ICTON 2016 Tu.A4.

Surface Enhanced Raman Scattering in Surgery and Forensics

Catalin Micsa1, Constantin Rizea2, Madalin Ion Rusu3, Nicolae Dan Becherescu-Barbu4, Raluca
Munteanu1, Mircea Virgil Udrea4, Bogdan Chiricuta4, Anca Parau3, Alain Tonetto5, Roger Notonier5,
Ioan Alin Birtoiu1, and Cristiana-Eugenia-Ana Grigorescu3*
1
Faculty of Veterinary Medicine-University of Agronomic Sciences and Veterinary Medicine, Bucharest, Romania
2
ROXY VETERINARY S.R.L., Varteju-Magurele, Romania
*3
National Institute of Research and Development for Optoelectronics INOE 2000,
PO Box MG-5, 77125 Magurele, Jud Ilfov, Romania
4
APEL LASER S.R.L., Bucharest, Romania
5
PRATIM Aix-Marseille Universite, Site Saint Charles, Marseille, France
Tel:+4034050791, Fax:+4021 4574522, e-mail:krisis812@yahoo.co.uk
ABSTRACT
This paper shows some results of our work on the use of Surface Enhanced Raman Scattering as an ex vivo and
in vivo diagnostic means in veterinary surgery and oncology. Also, as part of the involved research, interspecies
discrimination of adipose constituents and body fluids is illustrated. These later outcomes would supply efficient
instruments to forensics. An innovative system based on surface enhanced Raman scattering, primarily used in
small animal surgery, will add to the ongoing research on tumour margin assessment. Samples from animal
patients following therapeutic mastectomy have been investigated ex vivo using a LABRAM HR 800 micro-
Raman spectrometer. Spectra were collected from 100 cm-1 to 4000 cm-1 using a 632 nm HeNe laser, which to
date has been reported on in vitro human samples only. A surface enhanced Raman effect of up to 107 has been
obtained on areas of surgery instruments either raw or coated with silver or gold. This dramatically improves
spectral resolution and reduces the accumulation time down to 60 s, which would allow for real time diagnostic.
Keywords: surface enhanced Raman scattering, Raman laser excitation in the visible, surface plasmon, gold and
silver nano-coatings, oncologic surgery diagnostic, forensics.

1. INTRODUCTION
Raman spectroscopy is known as a sensitive and non-destructive tool to extract information on molecular
chemical structure, interactions between molecules and the surrounding environment, and the physical state and
condition of matter. Its sensitivity to many different functional groups such as C=C, S-S, CS bonds that are
weak in the IR, the highly selective fingerprint allowing to discriminate similar compounds and the high spatial
resolution make this technique exceptionally desirable for diagnostic and monitoring in biological environment.
Measurements can be done in vivo or in vitro. The drawback denounced by many groups consists in the
weakness of the scattered signal and auto-fluorescence when applying Raman spectroscopy to biological samples
such as body fluids, tissue, fat, etc.
An effective way to improve the weak signals is to use surface enhanced Raman scattering (SERS), where
metal nanostructures could provide up to 1017 signal intensity amplification due to the resonant interaction of
light with the surface plasmons excited at the surface of the structure. This not only increases the spectral
resolution but also shortens significantly the duration of acquiring the spectra. The high improvement of the
Raman scattering signal of molecules at ~ or close to the surface occurs through the amplification of
electromagnetic fields generated by the excitation of localized surface plasmons at metallic surfaces [1-3].
It actually relies on both electromagnetic and chemical mechanisms [4, 5].
The electromagnetic enhancement factor GemSERS [4] arisen from enhanced optical fields due to excitation of
electromagnetic resonances in the metallic structures can be expressed as:
4
E ( rm ,n )
G em
( rm ,n ) = (1)
Einc (n )
SERS

with: E(rm, n) – the total electric field at the molecule location rm, n – the laser excitation frequency and
Einc(n) is the incident excitation field.
Chemical enhancement would be due to a metal electron-mediated resonance Raman effect via a charge
transfer intermediate state at “active sites.” [4, 6].
Accounting for the local field enhancement owing to plasmon resonances [7] and using equation (1) the
intensity ratio between the SERS signal and the normal Raman (NR) signal is derived:
I SERS em
= GSERS (2)
I NR

