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

Forensic Investigations using Confocal Raman Imaging
the alpha500 microscope series add
automation and large scan area measuring
capabilities. The confocal Raman microscope
can be equipped with a variety of excitation
lasers. All measurements were performed
using an UHTS300 (ultra-high-throughput
spectrometer) and back-illuminated CCD
cameras. The distribution of chemical specieson the sample was obtained in Raman spectral
imaging mode. In this imaging mode acomplete Raman spectrum is recorded at every
imaging point, leading to a 2D array of Ramanspectra. By extracting individual features suchas Raman peak intensity, peak width or peak
position from the recorded 2D arrays ofExperimental Methods
spectra, Raman images can be calculated
All experiments were performed with thedisplaying a variety of information contents.
confocal Raman microscopes of the alpha300 Furthermore, fitting algorithms can beR or alpha500 R series. The alpha300
employed to extract additional images frommicroscope series is optimized for highthe recorded 2D arrays of Raman spectra. The
resolution imaging of samples and is able to combination of such images allow thecombine several analytical measuring
illustration of the distribution of various
techniques in one instrument such as:compounds in one image. The illustration
diffraction-limited confocal microscopy,
colors are coded in the same way for thechemically-sensitive Raman spectroscopy and corresponding spectra.The acquisition time
cantilever based scanning probe microscopy, for a Raman spectrum within a 2D spectralincluding AFM and SNOM. Using these
instruments it is possible to analyze the same
sample position with various measuring
techniques sequentially or even in parallel(a)(b)(Raman and AFM). The alpha300 R Plus andIntroduction
Crucial tasks in forensics include the microscopic
analysis of very small objects in order to accurately
verify the source of specific compounds of such
sample, or to compare the characteristics ofseveral samples. Confocal Raman Imaging is avery effective tool for such analysis as it provides
information on the chemical composition as wellas the spatial distribution of the components.
The samples typically do not require specificsample preparation and remain unaltered forfurther investigations with other methods since
Raman Imaging is a non-destructive, non-contact
and non-contaminating analysis technique.

(a)SulfurAmorphous Carbon
NitrateFig. 1: (a) Typical Raman spectra of explosive compounds as detected in the large area Raman
scan of a human fingerprint. (b) Raman Image of the fingerprint with the location of thesmall explosives residues.
1array was only a few milliseconds or 10s of
milliseconds, thus allowing the acquisition of
thousands of Raman spectra within minutes.
Residues of Explosives on a Fingerprint
In the following study Confocal Raman
Imaging was applied to the detection of
explosives residues on a fingerprint which
could be a hint that the person most recently
handled such material. A fingerprint on glass
was scanned using the large area scanningcapabilities of the alpha300 R+ system. Amongthe acquired spectra, three spectra related toexplosive materials could be easily detected
as shown in Fig. 1 a (red spectrum: Sulfur, blue
spectrum: amorphous carbon, turquoise:Nitrate). The fluorescence signal was used togenerate an image of the fingerprint outline(Fig. 1b) with the location of the explosives
residues additionally shown in the same color
code as the spectra in Fig. 1 a. The imaging ofa complete fingerprint allows theidentification of an individual person and theco-location of explosives compounds strongly
suggests that this person was in contact withexplosive materials.

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

Writing and Printing on Paper“Who signed first? – And with which pen?”
Two common questions in forensic document
forging investigation processes. Confocal
Raman imaging can provide evidence of theuse of various ballpoint pen marks on paper
and the order in which these inks (pastes)
were applied to the paper. Fig. 2a shows avideo image recorded at the intersection of
two different ballpoint pen marks. Paste 1 wasapplied on the right side of the video image
and paste 2 on the left side. The possibility ofan overlap of these two pastes cannot be
excluded, however this is not evident fromthe video image alone. The area marked withthe red frame (50x50 µm2) was scanned in(a)Raman spectral imaging mode by recording
an array of 128x128 complete Raman spectrawith an integration time of 100 ms/spectrum.
From the array of spectra, three different
Raman spectra were calculated. At somesurface areas only luminescence could bedetected (not shown as a spectrum), whereas
other areas showed the unique Raman spectrapresented in Fig. 2b. These two spectra showonly very small differences of which some arehighlighted with arrows in the figure. In thecolor-coded Raman image presented in Fig.
2c, blue denotes the luminescent areas while
the distribution of the two ballpoint-pastes
are represented in red and green. An area
where both pens can be seen is clearly visible.
To prove the order in which these ballpoint

