Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 151 (2015) 208–212
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa
Dielectric and FT-Raman spectroscopic approach to molecular identification of breast tumor tissues Rasha Abd El-Hakam a, Safaa Khalil a, Ragab Mahani b,⇑ a b
Spectroscopy Dep., National Research Centre, 33 EL Bohouth st. (former EL Tahrir st.), Dokki, P.O. 12622, Giza, Egypt Microwave Physics and Dielectrics Dep., National Research Centre, 33 EL Bohouth st. (former EL Tahrir st.), Dokki, P.O. 12622, Giza, Egypt
g r a p h i c a l a b s t r a c t The frequency dependence of dielectric loss tangent (tand) of N, F, ILC and IDC breast tissues of grade 2, measured at room temperature.
a r t i c l e
i n f o
Article history: Received 13 December 2014 Accepted 17 June 2015 Available online 22 June 2015 Keywords: Breast cancer FT-Raman Dielectric spectroscopy Permittivity
a b s t r a c t FT-Raman spectra and dielectric properties of benign and malignant women breast tissues in vitro were investigated. FT-Raman spectra for the malignant tissues showed a remarkably decrease in the lipid/protein ratio. Dielectric properties of women breast tissues measured in the low frequency range (42–106 Hz) were interpreted in spite of electrode polarization effect. Experimental results showed a contrast between the dielectric properties of malignant (Grade II) and benign tissues within the frequency range studied. The permittivity of malignant to normal breast tissue was found to be 160:1 while it could be 1.3:1 for fibrocystic breast tissues. These findings could contribute to distinguish between two breast tissues. The differences in spectral features between benign and malignant tissues may lead to breast cancer detection. Ó 2015 Elsevier B.V. All rights reserved.
1. Introduction Breast cancer is the most common malignant tumor among women in the western world [1]. Malignant tumor is cancerous while benign tumor is not usually harmful. The development of ⇑ Corresponding author at: El Buhouth St., Dokki, Cairo, Postal Code: 12311, Egypt. E-mail address: [email protected] (R. Mahani). http://dx.doi.org/10.1016/j.saa.2015.06.055 1386-1425/Ó 2015 Elsevier B.V. All rights reserved.
reliable and affordable diagnosis techniques may give chance of early cancer detection and hence increase the ability of treatment. Recently, many developments have been devoted to allow a high, sensitive and specific diagnosis to act within seconds, allowing guided biopsies and therapies during a single procedure [2–4]. For instance, FTIR spectroscopy is a non invasive analytical technique which provides information about the molecular composition and structure of the examined sample [5]. Many studies carried out on natural hard and soft tissues using FTIR
R. Abd El-Hakam et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 151 (2015) 208–212
spectroscopy to contribute in enhancing the early detection rate of cancer [6–8]. In our previous work on breast cancer tissues [9], FTIR – spectral features showed that malignant tissues have higher intensities comparing to the normal ones. Instead, electrical properties of the biological tissues have awakened some interest over a century because they determine the pathways of current flow though the body. Many studies have been shown that the malignant human breast cancer epithelial cells and benign breast epithelial cells have different electrical properties [10–12]. These properties are very important since their frequency dependence permits identification and investigation of number of completely different underlying mechanisms. Benign and cancerous cells are different in many aspects including proliferation, metabolism, cytoskeleton, and other functional categories [13,14]. Some of these differences can lead to distinctions in these cells’ electrical properties [15,16]. More evidence has been found that malignant breast tumors have significantly electrical properties higher than normal tissues [16–19]. However, studies of dielectric properties of biological tissues at low frequencies have been limited due to electrode polarization effect which masks the possible relaxation process. For this reason, our work proposed to find a discrimination between the different tissues by investigating their dielectric properties at the low frequency range (42–106 Hz. Also, to extend the area of diagnosis on the breast cancer. To achieve that, the measurements are carried out in vitro on the malignant (Grade II) and benign breast tissues, at room temperature.
