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Quantitative fiber-optic Raman spectroscopy for tissue Raman measurements

Article  in  Proceedings of SPIE - The International Society for Optical Engineering · February 2014
DOI: 10.1117/12.2039576

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 Quantitative fiber-optic Raman spectroscopy for tissue Raman
measurements
Shiyamala Duraipandian, Mads Bergholt, Wei Zheng, and Zhiwei Huang*
Optical Bioimaging Laboratory, Department of Biomedical Engineering, Faculty of Engineering,
National University of Singapore, Singapore 117576

ABSTRACT
Molecular profiling of tissue using near-infrared (NIR) Raman spectroscopy has shown great promise for in vivo
detection and prognostication of cancer. The Raman spectra measured from the tissue generally contain fundamental
information about the absolute biomolecular concentrations in tissue and its changes associated with disease
transformation. However, producing analogues tissue Raman spectra present a great technical challenge. In this
preliminary study, we propose a method to ensure the reproducible tissue Raman measurements and validated with the in
vivo Raman spectra (n=150) of inner lip acquired using different laser powers (i.e., 30 and 60 mW). A rapid Raman
spectroscopy system coupled with a ball-lens fiber-optic Raman probe was utilized for tissue Raman measurements. The
investigational results showed that the variations between the spectra measured with different laser powers are almost
negligible, facilitating the quantitative analysis of tissue Raman measurements in vivo.

Keywords: Quantitative, Raman spectroscopy, in vivo, fiber-optic

__________________________________________________________________________________
*Corresponding to: Dr Zhiwei Huang, E-mail: biehzw@nus.edu.sg, Tel: +65 6516 8856, Fax: +65 6872 3069

Biomedical Vibrational Spectroscopy VI: Advances in Research and Industry,

edited by Anita Mahadevan-Jansen, Wolfgang Petrich, Proc. of SPIE Vol. 8939,
89390V · © 2014 SPIE · CCC code: 1605-7422/14/$18 · doi: 10.1117/12.2039576

Proc. of SPIE Vol. 8939 89390V-1

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 1. INTRODUCTION

Optical spectroscopy methods that can probe changes in tissue morphology, intrinsic fluorophores and biomolecules are
promising candidates suited for biomedical diagnosis. Among various optical spectroscopic methods, fiber-optic Raman
spectroscopy is a unique analytical technique, and has recently opened a new pathway for fighting against cancer based
on early detection by providing quantitative and quality information of tissue composition [1-3]. The advancements in
modern Raman instrumentation, recent breakthroughs in the development of miniaturized fiber-optic Raman probe
designs, and robust multivariate data processing methods have opened a new frontier of real-time Raman spectroscopy
for early cancer screening in vivo in a number of organs (e.g., bladder, nasopharynx and larynx, cervix, lung, esophagus,
and stomach, etc) [2, 4-9]. The biomedical tissue samples are often inhomogeneous and the acquired tissue Raman
signals are extremely complex, containing absolute biomolecular concentration information in the tissue and its changes
associated with disease transformation. Thus the quantitative Raman spectroscopy has the promising potential to greatly
improve tissue diagnosis. However, producing analogues Raman spectra from inhomogeneous tissue present a great
technical challenge to the biomedical community that requires controlling and monitoring of the most common sources
of variances in fiber-optic quantitative analysis, including laser excitation power, fiber coupling efficiency and
measurement reproducibility, etc. [10]. Normalization to the peak intensity or area under the curve is the most
widespread method to detect the disease based on the spectral shape information, while disregarding the quantitative
(intensity) information [11]. Till date, very limited studies have been reported on quantitative tissue Raman spectroscopy
using fluorocarbon fibers, a fluorinated ethylene propylene copolymer cap on the excitation fiber of Raman probes, and
fiber-optic Raman probes with embedded diamond in the fiber-tip or distal lens [10, 12-14]. In this study, without
inserting additional reference materials or impurities into the fiber-optic Raman probe, we utilize sapphire Raman signals
generated from the distal ball-lens of confocal fiber-optic Raman probe as an internal reference for quantitative fiber-
optic tissue Raman measurements.