978-1-5090-1466-8/16/$31.00 ©2016 IEEE 1

ICTON 2016 Tu.A4.1

Elucidation of the full SERS mechanism is still under debate. However, there is no theoretical disagreement
concerning the two sources of enhancement, i.e. electromagnetic versus chemical [8].
Chemical enhancement requires chemical interactions between the probed samples and the metallic surface
while still accompanied by electromagnetic interactions. These latter ones would be obvious even in the absence
of the adsorbed sample [1, 9] thus proving the dominant role of the electromagnetic like amplification.
Polarization, excitation, as well as size and shape dependence were found to be qualitatively consistent with the
surface plasmon-based theory in SERS studies [8, 10].
View the above, SERS has the potential of a powerful spectroscopic tool with high sensitivity and high
specificity (down to molecule level) for the detection of chemical, biological, and medical analytes [8].
In the following Sections we show some results of our work concerning the use of SERS in veterinary surgery
and oncology aiming at a real time diagnostic tool and its possible use in forensics.

2. EXPERIMENTAL RESULTS AND DISCUSSION

The Raman measurements were run ex vivo using a LABRAM HR 800 (Horiba Jobin Yvon) micro-Raman
spectrometer, in the backscattering geometry with a λ = 632 nm HeNe laser for excitation source. Scalpel blades
with raw, Ag and Au nanostructured surfaces have served to collect the samples during surgery and as SERS
substrates to the end. The spectra were acquired from 100 cm-1 to 4000 cm-1. More details on the measuring
procedure are found elsewhere [11].
Twin samples (~60 µm thick) were investigated in vitro with an LSM 710 NLO Zeiss confocal microscope to
better understanding the complexity of the Raman spectra.
2.1 Diagnosing benign versus malignant tissues
Breast cancer has registered a worrying increase lately in both humans and other mammals, especially domestic
ones. The influence of surgical procedure on the survival time, disease free interval and period of new tumours
development has been evaluated recently in an ample study [12] showing that the simplest surgical technique
must be applied while achieving negative margins [13]. Accurate identification of tumour cells in body fluids
during surgery may substantially enhance the chance to realize a clean margin especially when excision of large
surfaces of tissue is required like in dog or cat Since SERS can discriminate single molecule [2] one could admit
that fresh smears from intraoperative malignant or benign tissue would provide exact fingerprints of tumour
markers or healthy margins. Those are obtained in Raman spectra as a result of photon inelastic scattering on
chemical bonds.
From the major Raman bands of the noncancerous and respectively cancerous human breast tissues assigned
from in vitro Raman imaging [14] we have chosen 1558 cm-1 (carotenoids, in benign only) and 3311 cm-1
(OH vibration, in malignant only) as markers for negative margins and tumour respectively (Fig. 1).

(a) (b)
Figure 1: (a) Confocal fluorescence image of malignant nodule; (b) Comparison between the Normal Raman
and SERS spectra of the nodule in the image (a). The 3311cm-1OH peak is noticed.
Figure 1a shows the image of smears from a tumour nodule whose Raman spectra (normal and SERS on
nanostructured silver coatings respectively) are displayed in Fig. 1b. The benefit of using SERS is obvious in
Fig. 1b: autofluorescence is counteracted owing to the metal surface plasmons, the spectral resolution is
amazingly improved along with a dramatic reduction of the collection time from 1800 s down to 120 s,
i.e. a factor of 15.
2.2 Potential of SERS in Forensics
Precise identification of traces through non-destructive analysis means is crucial in forensic science. To date the
common way to discriminate between various traces involves specific chemical tests for specific samples Raman
spectroscopy appears as an ideal technique for forensic investigations and advances has been reported especially

