation of
the tailored domain structures (domain engineering). It implies that the efficiency of nonlinear opticalinteractions in these crystals can be improved further by periodical poling for realization of quasi-phasematching conditions. The potassium titanyl arsenate (KTiOAsO 4, KTA) possesses the properties
outperforming KTP which resulted in creation of periodically poled KTA crystals (PPKTA) for various
nonlinear optical applications [2]. The periodical poling in KTA consists in application of electric field pulse to
the photolithographically defined stripe electrodes to form the precise periodical domain pattern [1]. The
undesirable pronounced domain broadening out of the electrodes in KTA crystals requires the deep
knowledge of domain kinetics during poling process which implies the possibility of domain structure imaging
in the bulk.
The confocal Raman microscopy (CRM) has shown to be a powerful technique for imaging of domain
structure in the bulk [3] based on the detection of the shifts of certain Raman peaks on the domain walls. It
has been used previously for domain structure imaging in other ferroelectrics but never applied to the KTA
crystals. The model quasi-regular streamer structure formed in KTA after partial polarization reversal (field
2.6 kV/mm, pulse duration 10 ms) has been visualized by CRM using Alpha AR300 (WITec, Germany). The
incident laser beam was linearly polarized along [010] crystallographic direction. The Raman spectrum in-1
KTA at the domain wall and in the bulk differed by the intensity and the position of the peaks near 675 cm-1-1
and 730 cm . We have used the shift of the peak 730 cm for domain wall imaging as the most sensitive.
The domain structure imaging with spatial resolution down to 350 nm has been demonstrated.
The equipment of the Ural Center for Shared Use “Modern Nanotechnology” Ural Federal University wasused. The research was made possible by support from President of Russian Federation Grant for young
scientists (grant No. МК-1217.2019.2) and by Russian Foundation for Basic Research (grant No. 18-2920077-mk).

[1] V. Ya. Shur et al, Ferroelectrics 496, 49(2016)[2] A. Zukauskas et al, Opt. Mater. Express 3, 1444(2013)[3] V. Ya. Shur et al, J.Appl.Phys. 116, 066802(2014)17

Advanced Materials Analysis
P-136

Dentin-Dental Adhesive Interface Characterization by Raman Microscopy and Atomic ForceMicroscopy
1

2

1

1

2

2

1

B. Anich , B. Dippel , H. Loll , A. Lopez , G. Mishra , M. Moos , C. Thalacker
2
3M Deutschland GmbH, 3M Oral Care, Seefeld, Germany, 3M Deutschland GmbH, Corporate Research Analytical
Laboratory, Neuss, Germany
1

Dental adhesives are used for dental restorations. In dental restoration, a dentin-adhesive interface typicallyforms a strong bond between the dentin and composite material. The quality of a dental restoration depends
strongly on the properties of a hybrid layer which is formed between the dentin and the adhesive. This hybrid
layer consists of a demineralized dentin zone which is infiltrated with adhesive. Demineralization can beaccomplished by an additional etching step (total-etch, TE) of the dentin or by self-etch (SE) adhesives.
In this study, we investigated the morphology and chemical nature of the dentin-adhesive interface of anexperimental and a commercial Scotchbond Universal (3M) adhesive.
Methods:
The labial surface of bovine incisors was ground to expose dentin and treated with the experimental or thecommercial Scotchbond Universal (3M) adhesive in total etch (TE) or self-etch (SE) modes according to
manufacturers’ instructions.
TM
A 1 mm thick layer of a composite material (Filtek Z250 (3M) A3) was placed on the adhesive and cured
TM(Elipar S10, 3M). Samples were ground perpendicular to the bonded surface.
The interfaces were investigated with confocal Raman microscopy (Witec 300R with a 100X objective, NA
0.9; excitation wavelength: 532nm) and Atomic Force Microscopy (AFM) (Dimension Icon, Bruker)
Results:
AFM images and Raman linescans were recorded from dentin-adhesive-composite interfaces. Ramanlinescans of the interface composite-adhesive layer-hybrid layer-dentin were recorded with high lateral
resolution. For each sample, a distinct adhesive and a hybrid layer were detected. Raman microscopy
revealed full adhesive penetration of the hybrid layer.
Conclusions:* Micro-Raman and AFM results complement each other.
* Raman Microscopy and AFM show that the experimental adhesive has a similar interaction with dentin asTMclinically proven Scotchbond Universal (3M) adhesive* Both adhesives achieved complete infiltration of the hybrid layer* The hybrid layer was significantly thicker for total-etch vs self-etch samples.
* There was no significant difference for hybrid layers between experimental and commercial adhesive whenused in the same etching mode.
* The experimental adhesive afforded similar adhesive layer thickness as the commercial Scotchbond
TM
Universal (3M) adhesive in TE mode, and lower adhesive layer thickness in SE mode18

Advanced Materials Analysis
P-143

Imaging the distribution of additives in different materials using Raman microscopy
J. Swart, C. van Hoorn
Nouryon, ECG-MAS, Deventer, Netherlands

At ECCD (Expert Capability Centre Deventer), part of Nouryon, a chemicals manufacturer, Raman imaging isused to provide insight in the distribution of organic and inorganic additives in all kinds of materials, e.g.
polymers, paints, coatings and chemicals. The distribution of components in the matrix plays a crucial role in
the functioning and end properties of a material.
Our poster will explore two different applications of using Raman microscopy to study the distribution of
additives in a material.
In the first example, we show how this technique can be used to study the migration properties of small wax
particles in a coating. These wax particles function as a supply source for migration of wax to the surface,herewith enhancing protection against fouling and mechanical damage.
The second example shows Raman imaging used as a tool to visualize the homogeneity of peroxidephlegmatizer particles. Phlegmatizers are chemical components used to reduce the hazards of highly
reactive peroxides during transport and while in storage. Preferably, the two components are mixed
homogeneously. Raman imaging offers a fast method to investigate the distribution of peroxide andphlegmatizer, on both the surface and the inside of the particles.
In both examples the ’cluster analysis’ data evaluation tool was used to identify the different components
present in the investigated materials. In the second example, a statistical method was applied to enable
comparison of different mixing technologies in a semi quantitative way.

19

Advanced Materials Analysis
P-145

Structural Study of PA66/PA612 Blends for Automobile Applications
O. Milikofu, A. Myalitsin, A. Tominaga, A. Kato
Nissan ARC, Ltd., Nanomaterial Analysis Group / Nanospectroscopy, Yokosuka, Japan

Common polyamides like PA6 and PA66 are attractive materials for automobile industry due to ease-toprocess, light weight and added mechanical strength in thermoplastic composites, lacking, however,
sufficient hydrolytic and chemical stability. In comparison to PA66, PA612 absorbs only 1/2 ~ 1/3 amount ofwater [1,2], but lacks strength and is much more expensive. Blends of PA612 with PA66 were reported tohave improved mechanical properties and stability [3,4]. Based on thermal studies, phase separation was
suggested, however, was never confirmed directly.
In this study, we used PA66:PA612 blends with weight ratio 10:0, 7:3, 3:7 and 0:10, kindly supplied by
AsahiKasei Corporation. Confocal Raman microscope alpha 300R (WITec GmbH, 532nm) in back-scatteringgeometry was used to obtain 2D and 3D maps of the samples. Data were processed and analyzed with
Project FIVE software.
-1
First, Raman spectra of pure PA66 and PA612 samples showed small differences in the 900-1700 cm
range (amide, CH2 and CCO groups). Instead, the shift of the spectral weight (center-of-mass) of CH2-1
stretching region (2850-2950cm ) was more prominent. This parameter was used in the blends to confirm
validity of the basis analysis with component de-mixing. Two basis components were identical to pure PA66
and PA612 spectra, and their calculated relative concentration was consistent with weight ratio. Clear seaisland structure was observed.
Second, we compared crystallinity of the samples. The 7:3 blend had denser and more regular hydrogenbond network than the 3:7 one. It appears that the minor component worked as a seed for crystallization of
the major component. Within the blends, PA66 domains had consistently higher crystallinity than PA612
ones.
Finally, the solubility parameter d, calculated from the cohesive energy and mole volume of the repeating
3 1/2
units, was found to be 27.975 and 24.887 (J/cm ) for PA66 and PA612, respectively. The values are close
but not the same, and taking into account hydrogen bonding, phase separation can be explained.
1 ARKEMA Corporation, Polyamide data sheet, 2015 (in Japanese).
2 S. Goto, H. Yamazaki, M. Nozaki, DENSO Technical review, 2009, 14, 89 (in Japanese).
3 D. Rusu, M. Rusu, Polyamide-based blends, in Handbook of Polymer Blends and Composites, 2003, 4a,201.
4 T.S. Ellis, Polymer, 1992, 33, 1469.

Structure (red: PA66, blue: PA612) and hydrogenbond network in 3:7 (a, b) and 7:3 (c, d) blends

20Advanced Materials Analysis
P-148

Individual flakes and films of graphene oxide patterned by laser using Raman spectroscopy
and atomic force microscopy
1

1

2

1

L. PEREZ , F. FIORAVANTI , N. BAJALES LUNA , G. LACCONIINFIQC-CONICET, Universidad Nacional de Córdoba, Dpto. Fisicoquimica, Facultad de Ciencias Quimicas, Córdoba,
2
Argentina, IFEG-CONICET, Universidad Nacional de Córdoba, Facultad de Matemática, Astronomía, Física yComputación, Córdoba, Argentina
1

Raman spectroscopy was used for quality control and identification of graphene oxide (GO) flakes, but it isable to generate a laser-induced reduction process during the spectroscopic characterization. However, only
few recent reports have advised about potential changes induced by laser irradiation, even at low power,
during the measurements of such systems. Thus, an overview of this phenomenon is urgently required
because it leads to new alternatives for obtaining hybrid surfaces of thin two-dimensional materials, which
are useful for diverse applications. Individual flakes and films of GO were deposited on silicon oxide andexposed to visible laser radiation during characterization by micro-Raman spectroscopy. Noticeable
topography modifications on both GO individual sheet and film surfaces were observed in the optical image,recorded after spectroscopic measurements. These surface morphology modifications together with anapparent thickness change were induced by the visible radiation in the graphene-based coating. Likewise,3
chemical changes of the oxygen functional groups (sp carbon defects in the GO structure) were recognized,
identifying the decrease of the oxygen content by means of X-ray photoelectron spectroscopy, whichevidenced the formation of reduced graphene oxide (rGO) in the trace generated by the laser, when asample movement control is available. Differences of characteristics between components GO and rGO in
the hybrid were supported also by SEM, dark field microscopy and NSOM images. Comparison between
laser-induced effects in supported GO films and GO flakes, as well as a correlation with changes observed in
Raman spectra and temporal evolution were performed. This study contributes to the quality control of
supported GO flakes and films because it provides information about physical damage produced by the laser
radiation during Raman measurements. In addition, the combined AFM-Raman spectroscopy researchstrategy here employed, introduces a simple lithographic method to obtain GO-rGO hybrid materials by using
a continuous visible laser to pattern GO flakes and films.
Keywords: Lithographic pattern; graphene oxide; microchannels; Raman imaging; atomic force microscopy.

21Advanced Materials Analysis
P-149

Characterization of thiolate-protected QDs on acrylic and silicone-based elastomers films
1, 2

1, 3

2, 4

1, 2

P. Jofré-Ulloa , M. Alegía , D. Guzmán de la Cerda , C. González-Henríquez
2
Universidad Tecnológica Metropolitana, Departamento de Química, Santiago, Chile, Universidad Tecnológica
Metropolitana, Programa Institucional de Fomento a la Investigación, Desarrollo e Innovación (PIDi), Santiago, Chile,
3
Universidad Tecnológica Metropolitana, Doctorado en Ciencias de Materiales e Ingeniería de Procesos, Santiago,
4
Chile, Universidad Mayor, Centro de Nanotecnología Aplicada, Santiago, Chile
1

The creation and development of new materials with highly adjustable properties is a rapidly expanding field
due to the significant technological advances in recent decades. Some smart materials, such as dielectric
elastomers (DE), can change their electromechanical characteristics by applying electric fields, which makes
them ideal for transducers.
The incorporation of Quantum Dots (QDs), semiconductor nanocrystals with optoelectronic properties
depending on their size, shape, and chemical composition could modify their optoelectromechanicalproperties. These properties provide a wide range of applications, especially in optoelectronic devices andbiomedicine, where the use of cadmium chalcogenides QDs have been intensively investigated. Modifying
polymers with nanoparticles (NPs) to form polymer nanocomposites (PNCs) capable of responding to
specific stimuli has been a major field of research.
In this work, we used PDMS and 3M VHB 4910 (AR) as substrates to obtain new PNC materials. Both DEs
materials have been modified by different methods with CdTe-MSA QDs obtained from organometallic
synthesis, but not by aqueous synthesis. The surface was modified following UV-ozone exposure andsubsequent formation of a Self-Assembled Monolayer (SAM) with (3-aminopropyl)trimethoxysilane (APTMS),
which served to anchor the thiol capped-QDs to the elastomer.
The covalent bond between the thiol coating and the SAM was verified by confocal Raman spectroscopy.
Considering the QDs fluorescence, samples for Raman spectra were characterized on gold surfaces, forquenching, and a lower energy laser (λ = 784 nm) resulting in the corresponding spectra of the modified
PNCs. The covalent bond was confirmed on samples APTMS-QDs of different sizes, corresponding to amide
signals.