2. Materials and Methods

2.1 Raman Instrumentation

The confocal Raman spectroscopy system utilized for in vivo tissue Raman measurements from oral cavity has been
reported in detail elsewhere [4]. Briefly, the Raman spectroscopic system consists of an electronically synchronized
spectrum stabilized near-infrared (NIR) diode laser (λex = 785 nm, maximum output: 300 mW, B&W TEK Inc., Newark,
Delaware), a transmissive imaging spectrograph (Holospec f/1.8, Kaiser Optical Systems Inc., Ann Arbor, Michigan), a
liquid nitrogen-cooled, NIR-optimized, back-illuminated, deep depletion charge-coupled device (CCD) camera (1340 
400 pixels at 20  20 µm per pixel; Spec-10: 400BR/LN, Princeton Instruments, Roper Scientific Inc., Trenton, New
Jersey) and a specially designed fiber-optic confocal Raman probe coupled with a ball-lens (1 mm in diameter, refractive
index n = 1.76) at the tip. The coupling of an NIR-coated sapphire ball-lens with the bifurcated fiber-optic Raman probe
offers a high degree of confocality for depth-selective tissue excitation as well as back-scattered tissue Raman photons
collection from the epithelial layer (~ 300 µm) [15]. These major advancements facilitate the acquisition of high-quality
tissue Raman spectra in the fingerprint region (800-1800 cm-1) within 1 sec with high signal-to-noise ratio (SNR) and a
spectral resolution of 9 cm-1, paving the way for moving the cutting-edge rapid Raman spectroscopy technique from
laboratory bench-top to bedside. The atomic emission lines of mercury-argon spectral calibration lamps (HG-1 and AR-1,
Ocean Optics Inc., Dunedin, FL) are used for wavelength calibration. All the wavelength-calibrated tissue Raman spectra
are also corrected for the wavelength-dependence of the system using a tungsten-halogen calibration lamp (RS-10,
EG&G Gamma Scientific, San Diego, CA).

2.2 Patients
A total of 15 normal healthy volunteers between 18 to 35 years of age were recruited for in vivo tissue Raman
measurements in the oral cavity. Prior to research investigation, all patients signed an informed consent form permitting
the collection of in vivo spectroscopic measurements from the oral cavity. Before taking the spectroscopic measurement,
all volunteers underwent extensive mouthwash to reduce confounding factors (e.g., food debris, microbial coatings etc.).
As a result, a total of 150 in vivo tissue Raman spectra were measured from the inner lip of volunteers, in which 80
spectra were acquired using laser power of 60 mW and 70 spectra were measured using the laser power of 30 mW.

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 Multiple tissue Raman spectra (n~5) were obtained from each subject for each laser power (of 30 and 60 mW on the
probe tip) to include inter- and/or intra-tissue variability for data analysis.

2.3 Data processing

The in vivo Raman spectra measured from inner lip in the 800-1800 cm-1 range represented a combination of prominent
tissue autofluorescence (AF) background, weak tissue Raman scattered signals, and noise. The measured tissue spectra
were preprocessed with adjacent five-points smoothing using a Savitzky-Golay filter to reduce the noise [16]. A fifth-
order polynomial was found to be optimal for fitting the broad tissue AF background in the noise smoothed spectrum
[17]. The polynomial was then subtracted from the measured spectrum to yield the pure tissue Raman spectrum alone.
The data preprocessing including setting of exposure time, CCD readout, hardware binning, subsequent integration time
adjustment to prevent signal saturation, probe background subtraction, outlier detection, wavelength and intensity
calibration, and real-time display of in vivo tissue spectra were performed using in-house developed graphical user
interface (GUI) under Matlab environment (Mathworks Inc., Natick, MA, USA). This framework has been integrated as
a software prototype and optimized for rapid spectral processing time of ~ 100 msec for continuous spectral acquisition
with auditory probabilistic diagnostic feedback [18].

