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APPLICATION NOTE

On the RISE

Correlative Confocal Raman and ScanningElectron Microscopy

The Raman principle

No need for compromises

The Raman effect is based on the inelastic
scattering of light by the molecules ofgaseous, liquid or solid materials. Theinteraction of a molecule with photons
causes vibrations of its chemical bonds,
leading to specific energy shifts in thescattered light. Thus, any given chemicalcompound produces a particular Raman
spectrum when excited and can be easily
identified by this individual “fingerprint.”
Raman spectroscopy is a well-established,label-free and non-destructive method foranalyzing the molecular composition of asample.

The Raman effect is extremely weak, so
every Raman photon is important forimaging. Therefore WITec Raman imaging
systems combine an exceptionallysensitive confocal microscope with anultra-high throughput spectrometer(UHTS). Precise adjustment of all opticaland mechanical elements guarantees thehighest resolution, outstanding speed andextraordinary sensitivity - simultaneously!
This optimization allows the detectionof Raman signals of even weak Raman
scatterers and extremely low material
concentrations or volumes with thelowest excitation energy levels. This isan unrivaled advantage of WITec systems.

Raman imaging
In Raman imaging, a confocal microscopeis combined with a spectrometer anda Raman spectrum is recorded at every
image pixel. The resulting Raman image
visualizes the distribution of the sample’scompounds. Due to the high confocality ofWITec Raman systems, volume scans and3D images can also be generated.

Resolution
Lateral resolution is physicallylimited to ~200 nm, dependingon the wavelength of theincident light.

Speed
The more sensitive a system is, the shorter
the acquisition time for a single spectrum.
WITec’s Ultrafast Raman Imaging reduces
acquisition times for single Raman spectra
down to well below 1 ms.

Sensitivity
A high confocality increases the signal-to-noise ratio by reducing
the background. With the UHTS Series, WITec developed lens-based,
wavelength-optimized spectrometers with a spectral resolution
down to 0.1 cm-1 relative wavenumbers.

Introduction
Correlative imaging techniques gain more
and more importance in many fields ofapplications due to the fact that morecharacteristics of a specimen can beanalyzed with one instrument. The efficient workflow of correlative microscopy
techniques is an additional advantage thatsaves time and money.
In this application note we present correlative RISE microscopy: The first instrument
combining a fully loaded SEM with a confocal Raman microscope including whitelight microscopy.
RISE: Raman Imaging and Scanning Electron Microscopy
Scanning electron microscopy (SEM)
uses the interaction of electrons with theinvestigated material to reveal the highest
lateral resolution images of a specimen
structure. The same focused electron beam
can be used to generate energy dispersive
X-ray spectra (EDX) for obtaining information on the chemical elements of the matter. This technique, though very powerful,cannot extract details of the bonding ofatoms, which would disclose the nature of
the molecules in a sample. This task can beachieved by fusing SEM with non-destructive Raman imaging.
Raman imaging relies on the interaction ofphotons with a sample. The tremendous
importance of the Raman effect is basedon the inelastic scattering of photons withmolecules caused by the excitation or annihilation of a specific molecular vibrationand is characteristic for the type and coordination of the molecules involved in thescattering process. Thus Raman imaging
reveals the molecular/chemical composition of the substance.
This powerful combination is realized in
one instrument for Raman Imaging andScanning Electron (RISE) microscopy. Dueto an intelligent positioning system theinstrument enables for the first time diffraction limited confocal Raman imaging
from exactly the same sample area as theSEM image.

Info Box: RISE Microscopy
RISE Microscopy is the combination of confocal Raman Imaging and Scanning Electron Microscopy. It incorporates the sensitivity of the non-destructive, spectroscopic
Raman technique along with the atomic resolution of electron microscopy. Raman
Imaging enables the identification of molecules, their allotropes and polymorphs, thedetermination of their orientation, purity and crystallinity, and the detection of strainstates. SEM allows for the imaging of surface structures on the nanometer scale. RISE
combines the advantages of both, thus facilitating the most in-depth characterization of the sample.