2. Materials and methods For Fourier Transform Infrared (FTIR) and atomic absorption (AA) spectroscopy studies [9], surgically excised human breast cancer specimens from fifty female patients aged from 26–74 years old, were analyzed (in parallel with normal tissues obtained from the specimens) and diagnosed 50 normal (N), 9 fibrocystic changes (F), 11 invasive lobular carcinoma (ILC) and 30 invasive duct carcinoma (IDC). All specimens were selected from Pathology Department, Kasr El Aini Hospital, Cairo University. Then, they were marked with India ink to indicate the region samples, fixed in formalin, routinely processed, paraffin-embedded and sectioned through the marked locations at 5 lm thickness and stained with hematoxylin and eosin. For the present study, formalin-free tissues samples were cut into thin sections 2–3 mm in thickness and 4 mm in diameters to be ready for the dielectric measurements. The measurements were carried out by placing the tissue specimen between two metallic parallel electrodes connected to the computerized LRC meter (Hioki model 3531 Z Hi Tester) which monitors the dielectric parameters such as resistance, capacitance, conductance, impedance, admittance and dielectric loss tangent in the frequency range (42–106 Hz). The electrical properties of each kind of tissue were measured at three different regions under the same conditions to ensure the accuracy and satiability of the measurements. Then the dielectric properties such as the permittivity (e0 ) and dielectric loss (e00 ) were calculated from the measured values of capacitance (C) and dielectric loss tangent (tand) as follows:
e0 ¼
Cd
eo A
e00 ¼ e0 tan d
209
The real part of ac conductivity (rac) was measured in terms of dielectric loss (e00 ) as follows:
rac ¼ eO xe00
ð3Þ
where, x was the angular frequency and is 2pt. The cell used for the dielectric measurements was calibrated using the non-polar standard materials with different thicknesses ranging from 1 mm up to 7 mm at 104 Hz. The permittivity error and the standard deviation were found to be ±1 and %0.04, respectively. 3. Results and discussion 3.1. FT-Raman spectroscopy qualitative analysis For FTIR spectroscopy measurements, Careful investigation of the spectra revealed that there is no observable shift in the position of peaks between benign; normal (N), fibrocystic (F), and the different malignant breast tissues; invasive lobular carcinoma (ILC) and invasive duct carcinoma (IDC) [9]. In contrast to FTIR spectra, conventional Raman spectra revealed all the scattering peaks that have a diagnostic marker without using mathematical treatment. The main frequencies of the FT-Raman scattering peaks of normal breast tissue together with their intensities and assignments are tabulated in Table 1. The stretching vibration of C@O stemming from ester linkage of the fatty acid tail and triglyceride polar head group of lipids is found at 1753 cm1 with medium intensities in N, F, ILC and IDC groups. The random turn secondary structure of proteins can be demonstrated by absorption peak at 1682 cm1. It is relatively medium in N, weak in F, while it appears strong in ILC and IDC samples. The stretching vibration of C@O of amide I and NAH in-phase bending are represented by an overlap absorption band at 1651 cm1 which is characterized by a-helix secondary structure of proteins. It is relatively medium in N, weak in F, while it appears strong in ILC and IDC samples. The peak at 1239 cm1 assigned to asymmetric phosphodiester stretching vibrational mode of nucleic acids tas(PO 2 ). This peak appears weak in N and F while, it appears medium in ILC and IDC groups. There are two peaks at 1280 and 1207 cm1 which can be attributed to amide III/CH2 wagging vibrations of collagen [20–24], who found some specific absorption peaks in the range 900–3600 cm1, that may help differentiating between healthy and diseased breast tissue and in which stage of cancer progression. 3.2. Dielectric properties Testing of the accuracy and stability of the dielectric measurements is first recommended. To achieve that, the measurements are carried out on three regions of each tissue under the same conditions. Then, the permittivity (e0 ) and dielectric loss (e00 ) of all tissues are plotted as a function of frequency as shown on the left hand side of Fig. 1. In all the cases, one can observe that properties
Table 1 The main frequencies of the FT-Raman scattering peaks of normal breast tissue together with their intensities and assignments. Raman shift (cm1)
Intensity (a.u)
Assignment
ð1Þ
1753
Medium
ð2Þ
1682 1651
Weak Medium
1280, 1207 1239
Medium Weak
C@O stretching vibration stemming from ester linkage of the fatty acid tail and triglyceride polar head group of lipids Random turn secondary structure of proteins C@O stretching mode of carbonyl group of ahelical secondary structure of amide I of protein Amide III/CH2 wagging vibrations of collagen Asymmetric phosphodiester stretching mode of vibration, mainly stemming from nucleic acids
where d was the sample thickness in meter (m), A was the cross section area of the electrode used in m2 and eo was the permittivity of free space (8.854 1012 F m1).