Scheme incorporation of quantum dots in elastomer films and the representation of the covalent bond forming
an amide between the organic coating and the SAM.

22

Advanced Materials Analysis
P-159

Nanofabrication of Metal Free SERS Substrate
A. Kundu, R. Rani, K. Hazra
Institute of Nano Science and Technology, Mohali, India

Black Phosphorous (BP), having intrinsic in-plane ferroelectric properties may have inherent capability of
SERS response and could be considered a replacement of metal nanoparticles based SERS substrate. A
simple one-step process has been demonstrated for controlled nano-structuring and rapid prototyping on BP
flake to develop metal free SERS substrate by low power focused laser irradiation. The effect of focusedlaser irradiation on the surface morphology of pristine BP flakes has been thoroughly investigated by realtime Raman spectroscopic measurements and corresponding AFM height profiling, which confirms that the
proposed laser irradiation technique has more advantages over conventional lithography process and freefrom undesired contamination. For 532 nm laser line, the minimum laser power needed to create a nano-void5
2
on BP flake is 25 mW (Power density ~ 15.62 x 10 W/cm ) with 5 sec exposure time where the etching rateis controllable by the laser power and exposure time. By analyzing the geometrical shape of the nano-void,
created due to laser irradiation, it is possible to identify the armchair and zigzag direction of BP flake.
Experimental results reveal that by controlling the exposure time and laser power it is possible to perform
layer by layer thinning of BP flakes. The proposed thinning process of the BP flake does not alter the pristinequality and no signature of oxidation is found in the Raman spectra, which signifies the reliability of this low
power laser irradiation technique towards the future of nano-fabrication of BP based devices. Controlledformation of nano-voids array on few layer BP flake induces enhanced local electric field (hot spots) at thevicinity of the nano-voids, resulting in ~ 30% Raman intensity enhancement. Such nano-voids induced
hotspots on BP flake open up a new species of metal free SERS substrate, demonstrating pronounced
6enhancement in Raman signal of Rhodamine B as high as of the order of ~10 and with a limit of detection(LOD) upto ~10 nM.

23Advanced Materials Analysis
P-161

Synthesis of silver nanoparticles using Silybin B or silibinin as reducing agents induced bylight and the raman characterization study
1

2

1

1

1

A. Solís-Gómez , R. A. Guirado-Lopez , J. G. Bañuelos-Muñetón , J. M. Saniger , M. E. Mata-Zamora
Universidad Nacional Autónoma de México, Instituto de Ciencias Aplicadas y Tecnología, Mexico city, Mexico,
2
Universidad Autonoma de San Luis Potosi, Instituto de Física, San Luis potosi, Mexico

1

Silybin is an antihepatotoxic polyphenolic substance isolated from the milk thistle plant, used for thetreatment of cirrhosis, hepatitis, and alcohol-induced liver disease which is also under study because of its
possible neuroprotective effect in Parkinson’s disease. Silibinin is a mixture of silybin A and silybin B in a 1:1-5-6
ratio. The reduction of silver nitrate with silybin B or silibinin (10 , 10 , M) without additional steps produced
silver nanoparticles can be used as SERS substrates. The SERS enhancement of these silver nanoparticles
was tested using the same reductors.
The structure and composition of the silver nanoparticles were characterized by UV−visible spectroscopy,
atomic force microscopy (AFM) and Raman spectroscopy, using a 532 nm laser. The evolvement of acharacteristic surface plasmon extinction spectrum in the range of 420 nm to 480 nm indicates the formationof silver nanoparticles after mixing silver nitrate solution and silybin B or silibinin. Raman maps wereacquired and analysed to identify silybin B or silibinin raman spectra. We also simulate Raman spectra ofsilybin B and silybin A with silver based on density functional theory (DFT) calculations .

References:[1] A Solís-Gómez, RY Sato-Berrú, ME Mata-Zamora, JM Saniger, RA Guirado-López. Characterizing theproperties of anticancer silibinin and silybin B complexes with UV-Vis, FT-IR, and Raman spectroscopies: A
combined experimental and theoretical study. Journal of Molecular Structure. 1182 (2019), 109-118.
[2] R. Y. Sato-Berrú, A. R. Vázquez-OlmosE, V. Mejía-Uriarte, et al. Synthesis of Silver Colloids with aHomemade Light Source. Journal of Cluster Science. 29 (2018), 719–724.
[3] R. Mohammadinejad, Sh. Pourseyedi, A. Baghizadeh, Sh. Ranjbar, G. A. Mansoori. Synthesis of Silver
Nanoparticles Using Silybum Marianum Seed Extract. Int. J. Nanosci. Nanotechnol., 9 (4) (2013), 221-226.

Acknowledgement: The authors acknowledge the support of the IN-111216 and IN-102917 PAPIIT-UNAM
project and CONACYT 2014-Fronteras de la Ciencia 2016 project.

24

Advanced Materials Analysis
P-162

Decoration of graphene oxide (GO) with metallic nanoparticles (NPs) for silybin detection at
low power laser
A. Solís-Gómez, J. G. Bañuelos-Muñetón, J. M. Saniger
Universidad Nacional Autónoma de México, Instituto de Ciencias Aplicadas y Tecnología, Mexico city, Mexico

Photodynamic therapy is one of the proposed ways to increase temperature locally in malicious cells.
However, the low power used in the photodynamic therapy lasers require, typically, metallic particles to aideabsorbing the heat. Another obstacle is the possible toxicity of the metallic particles. Silybin is anantihepatotoxic polyphenolic substance isolated from the milk thistle plant, used for the treatment for several
diseases. Carbon and its allotropic forms are subject to a longstanding tradition in research. All these
exhibits interesting open scientific questions and technological challenge to tackle. The present investigation
proposed a facile method for the decoration of graphene oxide (GO) with Gold (Au) and Silver (Ag)nanoparticles (NPs). The hybrid materials (Au–GO, Ag-GO) were prepared by in-situ reduction of HAuCl4
and AgNO3 into graphene oxide (GO) dispersions using sodium borohydride and sodium citrate as reductant;
the growth of gold and silver nanoparticles was induced by the joint effect of light and chemical reductor. The
resulting hybrid materials were characterized with UV–Vis, Raman spectroscopies and Atomic Forcemicroscopy. The characterizations revels the nanoparticles growth were induced by the joint effect of lightand chemical reductor. SERS substrates were used to detect silybin at low concentration using low power
laser.
Acknowledgment:
The authors acknowledge the support of the IN-111216 and IN-102917 PAPIIT-UNAM project and
CONACYT 2014-Fronteras de la Ciencia 2016 project25

Advanced Materials Analysis
P-170

Raman imaging as complementary technique for the study of molecular self-assembly on
surfaces
1

2

3

M. L. Para , O. E. Linarez Pérez , M. López Teijelo
INFIQC-CONICET-UNC, Departamento de Fisicoquimica, Facultad de Ciencias Químicas, Universidad Nacional de
2
Córdoba, Córdoba, Argentina, INFIQC-CONICET-UNC, Departamento de Fisicoquimica. Facultad de Ciencias
3
Químicas. Universidad Nacional de Córdoba, Córdoba, Argentina, Universidad Nacional de Córdoba, Departamento de
Fisicoquimica, Facultad de Ciencias Químicas., Córdoba, Argentina
1

The self-assembled monolayers (SAMs) are bidimensional arrays spontaneously generated onto the surfaceof different substrates in contact with molecules containing specific anchorage groups. Among usuallyemployed adsorbates, alkanethiols and aromatic molecules present special characteristics that confer
interesting properties to the surface for applications in electrochemistry, molecular electronics andbiochemistry. In the case of aromatic molecules, π−π attractive interactions between neighbor molecules
also appear, favoring formation of domains with an ordered packing, which are commensurate to and withthe same symmetry of the surface. Since the layers are often discontinuous, it is important to know the
distribution of the exposed areas that remain unchanged. Then, the uncovered areas may present
electroactive response and can be easily evaluated by electrochemical techniques.
In this work, we use Raman spectroscopy as a complementary technique for the study of isonicotinic acid
(INA) adsorption process on gold surfaces. Raman results allow obtaining conclusive evidences of both thestudy of chemical interaction between adsorbate and surface as well as monitoring the surface coverage
evolution by means of 2D mapping.

26

Advanced Materials Analysis
P-176

Magnetic graphene oxide-based composite: An attractive approach towards molecularsensing
1

2

2

2

3

4

D. M. Arciniegas Jaimes , P. Márquez , A. Ovalle , J. Escrig , O. Linarez Pérez , N. Bajales
2
IFEG, CONICET, Ciencia de Materiales, Córdoba, Argentina, Universidad de Santiago de Chile, Department of3
Physics, Santiago de Chile, Chile, CONICET, INFIQC - FCQ,UNC, Departamento de Fisicoquímica, Córdoba,
4
Argentina, IFEG, CONICET - FaMAF, UNC, Ciencia de Materiales, Córdoba, Argentina
1Magnetic nanostructures may be composited with graphene-like materials to obtain enhanced multiplexed
attributes. A current low-cost source of easy access and non-toxic nature, is focused on graphene oxide(GO). Epoxide and OH functional groups make the GO hydrophilic and dispersible in aqueous media withlong-lasting stability. Moreover, GO behaves as insulator or semiconductor, depending on the oxidation
degree. One of the most recent applications of GO is its use in molecular sensors, since it provides large
surface area and the presence of functional groups on the surface which serves as anchorage points to pin
molecules. Thus, functionalization of GO with metal or metal oxide nanoparticles, giving rise tomultifunctional composites, can enhance the electrochemical sensing properties. In this work, permalloy
nanowires (Py NWs)/GO composite was obtained by decorating thick GO multilayered films with ~30 nm indiameter and 1 µm long Py NWs, intercalated with aleatory distribution among the GO layers. Py NWs weresynthetized by electrodeposition inside alumina templates, and the carbonaceous matrix was prepared fromcommercial GO by applying sonication and sequential deposition steps. SEM images showed that the GOfilm surface is decorated with several decoupled Py nanowires arrays; most of them oriented parallel to theGO surface plane and probably overlapped among some GO layers. This indicates that Py agglomerates arelocated around the edges of the multi-layered GO thick films and also intercalated among their layers, which
is probably due to the anchorage of Py NWs to GO layers promoted by the functional groups in GO. SAXS
measurements provided additional evidence for the distribution of particle sizes in the composite. An
exhaustive comparative analysis between Raman spectra of GO and Py NWs/GO composite indicates thatthe chemical nature of GO in the composite is preserved. On the other hand, the bands expected for the Py−1
NWs are not resolved in the corresponding Raman spectrum when it is acquired from 250 to 3250 cm . Thisis probably due to the low density of decoupled nanowires in the Py sample. Identification of relevant bands−1
were positioned around 445 and 613 cm , and have been associated to the Ni–O and Fe–O, respectively.
This result suggests a certain degree of oxidation of the NWs in the sample that was also supported by XPS
measurements. Thus, Py NWs/GO composite arises as an attractive potential material towards molecularsensing.

Figure: (left) Py NWs/GOsketch, (center) Raman spectra of GO (green), Py NWs/GO composite (red), and Py NWs(blue), (right) Charge-transfer resistance of the different electrodes.