pens were applied to the paper, a depth scan(x-z scan) along the black line in the video
image (Fig. 2a) was performed. An array of
128x128 complete Raman spectra was acquired
over 90 µm in the x-direction and 50 µm inthe z-direction with an integration time of
100 ms/spectrum. The distribution along thedepth profile of the two ballpoint pens pastes
is shown in Fig. 2d, using the same color-code
as for Fig. 2c. This depth profile clearly showsthat the red marked paste was applied on topof the green marked paste, thus showing theorder in which these pastes were applied tothe paper. This result can only be obtained byusing a high confocal resolution Raman
imaging setup.

(b)paste 1paste 2

(c)
(d)

Fig. 2: Confocal Raman imaging of ballpoint writing on paper. Video image (a), evaluated Raman spectra (b), color-coded Raman image recorded fromthe paper surface, image area 50x50 µm2, 128x128 pixels, integration time: 100 ms/spectrum (c), and color-coded depth profile, image area 90x50 µm2,128x128 pixels, integration time: 100 ms/spectrum (d). The ballpoint writing is shown in red and green in the images.
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application noteLise-Meitner-Straße 6, D-89081 Ulm, Germany
Tel. +49 (0) 731 140 700, Fax. +49 (0) 731 140 70200
www.witec.de, info@witec.de

In the following examples a combined printing
and ballpoint mark on paper is analyzed toshow that by using Raman imaging it ispossible to distinguish printer ink fromballpoint pen ink. These experiments require
the analysis of large sample areas in the rangeof several millimeters and therefore thealpha500 R microscope equipped with a green
laser and a 50x air objective (NA = 0.5) wasused. In a first measurement the chemicalcomposition of the printer ink distributed on
the paper was examined. A sample area of
3x2 mm2 was imaged in Raman spectral

imaging mode by acquiring an array of 150x150
Raman spectra with an integration time of
160 ms/spectrum. From this array threedifferent spectra were calculated as shown in
red and yellow color in Fig. 3a, whereas thethird spectrum (not presented) showed strong
fluorescence represented as green spots in
Fig. 3b. The red spectrum is characteristic forthe coated printer paper, whereas the yellow
spectrum corresponds to the printing ink used
to produce a yellow printed line as shown inthe color-coded Raman image in Fig. 3b.
Additionally, in the printer ink, strongly

fluorescent particles (represented in green in
Fig. 3b) could be detected, which aredistributed randomly over the printed line.
In a second experiment, a ballpoint pen line
was drawn across the border between theprinted line and plain paper. A large area scanof 0.8x1.3 mm2 was performed over the border
between the paper, printed line and ballpoint
pen writing. The distribution of paper, printed
line and ballpoint pen mark is shown in Fig.
3c. The ink of the ballpoint pen (blue spectrumin Fig. 3a and blue color in Fig. 3c) shows
significant spectral differences compared tothe ink used in the printer.
The measurements presented in this section
show that confocal Raman imaging cancontribute to the analysis of forensic material,
providing information regarding printing
material used and the order in which theywere applied.

(a)

(c)

paper
(b)

Ballpoint-penFig. 3: Confocal Raman imaging of printing and ballpoint writing on paper: evaluated Raman spectra of paper (red), printer ink (yellow) and ballpoint
ink (blue) (a), color-coded Raman image recorded from the paper surface after printing, image area 3x2 mm2, 150x150 pixels, integrationtime: 160 ms/spectrum (b), and color-coded Raman image after printing and writing, image area 0.8x1.3 mm2, 128x128 pixels, integrationtime: 160 ms/spectrum (c).
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