210
R. Abd El-Hakam et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 151 (2015) 208–212
Fig. 1. The dielectric dispersion exhibited by normal, fibrocystic, lobular and duct tissues at 25 °C illustrated in terms of the change in dielectric permittivity (e0 ) and dielectric loss (e00 ). The right graphs show in details e0 (a1) and e00 (b1) as a function of frequency in the range between 6400 Hz and 106 Hz.
of the three regions of each tissue are closed to each other. Further, they decrease in the same manner as the frequency increases. This indicates good accuracy and stability of the measurements in accordance. After that, the average values of these properties are calculated and then studied in some details (see the right side hand graphs). From this one can observe that both properties decrease with increasing frequency. For the normal (N) and fibrocystic tissues (F), both propertied decrease at first with a slow rate and then followed by a significantly decrease as the frequency increases. As a result, one would be concluded that the benign tissues have almost similar dielectric behavior. In contrast, invasive lobular carcinoma (ILC) dielectric properties significantly decrease as the frequency increases up to 1.5 105 Hz and then kept constant at higher frequencies. Properties of the invasive duct carcinoma (IDC) exhibits a significant decrease over the whole frequency range studied. The decrease in e0 with frequency may be attributed to the electrical relaxation process, but at the same time the material electrode polarization [25–27] cannot be ignored as the tissues of our investigations are ionic conductors. The material electrode polarization gives rise to the significant values of the permittivity and a strong drop of conductivity (see Fig. 3) towards low frequencies, arises when the ions arrive at the metallic electrodes and accumulate in thin layers immediately beneath the sample surface forming the so-called space-charge region. The formed space charge region may mask the relaxation processes which is manifested by the disappearance of peaks in e00 spectra. Electrode polarization effect occurs in solid-state electrolytes as well as in
aqueous solutions, ionic liquids, and in many biological systems. Despite of the tissues electrode polarization effect, discrimination between the malignant and benign tissues in the frequency window can be easily observed. Since cells consist of layers of materials with largely different dielectric properties, the potential barriers can be formed at their interfaces thus current flow through the pathways is restricted. Subsequently, the charge carriers can be accumulated at these interfaces, providing an additional polarization which is manifested by the high values of e00 . This effect is known as interfacial polarization or Maxwell–Wagner–Sillars (MWS) polarization [25–27]. Further, charge carriers polarization; electrode polarization or/and MWS polarization do not occur instantaneously, and the associated time constant is called the relaxation time s. Relaxation of the electrons and small dipolar molecules is a relatively fast process, with relaxation times in the Pico- and nanosecond range whereas charge carriers polarization may give relaxation times of the order of seconds. Thus, the charge carriers will have enough time to accumulate at the interfaces causing increasing of the tissue permittivity. From Fig. 1, the significant increase in e0 and e00 values of the malignant tissues compared to that observed for the benign tissues is attributed to changes of the tissue structure and architecture [28–32]. The normal breast tissue is created mostly by fat whereas tumor resembles rather a scar or connective tissue. Among different types of soft tissue or malignant tissues, fat has the lowest permittivity and conductivity [33,34]. Therefore, the permittivity differences between the malignant and normal tissues
R. Abd El-Hakam et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 151 (2015) 208–212
are attributed to the differences in fat amount. Further, transformation of the normal tissues to the malignant tissues is always accompanied by changes in the cellular membrane composition and membrane permeability. This results in movement of the potassium, magnesium and calcium out of the cell and the accumulation of sodium and water into the cell [35]. As a consequence, the membrane potential in a cancer cell becomes consistently weaker than the membrane potential of a healthy cell. Hence, the electrical field across the membrane of the cancer cell is reduced. Fig. 2 shows variation of the dielectric loss tangent (tand = e00 /e0 ) with frequency of all tissues studied at room temperature. The loss spectra are characterized by a peak appearing at a characteristic frequency, suggest the presence of relaxation processes in all tissues. The first two relaxation processes positioned at 25 kHz and 72 kHz, are corresponding to the normal (N) and fibrocystic tissues (F), respectively. While the third relaxation process positioned at 110 kHz is corresponding to the invasive lobular carcinoma (ILC). The last one where its maximum is beyond the frequency window could be corresponding to the invasive duct carcinoma (IDC). Strength and frequency of the relaxation peaks depend on the characteristic property of the dipolar relaxation. In the present study, the dipolar relaxation is associated to the capacitive charging of the cellular membrane [36]. Its electrical property strongly depends on amount of the energy absorbed by tissue. The dense or malignant tissues absorb the most amount of electromagnetic radiation whereas the soft or normal tissues absorb the least. As a consequence, the dielectric spectra corresponding to the malignant tissues are characterized by high strength. The peak broadness may indicate that dielectric data is not only due to one relaxation process but it is due to superposition of different processes: a conductivity contribution charge carriers polarization as well as the capacitive charging of the cellular membrane. Furthermore, the relaxation time (s) may play an important role for discrimination between the malignant and benign tissues. Its values are calculated from the relation: s = 1/2pfm, where fm is the peak frequency indicated for each relaxation process. Hence, the relaxation time corresponding to N, F and ILC are 6.5 106 s, 2.3 106 s and 1.5 106 s, respectively. The relaxation time is noticed considerably longer for benign than malignant tissues. This indicates that the rotational motion of each dipole presents a characteristic relaxation time; those exhibiting
Fig. 2. The frequency dependence of dielectric loss tangent (tand) of N, F, ILC and IDC breast tissues of grade 2, measured at room temperature.
