Raman Spectroscopy for Tumor Diagnosis in Mammary Tissue

S. Pimenta

, M. J. Maciel

, A. Miranda

, M. F. Cerqueira

, P. Alpuim

and J. H. Correia

CMEMS-UMinho, Department of Industrial Electronics, University of Minho, Guimarães, Portugal

ICVS, School of Medicine, University of Minho, ICVS/3B’s - PT Government Associate Laboratory,

Braga/Guimarães, Portugal

International Iberian Nanotechnology Laboratory (INL), Braga, Portugal

{fatima.cerqueira, pedro.alpuim.us}@inl.int

Keywords: Raman Spectroscopy, ex-vivo Analysis, Normal vs Tumor Mammary Glands.

Abstract: This paper demonstrates the potential of Raman spectroscopy in the ex-vivo analysis of resected normal and

tumor mammary mouse glands, using a commercial confocal Raman microscope. The Raman spectra were

acquired with a 785 nm excitation laser at 40 mW and using an integration time of 2 s. The Raman spectra

for normal and tumor mouse mammary glands are presented and compared with literature results. The Raman

spectrum of normal mammary mouse gland is dominated by lipid signatures. On the other hand, the Raman

spectrum of tumor mammary gland is dominated by DNA and protein signatures. The molecular information

obtained by using Raman spectroscopy can be fundamental for a more precise and complete diagnosis of

tumors, for intraoperative assessment of tumors margins during surgeries and for tumors grade classification.

1 INTRODUCTION

Globally, cancer is a leading cause of death, being the

breast cancer one of the most common causes of

cancer death, accounting 627 000 cases in 2018

(WHO, 2018). Cancer early diagnosis decreases

healthcare costs and increases the probability to save

the patient life (WHO, 2017).

Raman spectroscopy is a non-invasive and

powerful tool for molecular analysis of disease. This

optical technique is based on inelastic scattering of

light by molecules. The shift in energy of the

scattered light is specific to the vibrational modes of

the molecules from a tissue. Thus, the molecular

information obtained by using Raman scattering can

be used to discriminate diseased and healthy tissues

(Stevens, Petterson, Day, & Stone, 2016).

The key idea behind Raman spectroscopy is that a

disease causes changes in the molecular composition

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of a tissue and these changes can be reflected in the

tissue Raman spectrum (Choo-Smith et al., 2002).

The changes are usually related with variations in the

relative concentrations of lipids, proteins, nucleic

acids, etc. (Stevens et al., 2016). The Raman spectrum

is a molecular fingerprint of the tissue (Kong,

Kendall, Stone, & Notingher, 2015).

Majumder et al. (Majumder, Keller, Boulos,

Kelley, & Mahadevan-Jansen, 2008) performed a

study comparing the capabilities of fluorescence,

diffuse reflectance and Raman spectroscopy for

discrimination of different human breast tissues.

Raman spectroscopy showed a superior performance.

The Raman spectrum is a set of narrow bands derived

from molecular vibrations of a large number of

biochemicals presented on the tissues. Thus, with

Raman is possible to acquire molecular information

with superior detail, comparing with fluorescence or

Pimenta, S., Maciel, M., Miranda, A., Cerqueira, M., Alpuim, P. and Correia, J.

Raman Spectroscopy for Tumor Diagnosis in Mammary Tissue.

DOI: 10.5220/0009093501310134

In Proceedings of the 8th International Conference on Photonics, Optics and Laser Technology (PHOTOPTICS 2020), pages 131-134

ISBN: 978-989-758-401-5; ISSN: 2184-4364

131

diffuse reflectance (Patil, Bosschaart, Keller,

Leeuwen, & Mahadevan-Jansen, 2008).

Raman spectroscopy is a non-destructive and

chemical-specific technique, being ideal for the

detection of microcalcifications in breast tissue

during biopsies, for example. Raman spectroscopy

can be executed with fiberoptic probes compatible

with biopsy needles (Barman et al., 2013).