RISE microscopes consist of a WITec confocal Raman microscope combined with an SEM (TESCAN or ZEISS).

RISE imaging of geological samples
A mineral phases of a rock section froma drill core was analyzed in the next example. The dominant rock type is Diorite.
The sample had no need to be treated orvaporized but was only sectioned with adiamond saw under water. This reduces
the risk of contamination of the surface,which is of great importance not only forgeological drill cores but also for objects
derived from the deep sea.
Fig. 1a shows the SEM image of a small
sample area using the back-scatteredelectron detector of the SEM. The same
area was analyzed using EDX (Fig. 1b). The
distribution of elemental groups indicatesthe presence of three distinct minerals.
Single Raman spectra could be acquired
from the three different areas (Fig. 1c)
three of which show the characteristic
Raman bands for quartz, epidote and plagioclase. This is in good agreement withthe elemental composition obtained byEDX. Raman spectra of the three primaryminerals were evaluated using cluster
analysis. In addition to these spectra, fourothers were detected, two of which could
indicate different grain orientations within
the phases of epidote and plagioclase
respectively. The spatial distribution of theminerals is shown in the color coded RISE
image (Fig. 1d) where the colors of the Raman image match the colors of the spectra. Not only mineral phase distribution
could be detected but also small grains
within a mineral phase. Fig. 1e shows thewhite light image acquired from the same
sample area as the SEM image. It clearly
emphasizes that high resolution opticalimages require proper focusing, whereas
SEM is completely insensitive to surface
roughness, which is why it is difficult toretrieve the sample area when using two
stand-alone instruments.
RISE imaging of non-conductive organic
materials
Non-conductive compounds can be examined with modern SEMs without specificpretreatment by using a low vacuum

abdce

mode that prevents the material from being electrically charged. Imaging polymer
blends or biological materials in this way
can reveal a contrast in the SEM images,
though without giving any clue regarding
the chemical composition of the variousphases. A film of a polymer blend consisting of polystyrene (PS) and polymethylmethacrylate (PMMA) was analyzed byRISE Microscopy. Both polymers are com-Fig. 1: A mineral phase of a diorite rock section. (a)SEM image (b) overlay of SEM and EDX image . Threeelement groups could be distinguished with EDX: Si,O: orange; Ca, Fe, Al: grey-purple; Na; green. (c) Raman
single spectra acquired from the three distinct
regions with the characteristic Raman bands ofquartz (brown), epidot (red) and Plagioclase (green).
(d): Raman spectral image overlaid with the SEM
image. (e) light-microscopy image from the same
sample area. Image parameters: 100 x 100 µm2, 150 x
150 pixels = 22,500 spectra, integration time 0.08 sec
per spectrum. Sample courtesy by Christiane Heim,
Geoscience Centre GZG, Dept. Geology, University ofGöttingen, Germany.

prised only of the elements carbon, oxygenand hydrogen. With EDX these elements
can be identified without revealing how
the elements are combined into molecules.
That is however precisely the strength ofRaman imaging as the spectra shed lighton the chemical nature (structural units,type and degree of branching, end groups,additives), conformational order (physicalarrangement of the polymer chain), stateabFig 2: (a) PMMA (red) and PS (green) show characteristic, distinguishable Raman
spectra. (b) Based on the spectra a Raman image of a PMMA-PS emulsion wastaken and overlaid with a SEM picture. Image parameters: 50 x 50 µm2, 300 x300 pixels = 90,000 spectra, integration time 0.035 sec per spectrum.

of the order (crystalline, mesomorphous,and amorphous phases), and orientation(type and degree of polymer chain andside group alignment in anisotropic materials). Fig. 2 shows an overlay of a color
coded RISE image of the PS (green) - PMMA(red) mixture. The SEM image reveals thefine structure of the two polymeric phases,
whereas the chemical identification of
the polymeric phases is provided by theconfocal Raman image. The Raman spectraof PMMA and PS are shown in Fig. 2b. Dueto their different hydrophilic properties
the two compounds do not mix but form adispersion of separated beads.

