APPLICATION NOTE

3D Raman Imaging

WITec GmbH, Lise-Meitner-Str. 6, 89081 Ulm, Germany
phone +49 (0) 731 140 700, fax +49 (0) 731 140 70 200
info@WITec.de, www.WITec.de

Best practice examples from various fields ofapplication

Confocal Raman microscopy is a
powerful technique for high-resolution, non-destructive and label-freechemical analysis. It is well-suitedfor three-dimensional investigationof samples from various fields ofapplication.

application noteLise-Meitner-Str. 6 • D-89081 Ulm, Germany
Tel. +49 (0) 731 140 700 • Fax. +49 (0) 731 140 70200
www.witec.de • info@witec.de

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.”

The Raman effect is extremely weak, soevery 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!

Raman spectroscopy is a well-established,label-free and non-destructive method foranalyzing the molecular composition of asample.

Raman imaging
In Raman imaging, a confocal microscopeis combined with a spectrometer anda Raman spectrum is recorded at everyimage 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.

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.

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.

application noteLise-Meitner-Str. 6 • D-89081 Ulm, Germany
Tel. +49 (0) 731 140 700 • Fax. +49 (0) 731 140 70200
www.witec.de • info@witec.de

3D confocal Raman imaging
2D Raman imaging visualizes the distribution of chemical compounds, e.g. on the sample’s surface or in a focal plane within the sample (x-y plane), while 3D Raman imaging enables more complex chemical analyses of component distribution throughout a volume. Thisis particularly advantageous for investigations of bulky samples, complex emulsions or mixtures, geological specimens and living organisms [1-6]. This application note presents a collection of best practice examples from various fields of research, such as pharmaceutics,
food science, semiconducting materials and geology.

Pharmaceutical emulsion
Figure 1A shows a large-area, high-resolution 2D Raman image of a pharmaceuticalemulsion. For more details, a zoom-in
image of the indicated area is shown aswell. As a complete Raman spectrum wasacquired at each pixel, the image resultsfrom evaluating 4,194,304 Raman spectra.
For image generation the raw data wasprocessed by applying a cosmic ray filter
and a constant background subtraction.
The characteristic spectra of the sample
were then identified and a basis analy-sis was performed. The resulting Ramanimage (Figure 1A) is color-coded according
to the spectra of the three identified components (Figure 1B). The emulsion consisted of water droplets containing an active
pharmaceutical ingredient (API, blue) inan oil-matrix (green). In addition to thewater/API and oil phases, a third component was found (red), which was present
in only a low concentration. Their distributions can be evaluated in more detail froma volume scan.

In order to generate a 3D image, confocal
2D Raman images from 50 successive focal
planes were acquired by automaticallyscanning through the sample along thez-axis. The resulting image stack was then
combined into a 3D representation of thesample’s chemical composition (Figure 1C).
This 3D Raman image of the pharmaceutical emulsion contains the informationof a total of 2 million Raman spectra andcan be used to assess the volume of thedroplets.

AC

B
Figure 1: Confocal Raman imaging of a pharmaceutical emulsion.
The emulsion consists of an oil phase (green) and a water phase with dissolved API (blue). A third
component (red) was present in a low concentration. A: 2D large-area, high-resolution confocal
Raman image, color-coded according to the spectra in B. The zoom-in image of the indicated area
reveals more details. Scan range: 175 x 175 µm2; 2048 x 2048 pixels = 4,194,304 Raman spectra; integration time: 2 ms per spectrum. B: Raman spectra of the three detected components. C: 3D Raman
image. The oil phase (green) is partially removed for better visibility of the other components (red andblue). Scan range: 25 x 25 x 20 µm3; 200 x 200 x 50 pixels = 2 million spectra; integration time: 10 msper spectrum.

application noteLise-Meitner-Str. 6 • D-89081 Ulm, Germany
Tel. +49 (0) 731 140 700 • Fax. +49 (0) 731 140 70200
www.witec.de • info@witec.de

Banana pulp
Pressed banana pulp mixed with water
was investigated with an alpha300 Ri inverted confocal Raman microscope. Figure
2 shows a 3D representation generated
from a stack of 45 2D Raman images anda total of 6,075,000 Raman spectra. Starchgrains are displayed in green while the cellwall components are shown in red.

Figure 2: 3D Raman image of squashed bananapulp. Starch grains (green) and cell wall components (red) are shown. Scan range: 300 x 200x 90 µm3; 450 x 300 x 45 pixels = 6,075,000
Raman spectra; integration time: 34 ms perspectrum.

Honey

Figure 3: 3D confocal Raman image of pollen in honey. The liquid honey phase (yellow) surrounds apollen grain (green) and different crystalline honey phases (red, blue, cyan). Scan range: 50 x 50 x 50
µm3; 150 x 150 x 50 pixels = 1,125,000 Raman spectra; integration time: 2 ms per spectrum.