Frank et al. (Frank, Redd, Gansler, & McCreery,

1994) performed one of the first studies about breast

cancer diagnosis using Raman spectroscopy. The

Raman spectra of normal and cancerous human

biopsy samples demonstrated Raman differential

features of lipids and carotenoids. In a second work

(Frankt & McCreey, 1995), the authors also studied

normal and diseased human breast tissues and

reported a weaker lipid band and a more pronounced

collagen band in diseased samples. Haka et al. (Haka

et al., 2005) reported the use of Raman spectroscopy

to diagnose benign and malignant lesions in ex-vivo

samples from human breast tissue. They developed a

diagnostic algorithm for distinguish cancerous tissues

from normal and benign tissues, with 94% sensitivity

and 96% specificity. In a second study (Haka et al.,

2006), the authors reported the in-vivo collection of

Raman spectra during partial mastectomy, suggesting

the potential of Raman spectroscopy for

intraoperative margin assessment (Cui, Zhang, &

Yue, 2018).

For breast tissues analysis is also reported the use

of an excitation laser of 785 nm and a maximum

power of 300 mW (Horsnell et al., 2010; Stevens et

al., 2016).

Along this paper, the potential of Raman

spectroscopy is shown with the ex-vivo analysis of

resected normal and tumor mammary mouse glands,

using a commercial equipment. Final considerations

were also discussed related to the potential of Raman

spectroscopy in routine clinical practice, associated

with intraoperative assessment of tumors margins

during surgeries and also with tumors grade

classification.

2 RAMAN SPECTROSCOPY OF

BIOLOGICAL TISSUE

2.1 Optical Considerations for Raman

Spectroscopy

The Raman system used in this study was the WiTec

Alpha300 R confocal Raman microscope, from Witec

Ulm Germany. This system combines a highly

sensitive confocal microscope with an

ultrahigh-throughput spectroscopy system. A high

spatial and spectral resolution are achieved by

combining several optical components such as filters,

lenses, objectives and a sensitive detector.

“Confocal” means “having the same focus” and

defines an optical microscope in which the sample is

illuminated with a point source. The “image” of this

point is detected using a pinhole in the optical fiber

that drives the signal to the spectrometer and then to

the detector. Thus, only the light originated from the

focal plane is detected and contribute to the “image”

(Giridhar, Manepalli, & Apparao, 2017).

The WiTec Alpha300 R confocal Raman

microscope has a lateral resolution of 200-300 nm, a

depth resolution of 500 nm, a spectral resolution of

approximately 1 cm

-1

and a spectrometer with a

variety of focal lengths.

The system allows different types of

measurements, but it was used for single spectrum

acquisition with a 785 nm excitation laser. Figure 1

represents a schematic of the WiTec Alpha300 R

confocal Raman microscope system.

Figure 1: Schematic of the WiTec Alpha300 R confocal

Raman microscope, from Witec Ulm Germany.

2.2 Raman Spectroscopy with WiTec

Alpha300 R

Raman spectra with WiTec Alpha300 R system were

collected from resected mammary mouse glands. The

main objective is to analyze the power of Raman

scattering to differentiate a normal mammary mouse

gland from a tumor mammary mouse gland. The

frozen mammary mouse glands (normal and tumor)

were sliced and put on a glass slide for the Raman

analysis.

The spectra were acquired with the Witec control

software, using a 785 nm excitation laser at 40 mW,

2 s of integration time and 100 accumulations. After

acquisition, the spectra were baseline corrected

PHOTOPTICS 2020 - 8th International Conference on Photonics, Optics and Laser Technology

132

considering a 4

order polynomial fitting and filtered

with a filter size of 3

order and a dynamic factor of 1.

The baseline correction and filtering were also

performed at the Witec control software.

Figure 2 shows the Raman spectra from literature

of an ex-vivo human breast sample, acquired with a

785 nm diode laser, a long-pass filter to reduce

Rayleigh scattering, a CCD camera and other optical

components (Patil, Bosschaart, Keller, Leeuwen, &

Mahadevan-Jansen, 2008).

Figure 3 shows the acquired Raman spectra, using

the WiTec Alpha300 R confocal microscope, for

normal and tumor mammary mouse glands. The

Raman spectra from the normal and tumor mammary

mouse glands are clearly different.

The obtained experimental results (Figure 3) from

normal and tumor mammary mouse glands, as

expected, presents the signature of lipids and proteins,

in the range of 800 to 1800 cm

-1

, and are very similar

to the literature spectra (Figure 2).