27

Advanced Materials Analysis
P-180

COMPARATIVE RAMAN SPECTROSCOPY STUDIES OF SOME CARBONACEOUS
MATERIALS
C. Pachiu
IMT Bucharest, L9, Bucuresti, Romania

Graphene is a carbonaceous family member with a single atomic layer of graphite, one of the mostinvestigated materials because of its excellent mechanical, thermal, optical and electronical properties.
Nanocrystalline graphite/graphene (NCG) and vertical graphene (VG) cannot reach the highperformance of large‐area graphene but these possess the attractive properties induced by its oriented
arrangement.
This work presents a systematic study of the structural characterization by Raman spectroscopy of somecarbonaceous material with graphene-like structure, such as single layer graphene, vertical graphene andnanocrystalline graphite/graphene. For Raman spectroscopy, a high- resolution Scanning Near-Field Optical
Microscope fitted with a Raman Module (Witec Alpha 300S) with 532 nm wavelength diode‐pumped solidstate laser and maximum power 145 mW. The incident laser beam (a 1.0 µm spot-size) was focused with a
100 x long working distance microscope objective and the spectra were collected with an exposure time of
20s accumulation and 600 grooves/mm grating. There are different ways in which carbonaceous materials
can be created, but by far the most popular way at this moment in time is by using a process called chemicalvapour deposition. In this study, the materials were growth by Plasma enhanced chemical vapor deposition(PECVD) - thermal method in CH4:H2 atmosphere at an optimized temperature on Cu or SiO2/Si substrates.
From the integrated intensities of the disorder-induced D and G Raman bands ID/IG ratio for eachcarbonaceous material, the lateral crystallite size of graphitic domains, number of graphene layer and defectdensity was determined. In particular, the parameters play decisive role in the quality determination ofgraphene-based materials and applications such as energy storage, environmental remediation, sensors andbiomedicals.

28

Correlative Imaging Applications

Correlative Imaging Applications
P-130

Correlative Raman imaging characterizes crystal properties of 2D materials
1

1

1

2

1

1

U. Schmidt , S. Gomes da Costa , E. Kallis , H. Hu , T. Dieing , O. Hollricher
1
2
WITec GmbH, Ulm, Germany, WITec Beijing Representative Office, Beijing, China

Due to their unique optical and electronic properties, two-dimensional (2D) materials have great potential for
application in optoelectronic devices. Diverse crystal properties such as the number of layers and growthdefects influence the optical and electronic properties of the 2D materials. Research, development andquality control thus require powerful and non-destructive imaging techniques for monitoring these crystal
properties and to evaluate synthesis processes. To comprehensively characterize the 2D material, it isadvantageous to combine several imaging techniques. Here, we present correlative Raman imaging as aversatile tool for investigating prominent examples of 2D materials, namely graphene and the transition metal
dichalcogenides (TMDs) molybdenum disulfide (MoS2) and tungsten disulfide (WS2). In combination withimaging techniques such as second harmonic generation (SHG), photoluminescence (PL), atomic force
microscopy (AFM) or scanning electron microscopy (SEM), Raman imaging is able to provide a thoroughcharacterization of the 2D materials. Crystal properties and features such as grain boundaries, surface
structure, layer number, defect density, doping and strain fields can thus be identified and visualized.

Correlative Raman and SHG imaging of mono-layer MoS2. The Raman image (A) shows thefrequency of the E2g mode, indicating strain fields. The SHG intensity image (B) reveals grainboundaries (low intensity).

30

Correlative Imaging Applications
P-163

Detecting Nanoplastic Particles using Correlative Microscopy
1

2

2

1

1

3

4

5

R. Schmidt , H. Fitzek , M. Nachtnebel , A. Zankel , H. Schröttner , M. Dienstleder , S. Mertschnigg , M. Poteser , H.-P.
5
6
Hutter , W. Eppel
1
2
Institute of Electron Microscopy and Nanoanalysis (FELMI), Technische Universität, Graz, Austria, Centre for Electron
3
4Microscopy, Graz, Austria, Center for Electron Microscopy Graz, Graz, Austria, Institute of Electron Microscopy and5
6Nanoanalysis (FELMI), Graz, Austria, Centre for Public Health, Medical University Vienna, Vienna, Austria, Medical
Universitty of Vienna, Department of Obstetrics and Gynecology, Vienna, Austria

Nowadays “microplastics” (MPs) is an already well-known term and micro-sized particles are increasingly
found in several consumer products [1]. Moreover, effects of micro- and nanoplastics (NPs) on human healthhave been investigated and discussed [2]. In this study, the focus is pointed to MPs smaller than 1 µm, witha specific focus on particles in the scale of a couple of 100 nm, which are referred here as NPs. A correlative
approach between scanning electron microscopy (SEM, high resolution) and Raman microscopy (chemical
identification) was tested to meet the challenges of finding and identifying these small particles. For thispurpose standardized polystyrene (PS) beads were mixed into various environments in different
concentrations, ranging from ideal (distilled water) to realistic (sea salt, human amniotic fluid), to proof thedetection limit of NPs with the so called RISE (Raman Imaging and Scanning Electron microscopy) system-3[3]. Promising results exhibit detection limits of 2·10 µg/L (distilled water), 20 µg/L (sea salt) and 200 µg/L
(human amniotic fluid).
[1] M. E. Iñiguez, J. A. Conesa, and A. Fullana, “Microplastics in Spanish Table Salt,” Sci. Rep., vol. 7, no. 1,
p. 8620, Dec. 2017, doi: 10.1038/s41598-017-09128-x.
[2] E. Sazakli and M. Leotsinidis, “Possible effects of microplastics on human health,” in Microplastics inWater and Wastewater, IWA Publishing, 2019, pp. 177–190.
[3] R. Schmidt, H. Fitzek, M. Nachtnebel, C. Mayrhofer, H. Schroettner, and A. Zankel, “The Combination ofElectron Microscopy, Raman Microscopy and Energy Dispersive X-Ray Spectroscopy for the Investigation ofPolymeric Materials,” Macromol. Symp., vol. 384, no. 1, pp. 1–10, 2019, doi: 10.1002/masy.201800237.

31

Correlative Imaging Applications
P-167

Correlating SEM and Raman Imaging of Nucleopore Filter Sampled Nanofibers
A. Meyer-Plath, J. Schumann, S. Plitzko
Federal Institute for Occupational Safety and Health (BAuA), FG. 4.5, Berlin, Germany

In the light of the “Global Asbestos Disaster” [1], human exposure to high concentrations of respirable
biopersitent fibers must be controlled. Reports on the asbestos-like pathogenic potential of rigid andbiodurable nanofibers of multi-walled carbon nanotube type [2,3] make it necessary to determine
concentrations of airborne nanofibers at a level of 10,000 fibers/m³. BAuA develops such techniques basedon sampling aerosols on nucleopore filter membranes, followed by SEM imaging. For every nanofiber
recognized and localized on an SEM image, Raman analysis is mandatory to distinguish natural fibers likecellulose from man-made biodurable fibers. The high workload on SE and Raman microscopes motivated touse not an SEM-integrated Raman but two independent instruments to parallelize the analysis. We havedeveloped a method to automatically position filters in the Raman with an accuracy better than 100 nm toaddress and Raman image all candidate fibers of a sample. This enables to check compliance withrecommended nanofiber exposure limits [4].
Using the Hitachi and WITec software APIs, a software was developed to control both the SU8230 SE andApyron Raman microcopes. The software first automatically acquires focused SEM images at statisticallyrandom locations of a position-calibrated filter. Its track-etched pores of 400 nm diameter can also be
resolved in our Apyron using a 100X objective. Convolution of both images with a “standard pore” kernel
results in pore position images that allow correlating the pore constellation in the vicinity of a nanoscale
object to analyze.
This pattern matching approach allows improving the initial filter positioning accuracy of about 5 µm to better
than 100 nm. This way, even optically non-resolvable objects can be localized.
WITec’s microscope controller API gives full access to all necessary hardware features (except stage
acceleration limits) to achieve not only image analysis and stage control but to implement even complex
analysis workflows.
1. Furuya et al. DOI 10.3390/ijerph15051000
2. Rittinghausen et al. DOI 10.1186/s12989-014-0059-z
3. Broßell et al. in DOI 10.20850/9781716632426
4. https://www.baua.de/EN/Service/Legislative-texts-and-technical-rules/Rules/TRGS/TRGS-527.html

32

Correlative Imaging Applications
P-168

Q-switched Nd:YAG laser on dental enamel with photoabsorber: a confocal Raman pilot
study
1

1

2

3

1

P. Castro , D. Pereira , P. Ana , C. Matos , D. Zezell
1
2
Nuclear and Energy Research Institute, Center for Lasers and Applications, Sao Paulo, Brazil, Engineering Modelling3
and Applied Social Sciences Center, Santo Andre, Brazil, MackGraphe - Graphene and Nanomaterials ResearchCenter, Sao Paulo, Brazil

Nd:YAG lasers emitting λ = 1064 nm at microsecond and nanosecond pulses are alternatives to prevent
dental caries and erosion in clinics. This wavelength allows most of photons to penetrate deep in the hardtissue due to low absorption of hydroxyapatite in the region. It is necessary to use photoabsorbers so most ofphotons are absorbed in the surface of the tissue preventing dental pulp necrosis. Currently the coal paste isused as a photoabsorber but the irradiated tissue turns darker what implies in the patients low adherence tothe treatment due to aesthetic reasons. [1,2]. Confocal Raman spectroscopy is a non-destructive opticalmethod to obtain detailed information about molecular composition of biological structures in depth. The
most prominent feature of Confocal Raman spectroscopy is the reliable capability to provide thebiomolecular data with no use of ionizing radiation to penetrate in the sample. This work aims to characterize
the dental enamel irradiated with Nd:YAG laser with nanoseconds pulses, in order to describe the depth
related changes promoted in the enamel, by the heat generated due to laser irradiation. For these2
measurements, 30 bovine enamel blocks of 8 mm , were randomized into 3 groups: G1 – enamel untreated;
2
G2 – enamel irradiated with Nd:YAG nanopulsed laser (1064 nm, 4 W; 1,05 J/cm ; 5 ns 20 Hz, Brilliant,
Quantel Laser) using a coal paste as photoabsorber; G3 -enamel irradiated with Nd:YAG nanopulsed laser (same parameters as G2) using squid ink as photoabsorber. The assessments of three different depth
regions of the cubic shaped samples were: region A- left corner above of the sample, region B-middle of the-1
sample and Region C- right corner below of the sample. The intensity map of phosphate (950 cm )regarding the position, were calculated [3,4] as shown in the Figure 01.

Figure 01: Phosphate intensity maps of (a) Enamel untreated - (b) Enamel with coal paste irradiated with Nd:YAG nanopulsed laser (c)Enamel with squid ink irradiated with Nd:YAG nanopulsed laser.

The comparative results in the Fig.1 demonstrated that application of coal paste associated with Nd:YAG(G2) can preserve the inorganic content better than the squid ink group (G3). These findings have crucial
clinical implications in the laser protocol development and it was possible to correlate the heat penetration
depth of the laser irradiation with photoabsorber using the images obtained by the confocal Raman.

REFERENCES[1] D. L. Pereira et al, Int J Mol Sciences 19, 433-447, 2018.
[2] P. A. Ana et al, Caries Res, 46, 441-451, 2012.
[3] J. B. Holly et al, Nat. Protrocols. 11, 664–687, 2016.
[4] H. C. Charles et al, Nat. Photonics 8, 627-634, 2014.

33

Correlative Imaging Applications
P-172

Microbiologically influenced corrosion (MIC) of steel – a study using correlative SEM, EDX
and Raman microscopy
1

1

2

1

3

1, 4

T. Planko , H. Fitzek , S. Eichinger , J. Rattenberger , G. Koraimann , H. Schröttner
1
2Graz Centre for Electron Microscopy, Graz, Austria, Graz University of Technology, Institute of Applied Geosciences,34Graz, Austria, University of Graz, Institute of Molecular Biosciences, Graz, Austria, Graz University of Technology,
Institute of Electron Microscopy and Nanoanalysis, Graz, Austria

Microbiologically influenced corrosion (MIC) is responsible for 20% of all corrosion damage [1]. In thiscontext, there is great interest in understanding MIC especially, since it has been shown that some microbes
slow down the rate of corrosion [2], while others speed it up [3]. During experiments in the Koralmtunnel,iron-oxidizing bacteria were found, to be part of a MIC causing, microbial community. The aim of theinvestigations is to observe the influence of MIC on steel on the macroscopic (corrosion rates CR, pitting
corrosion), and the microscopic side. A novel technique is applied for the latter, which combines Raman
imaging with scanning electron microscopy (SEM) and energy dispersive X-Ray spectroscopy (EDX) (Zeiss
Sigma 300 VP; Oxford X-Max 80; WITec RISE). An example of a correlative SEM-EDX-Raman (RISE)
measurement is shown in the figure.