211
rotations more effectively hindered by their environment will show higher s. From these in vitro permittivity measurement results, a common conclusion can be drawn that there are significant differences in the dielectric properties between the normal and malignant women breast tissues. 3.2.1. The Frequency dependence of ac conductivity The ac conductivity (rac) of breast tissues is plotted as a function of frequency as given in Fig. 3. This figure shows that rac significantly increases at low frequencies that followed by a plateau at high frequencies. The plateau becomes more pronounced for N, ILC and IDC tissues compared to that observed for F tissues. The observed plateau is corresponding to the contribution of dc conductivity (rdc) to the total conductivity measured. The origin of such conductivity comes from movements of the physiological ions. The dispersion detected in conductivity could be attributed to the electrode polarization effect. The conductivity value of the malignant tissues is in the range (3 103–102 S/cm) values relative to that observed for the normal ones (8.0 109– 3.4 108 S/cm). This means that conductivity of the malignant tissues is 104–106 fold of the normal ones. The observed increase in conductivity of the malignant tissues comparing with benign could be attributed to decrease of the lipids amount. Such result is in agreement with that reported elsewhere [33]. 3.2.2. The electrical properties ratios For discrimination between the normal and tumor breast tissues in terms of the dielectric properties, it is not important to know the exact values while the ratio between them is important. Table 2 shows ratios of the permittivity, loss tangent and ac conductivity (rac) of all tissues at 105 Hz. It indicates that the tumor to normal breast tissue permittivity can be 160:1 while it could
Fig. 3. The frequency dependence of the AC conductivity (rac) for each kind of tissues measured at three different regions.
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Table 2 The electrical properties of the malignant and normal breast tissues and their ratios selected at 105 Hz. Tissues
Normal Fibrocystic Lobular Duct
Loss Tangent
Conductivity [S/cm]
e0
Permittivity
e0ratio
tand
tandratio
r
rratio
3371 4376 75,089 535,368
1 1.3 22 160
7 15 30 74
1 2.1 4.3 10.5
2.5 108 1.8 105 2.8 104 6.7 103
1 720 11,200 268,000
[6] [7]
[8] [9]
[10]
be 1.3:1 for fibrocystic breast tissues. Such result seems in agreement with that reported in literature [37]. Further, apparently differences can be observed in the conductivity ratios between normal and tumor. For instance, the tumor to normal breast tissue conductivity can be 268,000:1 while it could be 720:1 for fibrocystic breast tissues. From above, a good relationship between the breast tissue and its dielectric properties is reported; this leads to possible discrimination between the malignant and benign tumors with higher accuracy.
[11] [12]
[13]
[14] [15]
4. Conclusion [16]
In summary, FT-Raman spectroscopy was used to investigate the differences of spectra between the normal and cancer breast tissues. Quantitative analysis of the conventional FT-Raman spectra for the malignant tissues (ILC & IDC) showed a remarkably decrease in the lipid/protein ratio. Dielectric spectroscopy was used to investigate the electrical properties differences between these tissues. Since the malignant tissue has a lower amount of lipid, its permittivity shows an increase. As a result, a clear difference was found in the permittivity behavior between the malignant and benign tissues in the frequency window despite of the electrode polarization effect. Further, the loss spectra corresponding to the malignant tissues were positioned at high frequencies and characterized by high strength. The peak broadness of these spectra may indicate that dielectric data is not only due to one relaxation process but it is due to superposition of different processes: a conductivity contribution, charge carriers polarization as well as the capacitive charging of the cellular membrane. From above, a good discrimination between the normal and breast cancer tissues was obtained using FT-Raman and dielectric measurements. This may extend the area of diagnosis on the biomedical treatment. Acknowledgments This work was supported by Scientific Research Ministry, Cairo, Egypt. The authors acknowledged Prof. Dr. Ahmed Ghoneim for revising the paper.