2
ICTON 2016 Tu.A4.1

in body fluid recognition [15, 16]. Use of visible light excitation and of SERS is still under debate [17, 18]
mainly due to the contact between the metallic surface providing the plasmon interaction and the possible
modification of the samples under the laser action. As previously reported on diagnosis in human surgery
[references for 2.1] and our work related to discrimination of smears through SERS, the laser would not modify
the biologic samples if appropriate excitation wavelength and incident power were chosen [14]. Similar to
oncologic surgery issues, more extended spectral ranges would provide more detailed information for forensics,
such as the presence of fat of either human or animal origin. Fat molecules are ever present in skin, fingerprints,
blood stains etc. An example of SERS application to discriminate between fat traces of different mammal species
is shown in Fig. 2. Confocal microscopy images of cat, dog and pig fat are included to support the SERS results.

Dog Cat Pig

(a)
cat
human
dog
pig
Relative intensity [arb.u.]

b
c

d
500 1000 1500 2000 2500 3000 3500 4000
Raman shift [cm-1]
(b)
Figure 2: (a) Confocal fluorescence microscopy images of dog, cat and pig fatty cells; (b) SERS spectra of
ex vivo samples of fatty tissues from the above species. Human fat spectrum is also shown for comparison.
The SERS spectra displayed in Fig. 2 were collected on gold-coated stainless steel surfaces with a RMS
roughness of 3.3 nm as resulted from AFM measurements. Their G factor has gone up to 107. The laser power at
the samples’ surfaces was 2 mW with a spot of 1 µm diameter. Micro-imaging check of the sample surface
following Raman measurements has shown no microscopic modification of the biologic material. Therefore, we
would argue pro SERS employment in forensics research [18].

3. CONCLUSIONS
We have shown that SERS with visible laser excitation (λ = 632 nm) could be used as a nearly “real time”
diagnostic means in veterinary oncologic surgery and in forensics research.
The extended to 4000 cm-1 spectral range provides information from fats and OH vibrations, useful in both
negative surgical margins assessment and inter-species discrimination.
Auto-fluorescence signalled in samples laid on glass substrates was absent when noble metal nanostructured
surfaces were used for substrates.
Since there has been no chemical interaction between the samples and the respective Ag and Au
nanostructured substrate we believe that in our experiments the scattering enhancement is of electromagnetic
origin only.
The low excitation laser power employed to acquire the spectra produces a high enhancement effect (up to 107)
without damaging/modifying the samples.

ACKNOWLEDGEMENTS
This work has been supported from PCCA 2013-UEFISCDI Romania -Contract No. 20/2014.
The donors (humans, dogs, cats and pigs) that consented for the samples whose SERS spectra are displayed in
Fig. 2 are greatly acknowledged.