RISE-measurement: rebar-steel (bio-corroded, cross-section); Top: SEM-image with Raman positions, and Raman spectra: Maghemite,
Goethit, amorphous carbon, Magnetite, Lepidocrite; Bottom: EDX-mappings: iron, oxygen, carbon, sulfur, manganese, silicon.

The result showed that the CR of those samples exposed to MIC was two to six times lower than thereference samples. This is because bacteria placed a thin layer of sulfur over the sample (see figure 1,
bottom left). A sulfur layer reduces the CR by acting as a solid diffusion barrier [4].
References:[1] Dubiel, M., et al. Appl. Environ. Microbiol. 68.3 (2002), p. 1440-1445.
[2] Javed, M. A., et al. International Biodeterioration & Biodegradation 125 (2017), p. 73-85.
[3] Wade, S. A., et al. International Biodeterioration & Biodegradation 121 (2017), p. 97-106.
[4] ZHENG, Yougui, et al. CORROSION/2015, 2015, p. 5933.
The authors gratefully acknowledge funding from the Austrian Cooperative Research (ACR) under the ACRProject:“KorronNet”: Avoidance of selective corrosion

34

Environmental and Geoscience

Environmental and Geoscience
P-112

Application of Raman microscopy to the study of the mineralogical composition of
pyroclastic rocks from Lesvos petrified forest
1

2

3

1

A. IORDANIDIS , J. GARCIA-GUINEA , N. ZOUROS , A. ASVESTA
1
2
University of Western Macedonia, Mineral Resources Engineering, Kozani, Greece, Museo Nacional Ciencias
3
Naturales, CSIC, Madrid, Spain, University of the Aegean, Geography, Mytilene, Greece

The Lesvos Petrified Forest was formed by silicification process of plants during the Lower Miocene era,
when intense volcanic activity occurred in that region. Situated in the western part of Lesvos island (NEGreece) and consisting of hundreds of standing and lying fossilized tree trunks, it is one of the mostimportant natural heritage monuments in the world. Despite its significance, the conduction of extensivemineralogical analyses on fossil plants and their hosting pyroclastic rocks has merely commenced, providing
information on the permineralization process. Sigri pyroclastic rock samples were collected within the Lesvos
Petrified Forest, and four polished block sections with embedded fossil plant material were created foranalytical purposes. A Thermo Scientific DXR Raman Microscope with a 780 nm laser beam was employed.
The power value of the sample irradiation was ranging from 6 mW to 12 mW. The average spectral-1-1resolution in the Raman shift range of 100-3000 cm was 5cm (grating 400 lines/mm, spot size 2μm).
-1Magnetite was revealed with its intensive band at 662 cm , along with anatase (titanium oxide) showing an-1-1
intensive band at 144 cm and secondary bands at 396, 516 and 637 cm . Apatite with its distinct strong-1
peak at 965 cm was also determined along with biotite from the mica group and labradorite and sanidinefrom the feldspars group. Pyrolusite (manganese oxide) revealed its characteristic peaks at 503 and 573 cm
1-1
and lepidocrocite showed two strong peaks at 256 and 440 cm and several secondary peaks at 170, 285,-1
-1
525 and 695 cm . Hematite (iron oxide) was also evident with three strong peaks at 220, 287 and 1315 cm-1and some weaker peaks at 402, 607 and 651 cm . Overall, the utilization of Raman micro-spectroscopy
provided substantial information for the identification of the mineral assemblages comprising the pyroclastic
rocks hosting the Lesvos petrified wood samples.

Polished block section of the MPT762 fossil plant/pyroclastic rock sample [left] and characteristic Raman spectra of an anatase mineralphase (red), comparing to the standard anatase mineral (purple) [right]

36

Environmental and Geoscience
P-115

Image of microplastics and nanoplastics
C. Fang, Z. Sobhani
UON, Newcastle, Australia

We demonstrate that Raman imaging can be employed to visualise and identify microplastics andnanoplastics down to 100 nm, by distinguishing the laser spot, the pixel size/image resolution, thenanoplastics size/position (within a laser spot), the Raman signal intensity.
We also examine the lateral intensity distribution of the Raman signal emitted by nanoplastics (diameters
ranging ~30–600 nm) within the excitation laser spot. We find that the Raman emission intensity, similar to
the excitation power density distributed within a laser spot, also follows a lateral Gaussian distribution. We
break down the diffraction limit of laser and cross-check multi-images simultaneously mapped at two or threecharacteristic peaks via either a logic-OR or a logic-AND algorithm. Thus the imaging uncertainty can besignificantly reduced from a statistical point of view.

37Environmental and Geoscience
P-123

Using Cryo-Raman spectroscopy to analyse the location and chemistry of micro-inclusions
in the EastGRIP ice core
1

1

1, 2

3

N. Stoll , j. Eichler , I. Weikusat , D. Dahl-Jensen
1
Alfred Wegener Institute, Helmholtz Centre for Polar and Marine Research, Geosciences, Bremerhaven, Germany,
2
3
Eberhard Karls Universität Tübingen, Geosciences, Tübingen, Germany, Physics of Ice, Climate and Earth, Niels Bohr
Institute, Copenhagen, Denmark

One of the major uncertainties regarding future sea-level rise is the physics behind the flow of ice, whichsignificantly impacts the amount of solid ice discharge into the oceans. Various physical properties of polar
ice are affected by impurities in ice, ranging from electrical conductivity to internal deformation and thus, ice
flow. Our goal is to better understand the impact of impurities on the deformation of ice by analysing the East
Greenland Ice Core Project (EastGRIP) ice core. The on-going EastGRIP drilling delivers the first deep icecore from a fast-flowing ice stream, the Northeast Greenland Ice Stream, enabling novel in-situ informationon the flow of ice. We analysed seven samples from various depths of the first 900 m of the ice core in ourcold lab (-15°C). First, we used an optical microscope to map the surface of the sample and the same area
focused 500 μm below the surface. These maps allow us to locate micro-inclusions, solid impurities of μmsize, inside the ice while preserving the original microstructure and preventing contamination . Following this,a WITec alpha 300 M+ confocal cryo-Raman system with a Nd:YAG laser (532 nm) is used to analyse thechemical composition of these inclusions.
We present impurity distribution and mineralogical data from seven different depths of the EastGRIP ice
core. Preliminary key findings are 1) a homogenous spatial distribution of impurities in the microstructure, 2)
distinct layers of impurity-rich grains, 3) a strong relationship between identified minerals and the bulk
chemistry, and 4) a diverse chemical range of micro-inclusions mainly consisting of sulfates (e.g., gypsum)and insoluble terrestrial minerals such as quartz, mica or feldspar.

Located impurities in an ice core sample from 277 m of depth. Colored dots represent identified
compounds, blue thick lines indicate grain boundaries at the sample surface. The arrow points towards
the surface of the ice sheet.

38

Environmental and Geoscience
P-128

Raman mapping of complex mineral assemblages: application on pegmatitic phosphates
sequences
F. Prado Araujo, N. Hulsbosch, P. Muchez
KU Leuven, Leuven, Belgium

Phosphate minerals form an important part of many geological and biological systems. In geologicalsystems, phosphates occur nearly in all rock types, being particularly abundant in sedimentary (e.g.
phosphorite) and magmatic rocks. In the latter, pegmatites are highlighted for their vast and exceptionallyrich phosphate mineralogy. Changes in physico-chemical conditions during crystallization of phosphates arereflected on the sequence of formation for these minerals, termed paragenesis. Raman imaging is able todetect those mineralogical changes in complex assemblages with high spatial resolution, successfully
determining the paragenesis. Raman spectroscopy is more advantageous than conventional methods
because it requires little sample preparation, it is non-destructive, and is capable of identifying hydrated
minerals and phases composed of light elements (e.g. H, Li). This study applies state-of-the-art Raman
imaging techniques to define the formation sequence of complex mineral assemblages in the Buranga raremetal pegmatite, a P-Li-Nb-Ta-rich magmatic rock from Western Rwanda. The Buranga dyke was chosen forthe development of the Raman mapping methodology of complex mineral assemblages because it hosts
more than 50 phosphate species, often with intergrowth or replacement textures. Our results show the
replacement of amblygonite [LiAl(PO4)F] by montebrasite [LiAl(PO4)OH]. Mapping of the OH-stretching peak-1
position around 3360 cm and the peak width identifies the mineral phases, illustrates their spatial relations,and allows to estimate the distribution of fluorine within the sample. We also investigate the replacement
textures of phosphate minerals in a complex assemblage. Each observed phase has unique spectralfeatures that allow their identification and classification in the Raman maps. The sequence trolleite
2+
2+
3+[Al4(PO4)3(OH)3] + rosemaryite [Na(Mn ,Fe )Fe Al(PO4)3)] → lazulite-scorzalite [(Mg,Fe)Al2(PO4)2(OH)2] →samuelsonite [Ca9(Fe,Mn)4Al2(PO4)10(OH)2] → wardite [NaAl3(PO4)2(OH)4 . 2H2O] is proposed for thisassemblage. Raman maps show that the Buranga pegmatite experienced a progressive hydration processduring cooling, followed by internal remobilization of elements, such as F, Li, Na, and Ca. Our data confirm
that phosphates in complex mineral assemblages can (1) be straightforwardly analyzed by Raman images,(2) record the evolution of the magmatic host rock, and (3) be used as proxies for pegmatite formation.

39

Environmental and Geoscience
P-141

Transport of diesel soot particles in structured and homogenized soil visualized byhyperspectral imaging and confocal Raman microscopy
12

1

M. Ortner , H. Buddenbaum , S. Thiele-Bruhn
1
2
University Trier / Regional and Environmental Sciences, Soil Science, Trier, Germany, University Trier / Regional andEnvironmental Sciences, Environmental Remote Sensing and Geoinformatics, Trier, Germany

Each year diesel motor-vehicle exhausts produce about 1.8 g soot/km along the roadsides in Germany.
Containing a high amount of hydrophobic pollutants, a transport of soot particles in soil could lead tocontamination of water and of soil itself. Knowledge about the retardation or transport in soil of deposited
soot are largely missing. This study aimed to elucidate whether deposited soot is accumulated on the soil
surface or whether it is transported, possibly through coarse soil pores, downwards.
To determine soot mobility in soil, diesel soot was applied on top of structured and unstructured soil,
respectively, contained in columns with a length of 12 cm and a diameter of 4.70 cm. A constant water flow
was established for one month to enable soot particles to move downwards. Subsequently, soil columns
were cut open and air-dried for further analysis. Hyperspectral imaging was used to identify clusters of sootparticles in the soil columns. To determine the depth distribution of soot particles, the hyperspectral data wasanalyzed by using Maximum Likelihood Classification (MLC), Spectral Angle Mapper and autoPartial Least
Squares Regression (aPLSR). The received hyperspectral images of soot concentration along the depth
gradients were used to identify soot by using Confocal Raman Microscopy (WITec alpha300 R).
Hyperspectral data enabled to separate pixels into several clusters according to the concentration of soot on
the soil, while Raman microscopy was able to unequivocally distinguish soot from soil organic matter orblackish minerals. Single soot particles were identified in structured as well as in unstructured soil columns.
The concentration of soot particles declined with increasing soil depth, while most soot particles remained on
the top of the soil columns. Those soot particles that were identified deeper in the soil were mostly foundalongside of larger cracks and pores of the columns indicating their function as preferential flowpaths forparticles in soils. Particularly in structured soil columns more soot particles were found in deeper soil, while in
the unstructured soil columns less soot particles were translocated but remained on the top of the soil.
The combination of hyperspectral imaging with confocal Raman microscopy effectively enables to localize,
quantify and identify soot in heterogeneous environmental samples such as soils.