3. Results

Figure 1 shows the background spectrum of a ball-lens fiber-optic Raman probe under the 785 nm laser excitation with a
laser power of ~30 mW. The Raman peaks of sapphire (Al2O3) distal ball-lens can be found at 417 and 646 cm-1 (phonon
mode with A1g symmetry), and 380 and 751 cm-1 (Eg phonon mode). The AF background and the Raman peaks arising
from fused silica fibers likely due to the metal ion impurities in the silica fibers were also noticed around 490 and
606 cm−1 (Figure 1) [10].

20000 -

500 1000 1500 2000

Raman shift (cm -1)

Fig.1. Background Raman spectrum of a ball-lens fiber-optic Raman probe when excited by a 785 nm laser
with an excitation power of ~30 mW.

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 50000 -
60 mW (n=80)
30mW (n=70)
40000 -

30000 -

20000 -

10000 -i

0 i i i

0 500 1000 1500 2000

Raman shift (cm -1)

Fig.2. The mean in vivo raw tissue Raman spectra ±1 standard deviation (SD) of the inner lip using 785-nm laser excitation power
levels of 30 and 60 mW.

0.008 -
60 mW (n=80) T-
LCD
0.007 - - - - 30mW (n=70) l't
0.006 -
.-.

xi 0.005 -
(D
0.004 -
>,
..
C 0.003 -
a) d'
--. O
0.002 - O
."
0.001 - I
V
0.000 j i i i

800 1000 1200 1400 1600 1800

Raman shift (cm-1)

Fig.3. The intensity-calibrated and autofluorescence (AF) -subtracted mean in vivo tissue Raman spectra ±1 SD of inner lip using
785-nm laser excitation powers of 30 and 60 mW.

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 We successively acquired in vivo Raman spectra from the oral cavity of 15 subjects with the laser excitation power of 30
and 60 mW and the mean in vivo raw Raman spectra ± 1 standard deviation (SD) is shown in Figure 2. The measured
tissue signals also contain the prominent fused silica and sapphire Raman peaks (380, 417, 490, 606, 646, and 751 cm-1)
from the fiber-optic Raman probe. The Raman intensity of the sample is linearly proportional to the laser excitation
power utilized and we have also reported in our previous study that the reference Raman signal generated from the distal
ball-lens of the self-referencing fiber-optic probe (i.e., intensity of the prominent sapphire Raman peak at 417 cm-1) is
linearly proportional to delivered laser power [10]. Hence, by scaling the measured tissue spectra with the peak Raman
intensity corresponds to the sapphire peak 417 cm-1 (after tissue AF background subtraction), we can approximate the
absolute tissue Raman spectral intensities independent of laser excitation power variations [10]. This is one of the
simplest methods to extract the variations in tissue Raman spectra due to laser excitation power changes and can be
easily implemented in the diagnostic framework for real-time in vivo clinical diagnosis.
Figure 3 shows the intensity-calibrated, laser power corrected, background-subtracted mean in vivo Raman spectra of the
inner lip ±1 SD, showing Raman peaks at around 853 cm-1 (v(C-C)), 1004 cm-1 (νs(C-C)), 1302 cm-1 (CH3CH2 twisting
and wagging), 1451cm-1 (δ(CH2) deformation), and 1655 cm-1 (amide I v(C=O) of proteins). From Figure 3, it is
apparent that the variation in the absolute tissue Raman spectral intensities due to the changes in laser excitation power
can be corrected using the magnitude of sapphire ball-lens self-reference signals, actualizing the quantitative tissue fiber-
optic Raman spectroscopy that can significantly improve the clinical diagnosis.

4. CONCLUSION
This study demonstrates that the internal reference Raman signals arising from the ball-lens of fiber-optic Raman probes
can be utilized for realizing quantitative analysis of tissue Raman measurements in vivo.

ACKNOWLEDGEMENTS
This work was supported by the National Medical Research Council and the Biomedical Research Council, Singapore.

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