Moreover, the experimental spectra (Figure 3)

reveal clear differences between the normal and

tumor tissue. From Figure 3, it is seen that the normal

spectrum is dominated by lipid signatures, namely

peaks at approximately 1070, 1300 and 1440 cm

-1

. On

the other hand, the tumor spectrum is dominated by

protein (peaks at approximately 1450 and 1660 cm

-1

)

and by DNA (peak at 1336 cm

-1

) signatures, usually

indicative of malignancy (Movasaghi, Rehman, &

Rehman, 2007; Patil et al., 2008; Stone, Kendall,

Smith, Crowa, & Barra, 2004).

Additionally, it is also seen on Figure 3: i) a

strongly decrease in the intensity together with

broadening of the modes in the tumor gland; ii) a

strongly decrease in intensity of the peaks at

approximately 1100 cm

-1

and the

doublet at 1300 cm

-1

for the tumor tissue; and iii) the

peak at 1750 cm

-1

is absent in the tumor gland. All

these effects can be indicative of malignancy.

Finally, comparing Figure 2 and Figure 3 for

normal tissues, it can be seen that several ratios

between peaks are very similar, for example: between

1440 and 1300 cm

-1

(ratio of approximately 1.8); and

between 1440 and 1650 cm

-1

(ratio of approximately

2). In the case of the tumor tissues, the ratios between

the peaks are not very similar, which can be related

with the degree of malignancy of each sample (for

Figure 2 and Figure 3). Despite this differences, the

most important feature is the clearly difference

between the Raman spectra of the normal and tumor

tissues, for both figures.

Figure 2: Raman spectra of an ex-vivo human breast sample,

considering a 785 nm excitation diode laser (Patil et al.,

2008).

800 1000 1200 1400 1600 1800

100

200

300

400

Intensity (a.u.)

Raman shift (cm

-1

)

Normal mammary gland

Tumor mammary gland

Figure 3: Raman spectra of resected normal mammary

gland and tumor mammary gland of a mouse. The spectra

were acquired with the Witec Alpha300 R system, with an

integration time of 2 s and 100 accumulations.

3 CONCLUSIONS AND FUTURE

GUIDELINES

The ex-vivo analysis of biological tissues using a

commercial Raman equipment was presented. The

results proved the potential of Raman spectroscopy in

differentiate the normal and tumor tissues, using a

785 nm laser with 40 mW total power, an integration

time of 2 s and 100 accumulations.

In Raman spectroscopy is crucial to take into

account the maximum permissible light exposure and

avoid the tissue temperature increase, relevant to

minimize tissue damage. This parameter is even more

important during in-vivo spectra acquisition, to ensure

patient comfort.

These preliminary tests with ex-vivo analysis of

resected normal and tumor mammary mouse glands,

prove the potential of the used Raman microscope to

differentiate normal and tumor tissues: the normal

mammary gland spectrum is dominated by lipid

signatures and the tumor mammary gland spectrum is

dominated by DNA and protein signatures. These

promising results open the future possibility to use

Raman Spectroscopy for Tumor Diagnosis in Mammary Tissue

133

this commercial equipment to other type of analysis,

for example, the analysis and differentiation of

normal and malignant human tissues, for a variety of

applications.

A great area of expansion of the Raman

spectroscopy technique is the use of the high Raman

molecular specificity for intraoperative assessment of

tumors margins during surgeries, in order to reduce

the re-operation procedures. If the patient

re-operation is avoided, the healthcare costs and

patient anxiety are also reduced. For this application,

it is required the implementation of a portable Raman

system to be use in the operation rooms.

Finally, the application of Raman spectroscopy in

routine clinical practice can also have a huge impact

in grade classification, essential for diagnosis and

prognosis of diseases, i. e., to predict how quickly a

tumor will grow and spread. This knowledge is

crucial for planning the best treatment for the patient,

increasing the probability of a success treatment and

cure. Again, for this last described application, it is

desirable the implementation of a portable Raman

system or the adaptation of a Raman probe to the

existing methods of diseases diagnosis in routine

clinical practice.

ACKNOWLEDGEMENTS

This work is supported by FCT with the project

reference PTDC/CTM-REF/28406/2017, operation

code POCI-01-0145-FEDER-028406, through the

COMPETE 2020

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