[17] [18] [19] [20] [21]
[22] [23] [24] [25] [26] [27] [28] [29]
[30] [31]
[32] [33]
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Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa
Dielectric and FT-Raman spectroscopic approach to molecular identification of breast tumor tissues Rasha Abd El-Hakam a, Safaa Khalil a, Ragab Mahani b,⇑ a b
Spectroscopy Dep., National Research Centre, 33 EL Bohouth st. (former EL Tahrir st.), Dokki, P.O. 12622, Giza, Egypt Microwave Physics and Dielectrics Dep., National Research Centre, 33 EL Bohouth st. (former EL Tahrir st.), Dokki, P.O. 12622, Giza, Egypt
g r a p h i c a l a b s t r a c t The frequency dependence of dielectric loss tangent (tand) of N, F, ILC and IDC breast tissues of grade 2, measured at room temperature.
a r t i c l e
i n f o
Article history: Received 13 December 2014 Accepted 17 June 2015 Available online 22 June 2015 Keywords: Breast cancer FT-Raman Dielectric spectroscopy Permittivity
a b s t r a c t FT-Raman spectra and dielectric properties of benign and malignant women breast tissues in vitro were investigated. FT-Raman spectra for the malignant tissues showed a remarkably decrease in the lipid/protein ratio. Dielectric properties of women breast tissues measured in the low frequency range (42–106 Hz) were interpreted in spite of electrode polarization effect. Experimental results showed a contrast between the dielectric properties of malignant (Grade II) and benign tissues within the frequency range studied. The permittivity of malignant to normal breast tissue was found to be 160:1 while it could be 1.3:1 for fibrocystic breast tissues. These findings could contribute to distinguish between two breast tissues. The differences in spectral features between benign and malignant tissues may lead to breast cancer detection. Ó 2015 Elsevier B.V. All rights reserved.
1. Introduction Breast cancer is the most common malignant tumor among women in the western world [1]. Malignant tumor is cancerous while benign tumor is not usually harmful. The development of ⇑ Corresponding author at: El Buhouth St., Dokki, Cairo, Postal Code: 12311, Egypt. E-mail address: [email protected] (R. Mahani). http://dx.doi.org/10.1016/j.saa.2015.06.055 1386-1425/Ó 2015 Elsevier B.V. All rights reserved.
reliable and affordable diagnosis techniques may give chance of early cancer detection and hence increase the ability of treatment. Recently, many developments have been devoted to allow a high, sensitive and specific diagnosis to act within seconds, allowing guided biopsies and therapies during a single procedure [2–4]. For instance, FTIR spectroscopy is a non invasive analytical technique which provides information about the molecular composition and structure of the examined sample [5]. Many studies carried out on natural hard and soft tissues using FTIR
R. Abd El-Hakam et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 151 (2015) 208–212
spectroscopy to contribute in enhancing the early detection rate of cancer [6–8]. In our previous work on breast cancer tissues [9], FTIR – spectral features showed that malignant tissues have higher intensities comparing to the normal ones. Instead, electrical properties of the biological tissues have awakened some interest over a century because they determine the pathways of current flow though the body. Many studies have been shown that the malignant human breast cancer epithelial cells and benign breast epithelial cells have different electrical properties [10–12]. These properties are very important since their frequency dependence permits identification and investigation of number of completely different underlying mechanisms. Benign and cancerous cells are different in many aspects including proliferation, metabolism, cytoskeleton, and other functional categories [13,14]. Some of these differences can lead to distinctions in these cells’ electrical properties [15,16]. More evidence has been found that malignant breast tumors have significantly electrical properties higher than normal tissues [16–19]. However, studies of dielectric properties of biological tissues at low frequencies have been limited due to electrode polarization effect which masks the possible relaxation process. For this reason, our work proposed to find a discrimination between the different tissues by investigating their dielectric properties at the low frequency range (42–106 Hz. Also, to extend the area of diagnosis on the breast cancer. To achieve that, the measurements are carried out in vitro on the malignant (Grade II) and benign breast tissues, at room temperature.