40Environmental and Geoscience
P-142

Detection of different microplastic materials in soil by using confocal Raman microscopy
12

1

M. Ortner , A. Stablo , S. Thiele-Bruhn
1
2
University Trier / Regional and Environmental Sciences, Soil Science, Trier, Germany, UniversityTrier / Regional andEnvironmental Sciences, Soil Science, Trier, Germany

Plastic particles are distributed worldwide in every ecosystem. For soils there are several possible input
pathways such as the application of manure, plastic mulching or by atmospheric deposition. With decreasing
particle size, the identification gets more difficult. Actually, several approaches are tested for quantitative andqualitative analysis of microplastic (MP) in soil. Physical and chemical methods are highly time consuming,
cost intensive and destructive while methods using optical or spectral approaches such as microscopy, FTIR
and – investigated in this study – Raman are less cost intensive and non-destructive.
Aims of this study were 1) to prove the suitability of confocal Raman microscopy to determine MP in soil and2) to show whether biofouling or staining with mineral particles masks MP, which would be a kinetic process
occurring in moist soil.
Different MP materials (PA, PP) were added in selected contents (1%, 2%, 5% w/w) to sieved air-dried soil.
Samples were analyzed without further treatment. A second batch of samples was spiked with 5% MP,
biologically reactivated by rewetting, and stored at 20°C for up to eight weeks. Water losses were repeatedlycompensated to keep a constant soil moisture. In order to obtain a time series, samples were taken biweekly
for further analysis. From each sample a randomly selected surface area was analyzed with confocal Raman
microscopy (WITec alpha300 R) using a 532 nm laser. For identification of MP particles in the samples large
area scan was done, covering a surface of 1000 μm × 1000 μm. Recorded spectra were treated by using
background subtraction and Savitzky-Golay smoothing. Afterwards spectra were clustered using k-means
algorithm allowing an identification of the applied microplastic materials.
Characterization of all MP materials was successful for all recorded spectra. Also, analysis of dry samples
allowed an identification of different contents of MP particles of each plastic material in soil. Numbers of
pixels identified as MP particles closely correlated with the spiking content of MP (R² = 0.77-0.99). Yet, timeseries showed a decrease of determined MP particles from 27% after two weeks down to 3% after eightweeks.
It is concluded that Raman microscopy is a suitable technique for fast and efficient identification andquantification of MP particles in soil. However, in bioactive, moist soil recovery of MP is increasingly
inhibited, most likely due to the formation of surface coatings.

41

Environmental and Geoscience
P-147

Raman spectroscopy at pressures exceeding 10 000 atm
D. Kurzydłowski , J. Rogoża , T. Chumak
1
Cardinal Stefan Wyszyński University in Warsaw, Faculty of Mathematics and Natural Sciences, Warsaw, Poland,
2
University of Warsaw, Faculty of Physics, Warsaw, Poland
1

2

1

Pressures used in laboratories and industry rarely exceed 300 atm (≈0,03 GPa). However due to thedevelopment of new measurement techniques, in particular the diamond anvil cell (DAC), it is now possible
6
to probe in situ materials at pressures exceeding 1 GPa (≈10 000 atm), and even reaching 400 GPa (≈4·10
atm). Experiments performed at high pressure enable observation of many intriguing phenomena, such as1
the transformation of nitrogen into a polymeric structure resembling that of diamond.
Here we present investigation into the high-pressure phase transitions of simple solids, including
dichloromethane (CH2Cl2), chloroform (CHCl3), and inorganic fluorides (ZnF2, AuF3) up to pressures of 45
GPa (≈ 450 000 atm). By performing Raman scattering measurements we were able to monitor the evolutionof the Raman-active vibrations as a function of pressure. Comparing information on the Raman band
positions and intensities with results of ab initio solid-state calculations enabled us to assign the evolution of
2,3
the crystal structure, and phase transition sequence in these systems.
An often overlooked aspect of high-pressure experiments is the homogeneity and purity of the sample. Thisis particularly important given the small dimensions of the DAC chamber (of an order of 100 µm). Thanks to
the high confocality and sensitivity of our Raman microscope (Witec Alpha 300) we were able to perform
Raman mapping of samples enclosed in the DAC with μm resolution, even at high pressures (Figure 1) .

Figure 1. Photograph of solid dichloromethane compressed in a DAC (left) together with the Raman
map of the sample (right). Color coding indicates different crystallite regions.

References
1. M. I. Eremets, et al., Nat. Mater. 2004, 3, 558.
2. D. Kurzydłowski, et al. Inorg. Chem. 2020, 59, 2584.
3. D. Kurzydłowski, et al. Chem. Commun. 2020, 56, 4902.

42

Environmental and Geoscience
P-160

Determining the Provenance of Planetary Analogs with Spatially Resolved Micro Raman
Imaging of Nanocrystalline Minerals
1

2

3

D. Bower , A. Steele , M. Fries
1
2
University of Maryland College Park, NASA Goddard Space Flight Center, Greenbelt, United States, Carnegie
3
Institution for Science, Washington, DC, United States, NASA Johnson Space Center, Houston, TX, United States

Raman maps: (A) 3.5 Ga chert with a combination of disseminated (green) carbon with layers of(blue) anatase (TiO2) in (red) quartz; (B) Recent lava with dominant minerals pyroxene (red) andplagioclase (blue & teal), and trace anatase (green).

Geologic deposits on Earth and other planetary bodies undergo several iterations of geochemical changeover time resulting in compositional differences that can obscure the origins of those deposits. Our ability toreconstruct habitable environments and their evolution on other planetary bodies relies on the accuratecharacterization of surface deposits. In particular, the origins of both terrestrial and planetary deposits may
be recorded in the mineral assemblages as barely discernible nanocrystalline phases. Micro Raman imaging
facilitates the identification of nanocrystalline materials (grain size ≤ 1 mm) and their spatial relationships withmicro-scale minerals for a comprehensive characterization. For many older deposits, nanocrystalline phases
occur as inclusions within or along the edges of larger mineral grains. In younger or recent deposits, thenanocrystalline phases may be ephemeral and more difficult to detect. Here we explore some of theapplications in which the ability to identify nanocrystalline minerals results in more robust interpretations of
the rock record providing a path forward for planetary exploration using Raman spectroscopy.

43

Environmental and Geoscience
P-164

CL and Raman imaging of metamorphic and kimberlitic diamonds
A. KORSAKOV, D. Mikhailenko
IGM SB RAS, NOVOSIBIRSK, Russian Federation

Cathodoluminescence image is most common tool for recovery internal morphology and growth history of
diamond crystals. But there are several disadvantages of this very useful technique. First of all diamond
crystal need to be polished to the central plane and at least half of the diamond crystal and the information,
containing in this part, are lost during sample preparation. Our previous study reveal that confocal Raman
imaging could be potentially a very good alternative of CL imaging in mineralogical study. In this study wefocus on CL and Raman imaging of the Kokchetav metamorphic and Yakitiyan kimberlitic diamonds. A
WITec Apyron (Automated Confocal Raman system) was equipped with a 488 nm and 635 nm lasers fiber
coupled to the instrument. A 100× (NA 0.9 air) objective was used for excitation and detection. In allmeasurements, the laser with a power of approximately 50 mW was employed. Micro-cathodoluminescence(SEM-CL) analyses of microdiamonds were performed using a scanning electron microscope LEO 1430 VP(Carl Zeiss) equipped with a CL spectrometer. The accelerating voltage for each analysis was 20 kV. The
polished microdiamonds exposed on the surface of the thin sections were analyzed. The diamond shows astrong change in the position of the diamond Raman line as well as the width of this line, which could befound both in the XY and in the YZ scan. Raman images are identical with the CL images, but in contrast to
CL almost no sample preparation is required for the Raman imaging, which can be done at different depth ofthe diamond crystal and without any destruction of the inclusions and host-mineral. Thus Raman imaging are
powerful and non destructive technique for recovery growth history and internal morphology of the diamond
crystals. This study was supported by the Russian Science Foundation (Grant 18-17-00186).

44

Life Sciences, Biomedical andPharma Research

Life Sciences, Biomedical and Pharma Research
P-117

Three-Dimensional Spectroscopic Chemical Imaging of Pharmaceutical Tablets
1, 2

2

2

1

1

H. Carruthers , D. Clark , F. Clarke , K. Faulds , D. Graham
1
2
University of Strathclyde, Glasgow, United Kingdom, Pfizer UK, Sandwich, United Kingdom

Pharmaceutical tablets are the most widely used solid dosage form for delivering therapeutic agents. There
are knowledge gaps in current pharmaceutical manufacturing processes; particularly those related to therelationship of components in the formulation, processing conditions and the final product characteristics. By
providing a means of visualising the microstructure of a tablet matrix these areas may be better understood.
There are several techniques that can generate high resolution chemical images of the component
distribution within a tablet system but generally cannot go beyond an exposed surface layer. Threedimensional imaging has been explored by using a Raman microscope to obtain a depth profile of a sample,
however, this is typically less than 50 microns. The particle size of common excipients and active ingredients
often exceed this depth range making it unsuitable for pharmaceutical products. Instead, this study explores
an alternative method to obtain three-dimensional images of a sample using Raman and near-infraredchemical imaging. This involves stacking two-dimensional chemical images obtained at different penetration
depths by physically milling the sample and may hold the key to improved fundamental understanding ofsolid dosage forms. This investigation has also assessed the differences in the chemical images obtained by
Raman and near-infrared chemical imaging. Both techniques are closely related tools for characterising thechemical composition of a sample. Scanning electron microscopy with energy dispersive X-ray microanalysis
were employed as alternative imaging techniques to evaluate which spectroscopic imaging method provided
the most comparable and therefore representative image of the sample.

46

Life Sciences, Biomedical and Pharma Research
P-121

Tracking heavy water incorporated confocal Raman spectroscopy for evaluating the effectsof PEGylated emulsifiers on skin barrier
Y. Liu, D. Lunter
Tuebingen University, Pharmaceutical Technology, Tuebingen, Germany

The class of PEGylated emulsifiers finds broad application in the pharmaceutical and cosmetic industry. Withlarger use, their irritation potential and diverse effects on skin have caught our attention. We target on one of
the categories of polyethylene glycol (PEG) alkyl ethers with different lipophilic and hydrophilic chain lengthand aim to comprehensively examine their effects on the skin. We focus on finding their interactions with skinlipid components and try to summarise the potential rules for their further appropriate selection in skinresearch. With these clear goals, confocal Raman spectroscopy (CRS) was employed for measurements ofskin spectra in-depth and imaging. A unique probe of heavy water (D2O) was incorporated which can betracked percutaneously and simultaneously monitor the effects caused by emulsifiers. According to theresults, different extent of influences including the lipid content and lateral packing order state were observed
after the treatments of PEGylated emulsifiers. The results obtained from the depth profiling analysis provided
the possibility to estimate the least penetration depth of emulsifiers. Among them, PEG-20 ethers displayed
the most penetration ability. Meanwhile, it is interesting to find that the treatment of emulsifiers can also
affect the spatial distribution of D2O whose differences were in line with the variations of skin lipid properties.
Therefore, this study provides a systematic analysis for PEGylated emulsifiers and supports the possibility to
use D2O as a straightforward and cost-effective probe to evaluate the skin barrier function.

47

Life Sciences, Biomedical and Pharma Research
P-122

Raman Imaging Analysis of Signaling Molecules in Mucoid and Non-Mucoid Strains ofPseudomonas aeruginosaT. Cao, N. Morales-Soto, K. Kramer, J. Shrout, P. Bohn
University of Notre Dame, Notre Dame, United States

Pseudomonas aeruginosa is an opportunistic human pathogen that can cause acute and chronic infections
and diseases including cystic fibrosis (CF), especially among immune-compromised individuals. One
characteristic of CF lung infection is the presence of a mucoid phenotype caused by alginate overproduction
and this phenotype is closely related to quorum sensing (QS), a signal network that P. aeruginosa utilizes tocoordinate cell group behaviors. Here, we focus on a clinical isolate mucoid strain of P. aeruginosa, named
FRD1. We have specifically performed Raman imaging analysis to investigate spatiotemporal variations inthe signal molecules (i.e. quinolones) of FRD1 quorum sensing system. By using this label-free technique,quinolone molecules were detected in situ and identified by the signature ring stretch vibrations within the1330-1380 /cm spectral range using Raman micro-spectroscopy. We studied FRD1 under culturingconditions that both promote and limit the mucoid phenotype. Moreover, we compared FRD1 with 4 related
strains (FRD1, FRD810, FRD440, FRD840 and FRD2), but with different mucoid/non-mucoid phenotypes
and all strains were analyzed to inform the understanding of quinolone signaling during the mucoid to nonmucoid transition. Our results indicate that the Pseudomonas quinolone signal (PQS), one of the quinolone
signal molecules, is universally activated at a longer stage across mucoid and non-mucoid strains, and mostof the time after the appearance of 2-alkyl-4-hydroxyquinoline N-oxides (AQNOs) in mucoid cultures. This
study highlights the distinct advantage of Raman imaging techniques in helping with passing the limitation of
traditional biological techniques to acquire abundant spatiotemporal molecular information from bacterial
communities. This approach will be useful to further examine PQS and other quorum sensing signaling
during the transition of P. aeruginosa to the mucoid phenotype and other behaviors important to infection.