RISE imaging of hamster brain tissueIn this application example RISE Microscopy was performed on a microtome slideof hamster brain tissue. In the SEM image
the white and gray brain matter can be
distinguished by their structural differenc-es. Both tissues can be also chemically distinguished by their typical Raman spectra(figure 3a). Overlaying the SEM with thecolor-coded Raman image reveals a more
precise view of the distribution of the grayand white matter (figure 3b).

baFig. 3: a) The Raman spectra of a hamster brain tissue sample reveal the different spectral characteristics of the white and gray brain matter. b) Raman-SEM image
overlay. In the color-coded Raman image the white brain matter is shown in Green and the gray brain matter in Red. 100 x 100 µm2, 300 x 300 pixels = 90,000 spectra,
integration time: 50 ms/spectrum

abcFig. 4: (a) SEM image of a graphene sample. (b) SEM
image overlaid with the color-coded confocal Raman
image. The colors display the graphene layers andwrinkles. Image parameters: 20 x 20 µm2, 150 x 150
pixels = 22,500 spectra, integration time: 0.05 s/spectrum, 1 layer (blue, dark green), > 1 layer (lightgreen, red), Si (yellow).

RISE analysis of carbon nano-materials
Thin-layered or single layer materials,
defined as 2D materials, have recently
attracted enormous research interest due
to their special electronic and optical properties which differ significantly from thatof their bulk precursors. Graphene is oneof the most popular materials extensively
studied in this context. In Fig. 4 the SEM(a) and RISE (b) images of a CVD graphenesheet deposited on Si/SiO2 substrate arepresented. The SEM image was acquiredwith low acceleration voltage (2 kV) using
the secondary electron (SE) detector. Thisimaging mode of the SEM is very surface
sensitive, thus revealing the fine structureof the CVD graphene sheet. The colors
used represent a single layer (blue, darkgreen) with different grade of defects andmore than one layer (light green, red). The
RISE image highlights the large amount ofdefects in the blue area, in good agreement with the high resolution SEM image.
The special properties of carbon nanotubes such as high thermal conductivity
make them interesting for applications inelectronics, nanotechnology, optics andmaterials science.
Single-wall carbon nanotubes (SWCNTs)
consist of a single atom thick, curved sheetof carbon. Their unique electronic and mechanical properties make them attractivefor electronics fabrication.
The Raman spectrum identifies SWCNTs
as single-walled by the presence of theircharacteristic RBM (radial breathing mode)
bands at low wavenumbers (fig. 5a). The
RISE image (fig. 5c) of SWCNTs embedded
in polyethylene (PE) was generated bymerging the Raman image (fig 5b) withthe SEM image.

abcFig. 5: (a) Characteristic Raman spectra of SWCNTs
and PE. (b) color-coded Raman image. (c) RISE image.
Image Parameters: 150 x 150 µm2 scan range, 80 x 80
pixels = 6.400 spectra, integration time: 60 ms/spectrum, PE (red), SWCNTs (green), SEM image (grey).

Volume scans are a valuable tool in providing information about the dimensionsof objects or the distribution of a certain
compound throughout the sample. In
order to generate 3D images, confocal 2D
Raman images of different focal planes
are acquired by scanning throughout thesample in the z-direction. The 2D images
are then combined into a 3D image stack.
To explore the 3D structure of the SWCNTs
sample, 16 Raman images were taken in
the z-direction intervals of 1 µm (fig. 6a).
From these data a 3D image was compiled.
It illustrates that the SWCNTs lie on top of
the PE matrix (fig. 6b).
RISE microscopy of MoS2
Inspired by progress in graphene research,other mono-layered materials such as
hexagonal boron nitride (h-BN) and transition metal dichalcogenides (TMDs) havealso received widespread attention. Recentwork has shown that exfoliated monolayer molybdenium disulfide (MoS2) is a 2D
direct bandgap semiconductor indicatingthat the material is suitable for optoelectronics and energy harvesting. Bulk MoS2
however is an indirect bandgap semiconductor. Thus detailed knowledge of thestructures and features of grains and grainboundaries are essential for understanding and exploring the materials’ properties
and its further applications. Here we present with MoS2 that RISE microscopy reveals
structure as well as crystalline and exciton
dynamics of thin-layered TMDs.
CVD grown monolayers of TMDs form
triangular two dimensional crystals. Twincrystals of MoS2 on SiO2/Si appear in theSEM image as star-shaped forms (fig. 7a).
The Raman spectra of these 2D crystals
show the characteristic E2g and A1g
Raman band modes of MoS2 (fig. 7c). Withan increasing number of layers the two
Raman bands drift apart due to inter-layer
and in-plane vibrations.
At the grain boundaries the Raman bands
not only show shifts, but additional bands
also appear (green spectra), indicating
defects or misaligned 2D crystals.