2. Materials and methods For Fourier Transform Infrared (FTIR) and atomic absorption (AA) spectroscopy studies [9], surgically excised human breast cancer specimens from fifty female patients aged from 26–74 years old, were analyzed (in parallel with normal tissues obtained from the specimens) and diagnosed 50 normal (N), 9 fibrocystic changes (F), 11 invasive lobular carcinoma (ILC) and 30 invasive duct carcinoma (IDC). All specimens were selected from Pathology Department, Kasr El Aini Hospital, Cairo University. Then, they were marked with India ink to indicate the region samples, fixed in formalin, routinely processed, paraffin-embedded and sectioned through the marked locations at 5 lm thickness and stained with hematoxylin and eosin. For the present study, formalin-free tissues samples were cut into thin sections 2–3 mm in thickness and 4 mm in diameters to be ready for the dielectric measurements. The measurements were carried out by placing the tissue specimen between two metallic parallel electrodes connected to the computerized LRC meter (Hioki model 3531 Z Hi Tester) which monitors the dielectric parameters such as resistance, capacitance, conductance, impedance, admittance and dielectric loss tangent in the frequency range (42–106 Hz). The electrical properties of each kind of tissue were measured at three different regions under the same conditions to ensure the accuracy and satiability of the measurements. Then the dielectric properties such as the permittivity (e0 ) and dielectric loss (e00 ) were calculated from the measured values of capacitance (C) and dielectric loss tangent (tand) as follows:
e0 ¼
Cd
eo A
e00 ¼ e0 tan d
209
The real part of ac conductivity (rac) was measured in terms of dielectric loss (e00 ) as follows:
rac ¼ eO xe00
ð3Þ
where, x was the angular frequency and is 2pt. The cell used for the dielectric measurements was calibrated using the non-polar standard materials with different thicknesses ranging from 1 mm up to 7 mm at 104 Hz. The permittivity error and the standard deviation were found to be ±1 and %0.04, respectively. 3. Results and discussion 3.1. FT-Raman spectroscopy qualitative analysis For FTIR spectroscopy measurements, Careful investigation of the spectra revealed that there is no observable shift in the position of peaks between benign; normal (N), fibrocystic (F), and the different malignant breast tissues; invasive lobular carcinoma (ILC) and invasive duct carcinoma (IDC) [9]. In contrast to FTIR spectra, conventional Raman spectra revealed all the scattering peaks that have a diagnostic marker without using mathematical treatment. The main frequencies of the FT-Raman scattering peaks of normal breast tissue together with their intensities and assignments are tabulated in Table 1. The stretching vibration of C@O stemming from ester linkage of the fatty acid tail and triglyceride polar head group of lipids is found at 1753 cm1 with medium intensities in N, F, ILC and IDC groups. The random turn secondary structure of proteins can be demonstrated by absorption peak at 1682 cm1. It is relatively medium in N, weak in F, while it appears strong in ILC and IDC samples. The stretching vibration of C@O of amide I and NAH in-phase bending are represented by an overlap absorption band at 1651 cm1 which is characterized by a-helix secondary structure of proteins. It is relatively medium in N, weak in F, while it appears strong in ILC and IDC samples. The peak at 1239 cm1 assigned to asymmetric phosphodiester stretching vibrational mode of nucleic acids tas(PO 2 ). This peak appears weak in N and F while, it appears medium in ILC and IDC groups. There are two peaks at 1280 and 1207 cm1 which can be attributed to amide III/CH2 wagging vibrations of collagen [20–24], who found some specific absorption peaks in the range 900–3600 cm1, that may help differentiating between healthy and diseased breast tissue and in which stage of cancer progression. 3.2. Dielectric properties Testing of the accuracy and stability of the dielectric measurements is first recommended. To achieve that, the measurements are carried out on three regions of each tissue under the same conditions. Then, the permittivity (e0 ) and dielectric loss (e00 ) of all tissues are plotted as a function of frequency as shown on the left hand side of Fig. 1. In all the cases, one can observe that properties
Table 1 The main frequencies of the FT-Raman scattering peaks of normal breast tissue together with their intensities and assignments. Raman shift (cm1)
Intensity (a.u)
Assignment
ð1Þ
1753
Medium
ð2Þ
1682 1651
Weak Medium
1280, 1207 1239
Medium Weak
C@O stretching vibration stemming from ester linkage of the fatty acid tail and triglyceride polar head group of lipids Random turn secondary structure of proteins C@O stretching mode of carbonyl group of ahelical secondary structure of amide I of protein Amide III/CH2 wagging vibrations of collagen Asymmetric phosphodiester stretching mode of vibration, mainly stemming from nucleic acids
where d was the sample thickness in meter (m), A was the cross section area of the electrode used in m2 and eo was the permittivity of free space (8.854 1012 F m1).