Figure 1. Raman imaging of FRD1
bacterial colony; (a) Raman image
intergrated over 1330-1380 /cm; (b)PCA mapping of the selected principal
component (c) PCA loading plot of theseletected principal component48

Life Sciences, Biomedical and Pharma Research
P-126

Confocal Raman imaging as a useful tool to understand the internal microstructure ofmulticomponent aerogels
1

1

1

2

I. Benito Gonzalez , M. Martínez Sanz , A. López Rubio , L. G. Gómez Mascaraque
1
2
IATA (CSIC), Food Safety and Preservation Department, Valencia, Spain, Teagasc Food Research Centre, Fermoy,
IrelandThis work shows the characterization of (nano)cellulosic aerogels prepared from Posidonia oceanica waste
biomass by means of confocal Raman microscopy (CRM). For this aim, aerogels were prepared using four(nano)cellulosic fractions with different purification degrees. These were then coated with polylactic acid
(PLA) in order to improve their hydrophobicity and subjected to oil sorption–desorption experiments. Bothunivariate and multivariate analyses were compared in terms of the quality and the accuracy of theinformation provided. Univariate analysis only provided accurate information in the simplest systems (native(nano)cellulosic aerogels), while multivariate analyses facilitated the detection of the different components
even for the most complex structures. Automatic identification of components was selected as the optimal
methodology, although it also underestimated the abundance of the components with the least intense
Raman spectra (cellulosic clusters) in the presence of more intense spectra such as PLA and oil. Micron‐sized regions of concentrated cellulose were detected using CRM, being more abundant in the denser
aerogels. Results also confirmed that PLA was preferentially located close to the surface, while oil could
penetrate deeper along the matrix. Overall, the results showed the potential of Raman imaging as a novel
approach for the characterization of complex biopolymeric aerogels.

49

Life Sciences, Biomedical and Pharma Research
P-129

3D quantitative Raman imaging of organoids
1

2

1

1

3

M. Barbero Mota , V. LaLone , A. A. Bharath , K. Ritzau-Reid , M. M. Stevens
12Imperial College London, Bioengineering, London, United Kingdom, Imperial College London, Bioengineering &3
Material Sciences, London, United Kingdom, Imperial College London, Bioengineering & Material Sciences & Institute of
Biomedical Engineering, London, United Kingdom

There is an increasing demand in biological sciences for a quantitative imaging tool that can unveil thebiochemical 3D structure of bioconstructs. Tissue engineering clinical transfer requires thorough biochemical
analysis in order to assess the exact physiomorphology of any biotechnology product. Raman
microspectroscopy has been postulated as the ideal technique to meet this need. The emergence of more
powerful hardware has enabled label-free non-invasive high-resolution imaging at the subcellular level. Until
now, most Raman efforts have been qualitative, and those that offer quantification are limited to 2D analysis.
One of the main reasons behind this remains the non-trivial computational task of quantitative spectra
deconvolution in 3D, for which several algorithms have been proposed. Finding each biomolecular
endmember contribution to the overall Raman spectrum is highly impaired by noise and depth interferences
ligated to 3D specimens. In this study, we developed a novel quantitative calibration method which makes
use of the Hybrid Least Squares (HLA) algorithm to provide reliable biochemical concentration quantification
at each x-y-z position of 4% paraformaldehyde fixated 40-day human embryonic stem cell-derived brainorganoids (hESC BO).

Figure 1 Brain organoid 80 µm deep z-stack scan rendered into a 100*100*80 µm representation. Merge, nucleic acids
and lipid, and protein 3D quantitative channels are shown along with the 2D stack slice at 28 µm z-value withbiochemical concentration

HLA was selected out of seven machine learning algorithms by 10-fold cross-validation (10f-CV), including
the chemometrics gold standards partial least squares and multivariate curve resolution alternative leastsquares. Artificially calibration datasets were derived from real Raman spectra contaminated with depthnoise levels simulating up to 80 µm inside brain tissue. 10f-CV was run using the noisy datasets, and soerror metrics vs depth profile was obtained for each model. HLA ranked top 2 on noiseless data (NRMSE =20.25, R = 0.935), and also showed smooth profiles with depth that indicates HLA outstanding handling of
stochastic shot noise. hESC BO 3D quantitative imaging results are shown in figure 1, nonetheless, thistechnique enables quantitative assessment of metabolic and structural phenomena underlying any 3D
biospecimens.

50

Life Sciences, Biomedical and Pharma Research
P-139

Raman spectroscopy of biomembranes for tissue engineering
1

2

2

1

1

S. Leal Marin , O. Pogozhykh , C. Figueiredo , B. Glasmacher , O. Gryshkov
1
Gottfried Wilhelm Leibniz University of Hanover, Institute for Multiphase Processes (IMP), Garbsen, Germany,
2
Hannover Medical School, Institute for Transfusion Medicine, Hannover, Germany

Extracellular matrix components (ECM), such as growth factors, collagen, glycoproteins and proteoglycans,are a key element in tissue engineering (TE) to support cell growth and proliferation. The inclusion of suchcomponents obtained from native tissues, such as the human amniotic membrane (hAM), into polymericscaffolds has an advantageous application for the production of TE biomembranes. However, different
chemical and physical hAM preservation methods as well as fabrication methods for the biomembranes,
such as electrospinning, could affect the ECM structure and functionality. In this work, freeze-drying wasevaluated as a promising approach for the preservation of ECM components in hAM, such as collagen andproteinoglycans. The biomembranes were produced using electrospinning from 5% (w/v) poly(ethyleneoxide) polymer and 2% (w/v) hAM dissolved in hexafluoroisopropanol. The analysis of the chemicalcomposition of the unprocessed and freeze-dried hAM as well as produced biomembranes was performed
using Raman spectroscopy (alpha300RA, Witec GmbH) using 532 nm laser, laser power 10 mW, 5s
acquisition time, and 20 accumulations per point. The results presented the preservation of the main
components of the hAM after freeze-drying such as collagen and some amides corresponding toproteoglycans. Besides, the mix of these components with the polymer was also traceable in thebiomembranes. No shift in Raman peaks for the respective components was observed indicating theabsence of chemical interaction between ECM components and the polymer. In conclusion, Raman
spectroscopy allowed a simple and precise analysis of the preservation of the ECM components obtained
from hAM after their inclusion in biomembranes obtained by electrospinning.

51

Life Sciences, Biomedical and Pharma Research
P-144

Multi-bacteria, Multi-antibiotic, Testing Using Surface Enhanced Raman Spectroscopy(SERS) for Urinary Tract Infection (UTI) Diagnosis
1

2

1

1

K. Hadjigeorgiou , E. Kastanos , C. Andreou , C. Pitris
1
2
University of Cyprus, Electrical and Computer Engineering, Nicosia, Cyprus, Montgomery College, Biology Department,
Rockville, United States

A urinary tract infection (UTI), a bacterial infection of the urinary tract, is one of the most common types ofinfections affecting more than 50% of women globally. Also, vulnerable to UTIs are chronically ill patientsand children younger than two years old. Current diagnostic methods require 24 hours for the identification of
the causative bacteria as well as an additional 24 hours for the determination of the most effective antibiotic.
Because of these delays, doctors tend to prescribe broad-spectrum antibiotics until the official results of thediagnosis become available. The use of broad-spectrum antibiotics inevitably leads to ineffective treatments,
recurrent infections, a significant financial burden to national healthcare systems and are responsible for
antibiotic resistance. The aim of this research is to develop a fast and accurate method, based on Surface
Enhanced Raman Spectroscopy (SERS), that could (i) detect if a sample is “positive” or “negative” for a UTI,(ii) classify the causative bacteria, found in the “positive sample”, according to their species, and (iii)
determine the bacterial susceptibility to various antibiotics, all in a short time period. It should also allow
correct classification of bacteria species coming directly from urine samples. The results of this research
project provide a clear indication that, utilizing SERS, it is feasible to (i) detect the concentration of bacteria
3
per sample, down to levels of 10 bacteria/ml, and determine the presence of a UTI, (ii) classify correctly thebacterial species with an accuracy of 93.75%, and (iii) identify antibiotic susceptibility within two to four hours
of exposure with a correct classification ranging from 81.25 to 100%. These preliminary results can serve as
the starting point for developing an innovative tool that could provide same day diagnosis and antibiogramfor UTI, leading to more effective treatment with a significant socioeconomic impact.

52

Life Sciences, Biomedical and Pharma Research
P-150

Silver nanoflower-coated paper for the determination of ketoprofen via surface enhaced
Raman spectroscopy and ambient pressure paper spray mass spectrometry
M. C. Díaz-Liñán, M. T. García-Valverde, Á. I. López-Lorente, S. Cárdenas, R. Lucena
Departamento de Química Analítica, Instituto Universitario de Investigación en Química Fina y Nanoquímica IUNAN,
Universidad de Córdoba, Campus de Rabanales, Edificio Marie Curie Anexo, E-14071, Córdoba, Spain

The use of paper has gained great attention in the last years due to its numerous advantages as support,
1such as low price, versatility and availability . Furthermore, the easy handling of this type of material
facilitates its coupling to different instrumental techniques, such as mass spectrometry or surface enhanced
Raman spectroscopy (SERS), among others. Regarding SERS, the use of plasmonic nanoparticles with2
sharp edges , e.g. gold and silver, has increased since a high number of the so called hot spots are formed,
which lead to strong electromagnetic confinement, thus significantly increasing the low interaction crosssections observed in vibrational spectroscopies such as Raman spectroscopy. Furthermore, the presence ofa metal is also important in more recent approaches like paper spray, since it is based on a difference ofpotential. This technique allows direct analysis of samples, thereby simplifying the analytical process, since3
sample pretreatment is avoided .
In this context, a dual substrate for both SERS and ambient pressure paper spray mass spectrometry (PSMS) based on the modification of filter paper with silver nanoflowers (AgNFs) has been developed. In theearly stage of the synthesis, filter paper was coated with nylon-6 and incubated in ethylenediamine, whichacts as an anchor point for the nanoflowers. The as synthesized substrate was characterized via scanningelectron microscopy and infrared spectroscopy. The densely packed petals of the AgNFs led to a greatamount of hot spots, resulting in an enhancement not only in the Raman signal, but also in the sensitivity ofthe mass spectrometric analysis compared to bare paper and nylon/Ag-coated paper.

1

Jiang, X.; Fan Z.H. Annual Rev. Anal. Chem., 2016, 9, 203.
2 Bibikova, O.; Haas, J.; López-Lorente, A.I.; Popov, A.; Kinnunen, M.; Meglinski, I.; Mizaikoff, B. Analyst,
2017, 142, 951.
3
Wang, H.; Liu, J; Graham Cooks, R.; Ouyang, Z. Angewandte, 2010, 122, 889.