A sample of natural honey was investigated. For the 3D image, 50 individual 2D
Raman images (x-y scans) were acquired
from different z-positions through thesample. In a first processing step the main
sample components were identified by
the cluster analysis function of the WITec
Software Suite. As biological samples
tend to show a strong fluorescence background signal, an automatic backgroundsubtraction filter was also applied. The
data was further processed and optimized
for 3D visualization by applying the basis
analysis algorithm for spectral demixing.
The resulting signal intensity values wereused to generate the 3D image stack fromthe 50 2D scans in which the identified
phases are color-coded (Figure 3). A pollen
grain (green) is surrounded by different
crystalline phases of the honey (red, blueand cyan) and the liquid honey phase(yellow). The complete 3D image consistsof 1,125,000 Raman spectra, which wererecorded in less than 40 minutes.

application noteLise-Meitner-Str. 6 • D-89081 Ulm, Germany
Tel. +49 (0) 731 140 700 • Fax. +49 (0) 731 140 70200
www.witec.de • info@witec.deGallium nitride crystals
3D Raman imaging can be used fornon-destructively monitoring the crystal
quality of semiconducting materials, as
strain and crystallinity influence the shapeof Raman spectra. Here it was applied togallium nitride (GaN) grown on a patterned sapphire substrate that featured
evenly-spaced hexagonal pits. A depthscan in the x-z plane was performed toget a cross-section along a line of thesepits (Figure 4A). Then, 20 2D scans in thex-y plane (Figure 4B) were recorded atdifferent focal planes and a complete 3Drepresentation was generated from thisset (Figure 4C).

ACD

Evaluation of the recorded Raman spectra
revealed three distinct areas in the sample(Figure 4A-C). High-quality GaN was present mainly at the surface of the sample(red). The GaN material directly above thesubstrate’s pits was characterized by lower
crystal quality (blue), indicated by strong
broadening of the A1(LO) Raman peak. An
enhanced fluorescence signal was detected around the walls of the substrate’s pits(green).

As strain leads to Raman peak shifts, strain
fields in the sample volume were visualized by quantifying and color-coding theposition of the E2high peak for each image
pixel (Figure 4D). The strain fields became narrower towards the surface, again
indicating that the GaN crystal quality
was higher at the surface than close to thesubstrate. However, the overall differencesin peak position, and thus in the crystal strain, were quite small (Figure 4D).
They could be detected due to the highsensitivity and resolution of the UHTS300
spectrometer connected to the alpha300
Raman microscope.

BFigure 4: 3D Raman imaging of a GaN crystal.
Sample courtesy of Dr. Eberhard Richter (Materials Technology Department of the Ferdinand
Braun Institute, Berlin, Germany). A: Depth scan(x-z plane) along a line of substrate pits. Scan
range: 60 x 20 µm2; 240 x 80 pixels. B: Example
2D Raman image (x-y plane) extracted from therecorded image stack (C). C: 3D Raman imageof the sample volume. Scan range: 60 x 15 x20 µm3; 180 x 45 x 20 pixels. At the front rightcorner, a part of the structure has been removed
for better visibility. D: Strain fields in the crystal,revealed by shifts of the E2high Raman peak. The
image in the bottom plane indicates the positions of the substrate’s pits.

application noteLise-Meitner-Str. 6 • D-89081 Ulm, Germany
Tel. +49 (0) 731 140 700 • Fax. +49 (0) 731 140 70200
www.witec.de • info@witec.de

Fluid inclusions in geological samples
Fluid inclusions in rock samples are quite
common but vary widely in their dimensions. Using 3D Raman imaging, the composition of many geological samples canbe characterized without damaging them.
Here a fluid inclusion in garnet was investigated. The 3D Raman image (Figure 5)
revealed the distribution of garnet (red),calcite (green) and mica (cyan) surrounding a water inclusion (blue).

Figure 5: 3D confocal Raman image of a fluidinclusion in garnet. Water inclusion (blue) ingarnet (red), calcite (green) and mica (cyan).
Scan range: 60 x 60 x 30 µm3.