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Fig. 1. The dielectric dispersion exhibited by normal, fibrocystic, lobular and duct tissues at 25 °C illustrated in terms of the change in dielectric permittivity (e0 ) and dielectric loss (e00 ). The right graphs show in details e0 (a1) and e00 (b1) as a function of frequency in the range between 6400 Hz and 106 Hz.
of the three regions of each tissue are closed to each other. Further, they decrease in the same manner as the frequency increases. This indicates good accuracy and stability of the measurements in accordance. After that, the average values of these properties are calculated and then studied in some details (see the right side hand graphs). From this one can observe that both properties decrease with increasing frequency. For the normal (N) and fibrocystic tissues (F), both propertied decrease at first with a slow rate and then followed by a significantly decrease as the frequency increases. As a result, one would be concluded that the benign tissues have almost similar dielectric behavior. In contrast, invasive lobular carcinoma (ILC) dielectric properties significantly decrease as the frequency increases up to 1.5 105 Hz and then kept constant at higher frequencies. Properties of the invasive duct carcinoma (IDC) exhibits a significant decrease over the whole frequency range studied. The decrease in e0 with frequency may be attributed to the electrical relaxation process, but at the same time the material electrode polarization [25–27] cannot be ignored as the tissues of our investigations are ionic conductors. The material electrode polarization gives rise to the significant values of the permittivity and a strong drop of conductivity (see Fig. 3) towards low frequencies, arises when the ions arrive at the metallic electrodes and accumulate in thin layers immediately beneath the sample surface forming the so-called space-charge region. The formed space charge region may mask the relaxation processes which is manifested by the disappearance of peaks in e00 spectra. Electrode polarization effect occurs in solid-state electrolytes as well as in
aqueous solutions, ionic liquids, and in many biological systems. Despite of the tissues electrode polarization effect, discrimination between the malignant and benign tissues in the frequency window can be easily observed. Since cells consist of layers of materials with largely different dielectric properties, the potential barriers can be formed at their interfaces thus current flow through the pathways is restricted. Subsequently, the charge carriers can be accumulated at these interfaces, providing an additional polarization which is manifested by the high values of e00 . This effect is known as interfacial polarization or Maxwell–Wagner–Sillars (MWS) polarization [25–27]. Further, charge carriers polarization; electrode polarization or/and MWS polarization do not occur instantaneously, and the associated time constant is called the relaxation time s. Relaxation of the electrons and small dipolar molecules is a relatively fast process, with relaxation times in the Pico- and nanosecond range whereas charge carriers polarization may give relaxation times of the order of seconds. Thus, the charge carriers will have enough time to accumulate at the interfaces causing increasing of the tissue permittivity. From Fig. 1, the significant increase in e0 and e00 values of the malignant tissues compared to that observed for the benign tissues is attributed to changes of the tissue structure and architecture [28–32]. The normal breast tissue is created mostly by fat whereas tumor resembles rather a scar or connective tissue. Among different types of soft tissue or malignant tissues, fat has the lowest permittivity and conductivity [33,34]. Therefore, the permittivity differences between the malignant and normal tissues
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are attributed to the differences in fat amount. Further, transformation of the normal tissues to the malignant tissues is always accompanied by changes in the cellular membrane composition and membrane permeability. This results in movement of the potassium, magnesium and calcium out of the cell and the accumulation of sodium and water into the cell [35]. As a consequence, the membrane potential in a cancer cell becomes consistently weaker than the membrane potential of a healthy cell. Hence, the electrical field across the membrane of the cancer cell is reduced. Fig. 2 shows variation of the dielectric loss tangent (tand = e00 /e0 ) with frequency of all tissues studied at room temperature. The loss spectra are characterized by a peak appearing at a characteristic frequency, suggest the presence of relaxation processes in all tissues. The first two relaxation processes positioned at 25 kHz and 72 kHz, are corresponding to the normal (N) and fibrocystic tissues (F), respectively. While the third relaxation process positioned at 110 kHz is corresponding to the invasive lobular carcinoma (ILC). The last one where its maximum is beyond the frequency window could be corresponding to the invasive duct carcinoma (IDC). Strength and frequency of the relaxation peaks depend on the characteristic property of the dipolar relaxation. In the present study, the dipolar relaxation is associated to the capacitive charging of the cellular membrane [36]. Its electrical property strongly depends on amount of the energy absorbed by tissue. The dense or malignant tissues absorb the most amount of electromagnetic radiation whereas the soft or normal tissues absorb the least. As a consequence, the dielectric spectra corresponding to the malignant tissues are characterized by high strength. The peak broadness may indicate that dielectric data is not only due to one relaxation process but it is due to superposition of different processes: a conductivity contribution charge carriers polarization as well as the capacitive charging of the cellular membrane. Furthermore, the relaxation time (s) may play an important role for discrimination between the malignant and benign tissues. Its values are calculated from the relation: s = 1/2pfm, where fm is the peak frequency indicated for each relaxation process. Hence, the relaxation time corresponding to N, F and ILC are 6.5 106 s, 2.3 106 s and 1.5 106 s, respectively. The relaxation time is noticed considerably longer for benign than malignant tissues. This indicates that the rotational motion of each dipole presents a characteristic relaxation time; those exhibiting
Fig. 2. The frequency dependence of dielectric loss tangent (tand) of N, F, ILC and IDC breast tissues of grade 2, measured at room temperature.