53

Life Sciences, Biomedical and Pharma Research
P-151

Gold Nanoparticles contaminated by Bacterial Endotoxin: biophysical characterization,
imaging and nanotoxicology
A. Verde, M. Mangini, P. Italiani, D. Boraschi, A. C. De Luca
National Research Council of Italy, Institute of Biochemistry and Cell Biology, Naples, Italy

Gold nanoparticles (AuNPs) are nanodevices that can have many uses in biomedical applications but
unfortunately they show in some cases nanotoxic effects on biological systems [1]. Among the different
AuNPs effects in cells, activation of immune responses is considered a central issue for assessing healthrisks of AuNPs [2]. The main goal of this study is to identify and analyse the activation of the inflammatory
response associated to gold nanoparticles and/or to the presence of bacterial endotoxin (orLipopolysaccharide, LPS) on the nanoparticles’ surface. To this aim, the interaction of AuNPs with LPS is
analysed, the presence of LPS molecules on NPs is quantified, and the interaction of AuNPs with humanprimary macrophages is investigated, in order to distinguish the intrinsic NPs biological effects from thoseinduced by LPS.
LPS dose-dependent adsorption on 50 nm AuNPs was studied by DLS and SERS in order to understand theamount of LPS that binds to NPs surface and quantify it. Internalization of bare and LPS coated 50 nm
AuNPs was studied in macrophages by Raman imaging [3] and their inflammatory effect was studied by invitro stimulation through evaluation of inflammatory cytokine production (TNF-α).
DLS results indicate that a uniform LPS corona (8712 molecules) is formed around all NPs (2 µg) whenincubated with doses greater than 500 ng, while analysis of SERS signals show a Limit of Detection (LOD)
for LPS amount of the order of fg. These promising results show how SERS technique can be a reliable
LPS-Sensor, while NPs imaging studies showed that NPs are localized in cytoplasmic vesicles insidemacrophages. Moreover, bare NPs do not induce the production of TNF-α cytokine in treated macrophages.
[1] Chen, YS., Hung, YC., Liau, I. et al. “Assessment of the In Vivo Toxicity of Gold Nanoparticles” Nanoscale
Res Lett (2009) 4: 858[2] Boraschi, D. and A. Duschl. 2012. “Immune System. In Adverse Effects of Engineered Nanomaterials”
edited by B. Fadeel, A.NANOTOXICOLOGY 1173 Pietroiusti, and A.A. Shvedova, 169–184.
Amsterdam:Academic Press.
[3] Managò, S. Migliaccio, N. Terracciano, M. Napolitano, M. Martucci, M. N. De Stefano, L. Rendina, I. De
Luca, A. C. Lamberti, A. Rea, I. “Internalization kinetics and cytoplasmic localization of functionalized
diatomite nanoparticles in cancer cells by Raman imaging” J. Biophotonics. 2018; 11: e201700207

54

Life Sciences, Biomedical and Pharma Research
P-152

Elucidating the composition of micro-scale polymer scaffolds for biomedical applications
by confocal Raman microscopy
T. Kielholz, F. Rohde, M. Walther, M. Windbergs
Institute of Pharmaceutical Technology and Buchmann Institute for Molecular Life Sciences, Goethe University, Frankfurt
am Main, Germany

Introduction:
Fiber-based scaffolds are gaining increasing importance as drug delivery systems as well as for tissue
engineering due to their easily tunable chemical composition, fibrous structure and favorable mechanicalproperties. However, especially in case of multi-component-systems, analytical characterization ischallenging with respect to differentiation of individual components. As integration of labels in such systems
is prone to modify the physicochemical behavior of the polymers and labeling after fabrication in threedimensional scaffolds is challenging, we aimed to evaluate confocal Raman microscopy in terms of its
applicability for multicomponent micro-scale polymer scaffolds.
Materials & Methods:
Samples with two distinct fiber types were fabricated via parallel electrospinning of organic polymer solutions
containing polycaprolactone (PCL) and poly(lactic-co-glycolic acid) (PLGA). An Alpha 300R+ confocal
Raman microscope (WITec GmbH, Ulm, Germany) equipped with a 532 nm laser was used to analyze thepristine fiber mats. Raman scans in x- and y-direction as well as for acquisition of multiple z-layer scans wereacquired to assess the three-dimensional organization of the sample. Adjacent evaluations were performed
with hierarchical clustering and basis analysis.
Results:
PCL/PLGA fiber mats were fabricated and Raman imaging was successfully used to differentiate theindividual fiber based on their composition. While PCL fibers showed a uniform appearance, individual PLGA
beads could be detected. Based on this information, the process parameters were optimized. Raman
analysis of the optimized fiber mat visualized uniform fibers without any bead formation. Interestingly, thecorresponding false-color image also showed significantly larger fiber diameters for PCL than PLGA. A threedimensional model was generated based on Raman data of z-stacks and proved the spatial homogeneouspolymer distribution up to the entire cross-section of the sample.
Conclusion:
Confocal Raman microscopy successfully visualized polymer distribution and existing manufacturing defects.
Visualizing the composition was crucial for gaining an in-depth understanding of the scaffold itself anddetermining subsequent steps for process improvement. The method offers great potential to analyze not
only fibers but also a plentitude of multi-component polymer specimens.

55

Life Sciences, Biomedical and Pharma Research
P-153

SERS imaging via metasurface made of silver-silicone periodic disks
A. Ivanov, I. Bykov, I. Ryzhikov, I. Boginskaya, A. K. Sarychev
Institute for Theoretical and Applied Electrodynamics, Russian Academy of Sciences, Moscow, Russian Federation

We investigate the light interaction with the regular metasurface made of silver-silicone periodic disk
resonators. Computer simulations as well as real experiment reveal an anomalous optical response of themetasurface, which is due to the excitation of various metal-dielectric surface plasmons. The enhancementof the local electric fields was measured from distribution of the Raman scattering signals over the surface.
We demonstrate SERS imaging of the metasurface made of silver-silicone periodic disks. Raman signals
excited by 785 nm laser radiation were recorded with a WiTec spectrometer (WITec 500 Alpha) using2
mapping regime. The Raman spectrum is collected with 100x objective (NA=0.9), mapping area 50x50 μm ,-1
step of scanning 0.56 μm, depth of focus 0.5 μm. We focused on crystalline silicone band 527 cm todemonstrate the enhancement of local electric fields over the metasurface.
-1
The optical mapping of the intensity distribution for the Raman line 527 cm of silicone is shown on Figure.
The result of the optical mapping was compared to the coordinate grid scanning electron microscopy (SEM)
to find the signal from each point of the surface. We demonstrate a significant enhancement of the intensity
distribution in the metasurface with respect to flat region. Moreover, the distribution of hot spots generated bysilver-silicon disks repeats the morphology of the surface, which is shown on SEM image. The metasurface
allows to reach the SERS enhancement factor of several orders of magnitude and very stable signal withuniform local electric field distribution over the entire surface. Therefore the system is much more thermallystable than most known SERS substrate, where molecules of an analyte degradate in the hot spots.

The work was partially supported by Russian Academy of Sciences (grant No. 20-21-00080) and Russian
Science Foundation (grant No. 16-14-00209).

56

Life Sciences, Biomedical and Pharma Research
P-155

Chemically-selective visualisation of organoids and their interaction with hydrogel matrices
1

2

2

2

1

N. Jung , T. Moreth , F. Pampaloni , E. H. K. Stelzer , M. Windbergs
1
Goethe University, Institute of Pharmaceutical Technology and Buchmann Institute for Molecular Life Sciences,2
Frankfurt am Main, Germany, Goethe University, Buchmann Institute for Molecular Life Sciences, Frankfurt am Main,
Germany

In vitro cell culture systems in 2D and 3D environments have experienced a surge in popularity over the last
decades in the growing field of alternative approaches to animal testing. Especially the cultivation oforganoids represents a promising approach in various clinical applications and with respect to personalised
medicine. Although the development of physiological relevant cellular systems is evolving steadily,
approaches for their comprehensive characterisation are stagnating, relying on bright-field based
visualisation and fluorescence-based imaging approaches. While the one delivers only limited information,
the other requires fixation and staining procedures, which are often impeded by the surrounding extracellularmatrix (hydrogels). In order to gain a comprehensive understanding of organoid cultures, their development
and especially their interaction with the surrounding matrix, which is neglected in the established analyses,
confocal Raman microscopy was implemented in the here presented study. Mouse pancreas organoids,
embedded in three different hydrogel matrices (commercial Matrigel® and two synthetic hydrogels), wereanalysed on single cell level as well as on whole organoid level to determine matrix-dependent
developmental alterations. We were able to show the sub-cellular organisation of single pancreas cells andthe distribution of organelles and nucleic acids and further evaluate the interaction of single organoids withthe surrounding matrix by imaging the interface between organoid and hydrogels. Based on these results,
suitable synthetic hydrogels could be identified as alternatives to the animal-derived, established Matrigel®
and a spectroscopy-based analysis procedure was established to allow for high-throughput analyses ofwhole organoid cultures. Due to the label-free and chemically-selective nature of the technique, we show for
the first time that it is possible to analyse 3D cell culture systems in their native state within a hydrogel matrix
and provide a glimpse into the possibilities of confocal Raman microscopy as an innovative approach for the
analysis of hydrogel-based 3D in vitro cell culture systems.

57

Life Sciences, Biomedical and Pharma Research
P-156

A Raman-based approach for detecting circulating tumor cells.
1

2

2

1

1

2

1

M. Mangini , M. A. Ferrara , G. Zito , S. Managò , A. Luini , G. Coppola , A. C. De Luca
1
2
Institute of Biochemistry and Cellular Biology, Naples, Italy, Institute of Applied Sciences and Intelligent Systems,
Naples, Italy

Circulating tumor cells (CTCs) are a rare subgroup of cells that detach from the primary solid tumor andcirculate in the bloodstream of cancer patients. These cells act as a seed for metastases; moreover, they
maintain the primary tumor heterogeneity and mimic tumor properties. All these features make CTCs goodcandidates to be used as clinical biomarker for patient diagnosis, prognosis and treatment. Indeed, theisolation and characterization of CTCs can smooth the way to apply precision medicine approach in cancer
patients. However, the current methods to detect CTCs are mainly based on the recognition of epithelial celladhesion molecule (EpCAM) expressed on cancer cell membrane, but they typically fail to detect tumoral
cells that are losing epithelial phenotype and starting invasion/metastatic process. Here, we used deuteriumas vibrational tag to develop a new CTCs detection method based on Raman spectroscopy potentially able
to detect any type of CTCs. In particular, we exploited the capacity of cancer cell to internalize andmetabolize glucose 5-10 faster than normal cell, the so-called Warburg effect. Normal prostatic cells (PNT2),
cancer prostatic (PC3) and hepatic (HepG2) cells were used as in vitro model. Cells were cultured inpresence of 25mM deuterated glucose for 48h and then analyzed by Raman spectroscopy (1). The typical-1
deuterium Raman band at 2100 cm was present in the spectra of PC3 and HepG2 cells, but not in PNT2
spectra. These results indicate the presence of Warburg effect in our cellular model and that cancer cells canbe differentiated from normal cells following glucose metabolism. To simulate the presence of CTCs in blood,
PC3 and HepG2 cells were co-cultured with white blood cells isolated from healthy donor blood in presenceof deuterated glucose. The deuterium Raman signal was observable only in the spectra of PC3 and HepG2
cells. Our data demonstrate that cancer cells can be distinguished from healthy cells independently from
EpCAM expression just exploiting the glycolytic metabolism also when they are in the same media. These
results shed a light on the possibility to develop new CTCs detecting methods using label-free approachbased on Raman spectroscopy.

1. Managò S., Valente C., Mirabelli P., Circolo D., Basile F., Corda D. and De Luca A.C, 2016. A reliable
Raman-spectroscopy-based approach for diagnosis, classification and follow-up of B-cell acutelymphoblastic leukemia. Sci Rep. 19, 6:24821.

58

Life Sciences, Biomedical and Pharma Research
P-165

Raman imaging of the functional adipose tissue
Z. Majka, K. Czamara, A. Kaczor
Jagiellonian University, Kraków, Poland

For many years, the adipose tissue, particularly the perivascular adipose tissue (PVAT), was disregarded by1
anatomopathologists as incoherent and irrelevant. Nowadays, it is certain that PVAT is markedly involved in
2
vascular homeostasis. Depending on its phenotype, PVAT has different functions, inter alia, anti3
inflammatory action and regulation of insulin sensitivity. PVAT can be reminiscent of interscapular brown
adipose tissue (iBAT) in the thoracic aorta (TA PVAT) or epididymal white adipose tissue (eWAT) in theabdominal aorta (AA PVAT). Moreover, PVAT dysfunctions may lead to inflammation, dyslipidemia and3
insulin resistance. All these diseases can contribute to the increased cardiovascular risk associated with
2,3
obesity, which is a common cause of mortality in the world. Therefore, it is important to find an effective
method to determine pathological changes in the adipose tissue. Due to large Raman scattering cross4
section of lipids, Raman spectroscopy is well fitted tool to investigate the adipose tissue.
The aim of this work was to investigate the processes involved in obesity development i.e. inflammation andexcessive lipid accumulation. We developed the in situ adipose tissue model for investigation of chemicalchanges in the functional adipose tissue stimulated by various factors. The chemical composition of theadipose tissue of healthy mice isolated from different depots (iBAT, eWAT, TA PVAT, AA PVAT) was
visualized in situ using Raman imaging after 24 and 48 hours incubation in medium supplemented withsaturated fatty acids or inflammatory factor i.e. TNF-alpha. The analysis of Raman images enabled to show
individual adipocytes and the intercellular matrix and to study changes in lipid content and lipid unsaturation
based on Raman marker bands. This work demonstrates the potential of Raman imaging in studies of single
adipocytes in situ in the functional adipose tissue.