3
ICTON 2016 Tu.A4.1

REFERENCES
[1] E.C. Le Ru, P.G. Etchegoin: Principles of Surface-Enhanced Raman Spectroscopy and Related
Plasmonic Effects, first ed., Elsevier, Amsterdam, 2008.
[2] J.P. Camden, J.A. Dieringer, Y. Wang, D.J. Masiello, L.D. Marks, G.C. Schatz, et al.: Probing the
structure of single-molecule surface-enhanced Raman scattering hot spots, J. Am. Chem. Soc. vol. 130,
pp. 12616–12617, 2008.
[3] B. Sharma, R.R. Frontiera, A.I. Henry, E. Ringe, R.P. Van Duyne: SERS: Materials, applications, and
the future, Mater. Today, vol. 15, pp. 16–25, 2012.
[4] K. Kneipp, Yang Wang, H. Kneipp, L. T. Perelman, I. Itzkan, R. R. Dasari, and M. S. Feld: Single
molecule detection using surface-enhanced Raman scattering (SERS), Phys. Rev. Letts., vol. 78,
pp. 1667–1670, 1997.
[5] A. Otto, in Light Scattering in Solids, edited by M. Cardona and G. Guentherodt, p. 289, Springer,
Berlin, 1984.
[6] S.L. Kleinman, R.R. Frontiera, A.I. Henry, J.A. Dieringer, R.P. Van Duyne: Creating, characterizing,
and controlling chemistry with SERS hot spots, Phys. Chem. Chem. Phys., vol. 15, pp 21–36, 2013..
[7] E.C. Le Ru, M. Dalley, P.G. Etchegoin: Plasmon resonances of silver colloids studied by surface
enhanced Raman spectroscopy, Current Applied Physics, vol. 6, pp. 411–414, 2006.
[8] Zheng Zeng, Yiyang Liu, Jianjun Wei: Recent advances in surface-enhanced Raman spectroscopy
(SERS): Finite-difference time-domain (FDTD) method for SERS and sensing applications, Trends in
Analytical Chemistry, vol. 75, pp. 162–173, 2016.
[9] S.A. Maier: Plasmonics: Fundamentals and Applications, first ed., Springer US, New York, 2007.
[10] Z. Yang, Q. Li, F. Ruan, Z. Li, B. Ren, H. Xu, et al., FDTD for plasmonics: Applications in enhanced
Raman spectroscopy, Chin. Sci. Bull. vol. 55, pp. 2635–2642, 2010.
[11] I.A. Birtoiu, M.I. Rusu, N.D. Becherescu-Barbu, B.A. Vitalaru, D. Togoe, M. Pascal, M.V. Udrea,
B. Chiricuta, C. Rizea, A. Parau, C.E.A. Grigorescu: Micro-Raman spectroscopy in the visible range:
A Tool for rapid investigation of mammary tumours, Romanian Reports in Physics, vol. 67, pp. 1525–
1536, Dec. 2015.
[12] R. Dos Santos Horta, G. Eunice Lavalle, R. M. De Castro Cunha, L. L. De Moura, R. B. De Araújo, G.
Dantas Cassali: Influence of surgical technique on overall survival, disease free interval and new lesion
development interval in dogs with mammary tumors, Advances in Breast Cancer Research, vol. 3,
pp. 38–46, 2014.
[13] F. T. Nguyen, A. M. Zysk, E. J. Chaney, J. G. Kotynek, U. J. Oliphant, F. J. Bellafiore, K. M. Rowland,
P. A. Johnson, and S. A. Boppart: Intraoperative evaluation of breast tumor margins with optical
coherence tomography, Cancer Res., vol. 69, pp. 8790-8796, 2009.
[14] J. Surmacki, J. Musial, R. Kordek, H. Abramczyk: Raman imaging at biological interfaces, Molecular
Cancer, http://www.molecular-cancer.com/content/12/1/48, 2013.
[15] K. Virkler, I.K. Lednev: Analysis of body fluids for forensic purposes: From laboratory testing to non-
destructive rapid confirmatory identification at a crime scene, Forensic Sci. Int. vol. 188, pp. 1–17,
2009.
[16] G. McLaughlin, I. K. Lednev: A modified Raman multidimensional spectroscopic signature of blood to
account for the effect of laser power, Forensic Sci. Int., vol. 240, pp. 88-94, 2014.
[17] K. Virkler, I.K. Lednev: Raman spectroscopic signature of blood and its potential application to forensic
body fluid identification, Anal. Bioanal. Chem., vol. 396, pp. 525–534, 2010.
[18] S. Boyd, M.F. Bertino, D. Ye, L.S. White, S.J. Seashols: Highly sensitive detection of blood by surface
enhanced Raman scattering, J. Forensic Sci., vol.58, pp. 753– 756, 2013.