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rotations more effectively hindered by their environment will show higher s. From these in vitro permittivity measurement results, a common conclusion can be drawn that there are significant differences in the dielectric properties between the normal and malignant women breast tissues. 3.2.1. The Frequency dependence of ac conductivity The ac conductivity (rac) of breast tissues is plotted as a function of frequency as given in Fig. 3. This figure shows that rac significantly increases at low frequencies that followed by a plateau at high frequencies. The plateau becomes more pronounced for N, ILC and IDC tissues compared to that observed for F tissues. The observed plateau is corresponding to the contribution of dc conductivity (rdc) to the total conductivity measured. The origin of such conductivity comes from movements of the physiological ions. The dispersion detected in conductivity could be attributed to the electrode polarization effect. The conductivity value of the malignant tissues is in the range (3 103–102 S/cm) values relative to that observed for the normal ones (8.0 109– 3.4 108 S/cm). This means that conductivity of the malignant tissues is 104–106 fold of the normal ones. The observed increase in conductivity of the malignant tissues comparing with benign could be attributed to decrease of the lipids amount. Such result is in agreement with that reported elsewhere [33]. 3.2.2. The electrical properties ratios For discrimination between the normal and tumor breast tissues in terms of the dielectric properties, it is not important to know the exact values while the ratio between them is important. Table 2 shows ratios of the permittivity, loss tangent and ac conductivity (rac) of all tissues at 105 Hz. It indicates that the tumor to normal breast tissue permittivity can be 160:1 while it could
Fig. 3. The frequency dependence of the AC conductivity (rac) for each kind of tissues measured at three different regions.
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Table 2 The electrical properties of the malignant and normal breast tissues and their ratios selected at 105 Hz. Tissues
Normal Fibrocystic Lobular Duct
Loss Tangent
Conductivity [S/cm]
e0
Permittivity
e0ratio
tand
tandratio
r
rratio
3371 4376 75,089 535,368
1 1.3 22 160
7 15 30 74
1 2.1 4.3 10.5
2.5 108 1.8 105 2.8 104 6.7 103
1 720 11,200 268,000
[6] [7]
[8] [9]
[10]
be 1.3:1 for fibrocystic breast tissues. Such result seems in agreement with that reported in literature [37]. Further, apparently differences can be observed in the conductivity ratios between normal and tumor. For instance, the tumor to normal breast tissue conductivity can be 268,000:1 while it could be 720:1 for fibrocystic breast tissues. From above, a good relationship between the breast tissue and its dielectric properties is reported; this leads to possible discrimination between the malignant and benign tumors with higher accuracy.
[11] [12]
[13]
[14] [15]
4. Conclusion [16]
In summary, FT-Raman spectroscopy was used to investigate the differences of spectra between the normal and cancer breast tissues. Quantitative analysis of the conventional FT-Raman spectra for the malignant tissues (ILC & IDC) showed a remarkably decrease in the lipid/protein ratio. Dielectric spectroscopy was used to investigate the electrical properties differences between these tissues. Since the malignant tissue has a lower amount of lipid, its permittivity shows an increase. As a result, a clear difference was found in the permittivity behavior between the malignant and benign tissues in the frequency window despite of the electrode polarization effect. Further, the loss spectra corresponding to the malignant tissues were positioned at high frequencies and characterized by high strength. The peak broadness of these spectra may indicate that dielectric data is not only due to one relaxation process but it is due to superposition of different processes: a conductivity contribution, charge carriers polarization as well as the capacitive charging of the cellular membrane. From above, a good discrimination between the normal and breast cancer tissues was obtained using FT-Raman and dielectric measurements. This may extend the area of diagnosis on the biomedical treatment. Acknowledgments This work was supported by Scientific Research Ministry, Cairo, Egypt. The authors acknowledged Prof. Dr. Ahmed Ghoneim for revising the paper.
[17] [18] [19] [20] [21]
[22] [23] [24] [25] [26] [27] [28] [29]
[30] [31]
[32] [33]
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