1. C. M. Pond, Front. Physiol., 2017, 8, 8–10.
2. K. Schäfer, I. Drosos and S. Konstantinides, Int. J. Obes., 2017, 41, 1311–1323.
3. M. S. Fernandez-Alfonso, B. Somoza, D. Tsvetkov, A. Kuczmanski, M. Dashoowd and M. Gil- Ortega,compregensive Physiol., 2018, 8, 23–59.
4. K. Czamara, Z. Majka, M. Sternak, M. Koziol, R. B. Kostogrys, S. Chlopicki and A. Kaczor, Int. J. Mol. Sci.,
2020, 21, 1–14.

59

Life Sciences, Biomedical and Pharma Research
P-169

Glioma Tumors Classified using Visible Resonance Raman Spectroscopy and Machine
Learning
1

2

1

3

4

5

4

6

7

5

5

Y. Zhou , S. Zhang , L. Zhang , B. Wu , K. Zhu , C. Zhang , Y. Yang , X. Yu , H. Hu , C.-h. Liu , R. Alfano
1
2
3
Air Force Medical Center, Beijing, China, JRME Co., Ltd., Taizhou, Jiangsu, China, Southern Connecticut State
4
5
University, Physics, New Haven, United States, Chinese Academy of Sciences (CAS), Beijing, China, City College of6
7
New York, New York, United States, General Hospital, Beijing, China, WITec GmbH Beijing Rep. Office, Beijing, China

Human glioma is an advanced disease which accounts for 75% of all newly diagnosed malignant primarybrain tumors, with an average survival time of about one year. Early diagnosis and detection of glioma is still
desired for initial treatment.
In this study, we report a new optical molecular histopathology method based on visible resonance Raman(VRR) spectroscopy combined with confocal Raman microscopy (CRM) for diagnosing human brain glioma
and distinguishing gliomas of grades II through IV from normal brain tissues.
VRR is a novel label-free technique for optical molecular histopathology assay started in 2011. VRR Raman
signals are enhanced due to “magic” 532nm excitation wavelength which matches a large group of natural
biological molecular absorption bands. CRM offers a new way to identify cancers from normal tissues withhigh resolution, non-destructive molecular mapping and provided hyperspectral images with highly complex
biochemical and physiological information of biological tissues. This is different from traditional opticalimaging.
VRR images from twenty-five specimens including healthy tissues and gliomas of grades II to IV were
investigated ex vivo using WITec300R confocal micro Raman imaging system with laser excitation of 532nm.
Two-dimensional (2D) RR spectral mappings performed in 20μm×20μm with a step size of 1μm generated adataset of 400 spectra which is essentially an image stack with Raman shift as the third dimension andshows the distribution of various biochemical bonds in image slices. A three-dimensional (3D) plot was used
to demonstrate the spatial distributions of three selected sets of molecular biomarkers and revealed
significant differences in the components between normal and different grades of gliomas due to thechemical composition changes. The RR molecular spectral fingerprints showed (a) a clear enhancement of-1
vibrational modes at 1129 and 1338cm which are believed to arise from lactate and DNA; (b) an evident-1decrease in the RR vibrational modes at 1442 and 2854/2885cm due to saturated fatty acids bonds(proteolipid) in all-grades of glioma tissues compared with normal tissues; and (c) an enhancement in the RR-1
spectral modes of 1129 and 2938cm (saccharolipids) which suggests the contribution from lactate. Thesefindings may provide a novel proof for anaerobic glycolysis metabolic process in brain gliomas which has
been explained by Warburg effects. An example of VRR imaging result is shown in Fig. 1.

Fig. 1. VRR imaging of grade II glioma. From left: brightfield image; VRR 2D image for 1129cm-1; VRR image using 3D plot; 3D image
zoomed-in; color-coded image based on spectra of the components and the corresponding spectra of lactate and lipids

60

Life Sciences, Biomedical and Pharma Research
P-173

Biopharmaceutial structural characterization by Confocal Raman methodology: Defining
critical parameters to method development
J. Silva, J. Cristóvão, J. Pereira
Hovione Farmaciência S.A., Estrada do Paço do Lumiar, Campus do Lumiar, Edificio S, 1649-038 Lisboa, Portugal, R&D- Analytical Development, Lisbon, Portugal

Confocal Raman microscopy and spectroscopy are powerful techniques for solid-state protein
pharmaceutical analysis that can be used for identity and stability testing, being useful to determine changes
1in the secondary and tertiary structure of proteins in different matrices . Raman spectrometry has significant
potential advantages in Biopharmaceutical industry, as intrinsically high molecular specificity, flexibility ofsampling configurations and the requirement for minimal (or no) sample pre-treatment and can be exploited
to deliver unique and useful analytical solutions. However, Raman-based methods, when applicable toproteins, can be limited by the chemical complexity of those. Nevertheless, with enough knowledge on theproteins structure and good experimental design, robust analytical methods can be developed. The present
work aims to define a systematic approach for method development of Confocal Raman spectroscopy bymeans of Analytical Quality by Design (AQbD) that could be used to a wide range of protein-based
molecules aiming at detecting discrete changes in conformational structure. In this presentation, the work
conducted regarding the development of an ID method for proteins, following an AQbD approach, is shown.
To develop an analytical method for the identification of proteins secondary structure, Bovine Serum Albumin(BSA), was defined as model protein. The goal is to later on apply this method for Amide I, Amide III and
2,3
Skeletal Stretch – hence the typical wavelengths regions of these proteins were selected for fingerprint.
Spectra accumulations and integration time were identified as Critical Method Parameters (CMP) (Figure1a); normalized areas of the zones of interest, as well as corresponding standard deviations were found tobe critical method attributes. With the defined success criterion, we were able to achieve a Method Operable
Design Region (MODR) (blank region on Figure 1b). To confirm the robustness of the method and theconsistency of the analytical target profile, a second DoE (Figure 1c) was performed - results obtained werein line with expectations (Figure 1d).
AQbD methodology was successfully used as an effective and comprehensive tool for Raman spectroscopy
ID method development of complex systems. In fact, with this approach, it was possible to optimize thecritical method attributes and define the most robust conditions of analysis.

Figure 1 - a) DoE to define CMP; b)
Experiment prediction in Fusion
QbD® software; c) DoE to proverobustness; d) Resulting spectra withzones of interest (red: 30 acc./1.4s
int. time; green: 40 acc./1.6s int. time;
blue: 35 acc./1.5s int. time)61

Life Sciences, Biomedical and Pharma Research
P-175

Visualization of extractives and xylan-type polysaccharides in ivory nut endosperm by
Raman imaging and principal component analysis
1

2

1

Y. Ferreira da Silva , R. Nunes Oliveira , R. Antoun Simão
1Federal University of Rio de Janeiro, Department of Metallurgical and Materials Engineering, Rio de Janeiro, Brazil,
2
Federal Rural University of Rio de Janeiro, Department of Materials Engineering, Seropédica, Brazil

In advance of World War II and the widespread consumption of synthetic polymers, ivory nuts were aprevalent source of income to South American countries such as Ecuador, Colombia and Brazil. These
seeds, produced by palms from the genus Phytelephas, present a rigid endosperm that was exported as araw material for the fabrication of buttons. However, the economic impacts of the war reduced theirinternational trade, which was not reestablished due to popularization of plastics. Nowadays, the awarenessof environmental issues caused by synthetic polymers has restored the interest in natural materials, andivory nuts have been lately investigated due to their noteworthy mechanical properties and eco-friendly
appeal. Their endosperm, previously characterized by wet chemical methods, has been reported as
constituted by pure mannan, biopolymer based on mannose with a low degree of substituents, and a minor
amount of cellulose. However, the characterization by chemical extractions may modify the original cell wall
structure and add artifacts to the results. Hence, in this work, ivory nut endosperm was characterized by
Raman imaging to identify cell wall constituents non-destructively and visualize their spatial distribution. Ivory
nut disks with polished surfaces were analyzed in a Witec Alpha 300 Raman spectrometer using a 785 nm
laser and a 10 x objective lens with a 0.30 numerical aperture. Hyperspectral imaging maps were generated
2
in a 50 x 50 µm region and spectral processing was performed using Witec Project 2.10 and RStudio, as
illustrated below.

Diagram illustrating ivory nut sample, an optical microscopy image and an average intensity map for
Raman imaging in the analyzed region. In addition, PCA average intensity score maps are provided.
-1

The average spectrum in the 1000 to 1750 cm range mainly contained peaks attributable to xylan-type-1-1
polysaccharides (1021, 1081, 1106, 1134, 1246, 1311 and 1364 cm ), cellulose (1454 cm ) and lipids (1466-1-1cm ). Besides, overlapped contributions from β-mannose units (1081, 1106 and 1134 cm ) and β-glucose-1
units (1106 cm ) could also be present. In order to evidence subtle spectral contributions and distinguishoverlapped peaks, principal component analysis (PCA) was performed using RStudio and hyperspec
package. PCA results for the first four principal components evidenced the accumulation of xylan andextractives, specially waxes and polar compounds, in endosperm tubules. These results indicate that,besides mannan and cellulose, ivory nut cell walls are constituted by xylan-type polysaccharides whileendosperm tubules contain extractive substances, probably present as a defense to biodeterioration.

62

Life Sciences, Biomedical and Pharma Research
P-179

Customizable SERS substrates dedicated for life science and diagnostic research
P. Albrycht , J. Borkowska , M. Księżopolska-Gocalska , Ł. Kozoń , R. Hołyst
12Institute of Physical Chemistry PAS, Soft Condensed Matter, Warsaw, Poland, Mossakowski Medical Research Centre
3
Polish Academy of Sciences, Warszawa, Poland, Institute of Physical Chemistry PAS, Warsaw, Poland
1

2, 3

3

3

3

Surface-Enhanced Raman Spectroscopy (SERS) is gaining popularity in life sciences as it allows us toobtain the fingerprint of a specific substance or even of a cell type. Thus, SERS is now intensively studied asa promising tool for diagnostic and pathogen identification. SERS-based biosensors and microfluidic systems
may be fast (a few seconds measurements) and accurate platforms for detecting cancer, foodcontamination, and even viruses identification.
We have recently developed sensitive and repeatable silver and silver-gold SERS substrates dedicated topotential use in biosensors and microfluidic systems for diagnostic, forensics, and life science research.
Substrates are made by the electrodeposition of silver and gold nanoparticles on ITO glass. Our substrates
have dimensions dedicated to 96-well plates and give excellent (ppm to ppb) enhancement after compounddeposition in a small volume of analyzed solution. Additionally, thanks to the use of a precise laser plotter,
we can obtain defined dimensions of substrates. It is especially desirable for the development of “Lab-on-aChip” type platforms. Our SERS substrates can have hydrophilic or hydrophobic properties (on demand).
Substrates with a hydrophilic surface give excellent enhancement of aqueous solutions, while hydrophobic
substrates are dedicated to alcoholic solutions.

63

Confocal Raman
Microscopy
2nd Edition

Confocal Raman Microscopy, edited by members of the WITec team and partof the Springer Series in Surface Sciences, has been thoroughly revised andexpanded for its 2nd edition. The book provides a comprehensive overview
of the fundamentals, practical considerations and real-world applicationsof Raman microscopy.
With 23 chapters it also includes sub-sections on theory and technology,
novel materials, geosciences, life and pharmaceutical sciences, materials
science and a historical overview of the Raman effect.
Additionally, it explores the rapidly evolving field ofcorrelative microscopy by detailing how 3D Raman
imaging can be integrated with other investigative
methods to achieve a far greater understanding ofa sample’s properties.

The 2nd edition of Confocal Raman Microscopy,
edited by WITec scientists Dr. Olaf Hollricher,Dr. Thomas Dieing and Dr. Jan Toporski, can bepurchased in print or e-book formats directlyfrom Springer or through online shops.

Order your copy today and havethe current state of the art inConfocal Raman Microscopy at
your fingertips.

Confocal Raman Microscopy
Editors: Jan Toporski, Thomas Dieing, Olaf Hollricher
ISBN: 978-3-319-75380-5
www.witec.deCorrelative Raman-SEM (RISE) image of graphene.

3D Raman Imaging
Turnurn ideas into
in discoveries
Let your discoveries lead the scientiﬁc future. Like no othersystem, WITec’s confocal 3D Raman microscopes allow for
cutting-edge chemical imaging and correlative microscopy
with AFM, SNOM, SEM or Proﬁlometry. Discuss your ideas
with us at info@witec.de.

Raman • AFM • SNOM • RISE
MADE IN GERMANY

www.witec.de