aFig 6: 3D imaging of SWCNTs on PEfilm. For a 3D image of the SWCNTs
on PE sample 16 equidistant Raman
images were taken while the focus
was shifted by 1 µm for each image.
The individual images are displayed
as RISE images in (a). The compiled 3D
Raman image of the analyzed sample
area shows the SWCNTs located on thetop of the PE filter (b).
Image parameters: 200 x 200 µm2
scan range, 70 x 70 pixels = 4,900
spectra, integration time: 0.037 s/spectrum. PE (red), SWCNTs (green),
SEM images (grey).

babcFig. 7: (a) SEM image of MoS2 twin crystals (b) Raman spectra of MoS2 monolayers (red), two or more layers(green) and silicon (purple). (c) corresponding color-coded Raman image derived from the spectral information
overlaid on the SEM image to give the RISE image. Image parameters: 22 x 17 µm2 scan range, 65 x 50 pixels =3,250 spectra, integration time: 0.037 s/spectrum.

They probably result from adjacent crystals colliding out at their boundaries. Thespectral information was used to createa Raman image which was overlaid onthe SEM image (fig. 7b). The overlapping
boundaries identified by Raman imaging
correlate perfectly with the dark edges
visible in the SEM image.
RISE analysis of polymorphs: Correlating
structure with chemical phasesWith SEM it is possible to identify materials consisting of different atoms using
a common combination with EDX (energy-dispersive X-ray spectroscopy). It cannot
however distinguish between different
modifications of chemically identicalamaterials (polymorphs). As the mannerof atomic bonding greatly influences thestructure and properties of a material,visualizing not only morphology but also
identifying its molecular architecture isimportant. RISE microscopy accomplishes
both of these tasks as demonstrated withthe analysis of TiO2 polymorphs (fig. 8). TiO2
is studied intensively because of its interesting chemical and optical properties andis widely employed in photo-catalysis, electrochemistry, photovoltaics and chemicalcatalysis. It is also used as white pigment
in tooth paste, sun screen and wall paintand as anode material for lithium-ionbatteries. Depending on the application,
one crystalline form or the other gains inimportance. TiO2 occurs in eight modifications, two of which – anatase and rutile– were examined. For RISE microscopy an
SEM image taken of a 1:1 anatase/rutile
powder mixture (fig. 8a) The corresponding confocal Raman image from the same
sample area was merged with this SEM
image (fig. 8b). It was demonstrated thatthough the elemental compositions ofthe modifications anatase and rutile areidentical, they can be distinguished fromone another by their characteristic Raman
spectra at relative wavenumbers between
300 and 800 cm-1 (fig. 8c). Both phases
were combined in agglomerates, in whichrutile accumulated in larger particles thananatase.

bc

Figure 8: Two modifications of TiO2, anatase and rutile, were mixed 1.1, ground, dissolved in water and imaged with an SEM (a) and a confocal Raman microscope. (b) SEM
and Raman images overlaid. (c) In the Raman spectrum anatase (blue) can be easily distinguished from rutile (red).
Image parameters: 12 x 12 µm2 scan range, 150 x 150 pixels = 22,500 spectra, integration time: 0.037 s/spectrum.

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