Asbestos fibers in mineral matrices
Asbestos refers to a group of six silicate
minerals which form fibers. Due to its
flexibility, strength and heat-resistance,
asbestos was a very popular building material. However, its use is now banned inmany countries because all asbestos forms
are highly carcinogenic. Therefore thedetection of asbestos fibers is necessary
in materials intended for construction,such as stone from quarries, and also thepreviously installed materials in old buildings. Their detection is often challenging
because the fibers’ diameters are usually
only a few hundred nanometers [6].
Together with the BRGM, the French national geological survey office, we recently
demonstrated the utility of combined 3D
Raman imaging and scanning electron
microscopy (SEM) for identifying and characterizing asbestos-like fibers in different
rock samples [6]. 3D Raman measurements revealed the shape of the fibers

Figure 6: Volume analysis of asbestos fibers ina mineral matrix [6]. Actinolite asbestos fibers(red) are surrounded by a matrix comprised oforthoclase (blue) and calcite (green). Image size
60 x 60 x 20 µm3.

within the mineral matrices and identified
the chemical composition of the fibers andthe matrix [6]. Such a 3D analysis allows
for the calculation of the aspect ratios andvolume fractions of the asbestos fibers.
Figure 6 shows actinolite asbestos fibers(red) in an orthoclase (blue) and calcite(green) matrix [6]. With an integration
time of 100 ms per spectrum, the entire 3D
image was acquired in only about 2 hours.

The measurements were performed using
a combined Raman Imaging and ScanningElectron (RISE) microscope (WITec andTescan). Thus, SEM and Raman images
could be obtained from the same sample
position, which enabled the correlation of
the structural information from SEM withthe chemical information from Raman
imaging [6]. More information on RISE
microscopy can be found in our Application Note [7].

application noteLise-Meitner-Str. 6 • D-89081 Ulm, Germany
Tel. +49 (0) 731 140 700 • Fax. +49 (0) 731 140 70200
www.witec.de • info@witec.deEmulsion with CCl4
Carbon tetrachloride (CCl4) was emulsified
with an alkane, water and oil and Raman
imaging revealed the distribution of theseingredients. Three different components
were identified in the mixture according
to their specific Raman spectra (Figure 7A):
the alkane (green), water (blue) and a mixture of CCl4 and oil (yellow). The Raman
images visualize the spatial distribution ofthese components in one image plane athigh resolution (Figure 7B) and in a recorded sample volume (Figure 7C).

AC

CCl4 can serve as a reference sample for
evaluating the spectral resolution of a
Raman spectroscopy system. The characteristic band at 460 cm-1 should be clearly
resolved into three peaks at room temperature, as can be seen in Figure 7A (inset).
The data was recorded with an alpha300
apyron fully automated microscope, whichallows for fast Raman imaging at highspatial and spectral resolution due to itsconsistently optimized optical throughput.

BFigure 7: Confocal Raman image of a CCl4 emulsion. A: Raman spectra of the identified components
alkane (green), water (blue), CCl4 in oil (yellow). The zoom-in image of the CCl4 spectrum shows thatthe peak at 460 cm-1 is clearly resolved. B: Example high-resolution 2D Raman image (100 nm perpixel), color-coded according to the spectra in A. C: 3D confocal Raman image of the emulsion. Scanrange: 200 x 200 x 20 pixels, 100 x 100 x 10 µm3; integration time: 60 ms per spectrum.

References[1] B. Prats Mateu et al. Label-free live cell imaging byconfocal Raman microscopy identifies CHO host andproducer cell line. Biotechnol. J. 12: 1600037 (2017). DOI:
doi:10.1002/biot.201600037.

[3] E. Tolstik et al. Studies of silicon nanoparticles uptake andbiodegradation in cancer cells by Raman spectroscopy. Nanomedicine 12: 1931-1940 (2016). DOI: 10.1016/j.
nano.2016.04.004.

[2] D. M. Bower et al. Raman imaging spectroscopy of a putative microfossil from the ~3.46 Ga Apex chert: Insightsfrom quartz grain orientation. Astrobiology 16: 169-180(2016). DOI: 10.1089/ast.2014.1207.

[4] C. Kallepitis et al. Quantitative volumetric Raman imaging of three dimensional cell cultures. Nat. Commun. 8:14843 (2017). DOI: 10.1038/ncomms14843[5] K. Czamara et al. Unsaturated lipid bodies as a hallmark
of inflammation studied by Raman 2D and 3D microscopy. Sci. Rep. 7: 40889 (2017). DOI: 10.1038/srep40889[6] G. Wille et al. Coupling SEM-EDS and confocal Raman-inSEM imaging: A new method for identification and 3Dmorphology of asbestos-like fibers in a mineral matrix.
J. Hazard. Mater. 374: 447-458 (2019). DOI: 10.1016/j.jhazmat.2019.04.012.
[7] WITec Application Note “On the RISE: Correlative Confocal
Raman and Scanning Electron Microscopy” www.witec.
de/assets/Literature/Files/WITec-AppNote-RISE-Raman-SEM-web.pdf

WITec Microscope Seriesalpha300 S: Scanning
Near-field Optical Microscopealpha300 A:
Atomic Force Microscopealpha300 R:
Confocal Raman Microscopealpha300 apyron: AutomatedConfocal Raman Microscopealpha300 Ri: Inverted
Confocal Raman Microscope

RISE: Raman Imaging –Scanning Electron Microscopealpha300 access: Confocal
Micro-Raman System

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