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The effect of proton-irradiation on the Raman spectroscopy of tissue samples
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The effect of proton-irradiation on the Raman spectroscopy of tissue samples J. de Boera, A. Synytsya b, P. Alexab, J. Besserera, S. Froschauera, a a b a M. Loewe , M, Moosburger , K. Volka , M. WQrkner Sektion Physik, Universit~t M0nchen, D-85748 Garching, Germany E-mail: deboer~,physik.uni-muenchen.de b
Institute of Chemical Technology, Technick& 5, 166 28 Prague 6, Czech Republic E-mail: Petr.Alexa~vscht.cz
Summary Tumor and healthy tissue samples were irradiated by 24 MeV protons. The samples were exposed to doses from 0 to 50 Gy and subsequently examined by Raman spectroscopy. The analysis of the intensity of characteristic peaks as a function of radiation dose exhibits different trends for the two types of tissue.
Keywords: tissue samples; proton irradiation; radiation dose; Raman spectroscopy 1. Sample Preparation Normal (healthy) swine skin samples (batch No. I) about 5x5 mm each were extracted from the frozen swine skin provided by the Veterinary Institute of the Munich University. After thermalization up to room temperature the samples were cut into four pieces each of about 1 mm thickness (in controlled atmosphere) and sealed in thin wrappers of plastic foil: without any solution or in the physiological solution (0.9% Sodium Chloride) or in two different formulations of DULBECCO's cell culture medium. Two human skin samples (batch No. II) 5x5 mm each were extracted from the skin of the scalp with the appropriate patient consent at the Clinic of Dermatology, Hospital Kralovsk~ Vinohrady, Prague. They comprise a piece of normal skin and a central region of a skin basalioma. After transportation from Prague to Munich in a thermos bottle at 2 °C, the samples were cut into four pieces each of about 1 mm thickness (in controlled atmosphere) and sealed in thin wrappers of plastic foil without any solution.
2. Irradiation and Transportation Protons were accelerated to an energy of 24 MeV by the Munich tandem accelerator and piped to a vertical-beam facility [2]. After leaving the vacuum through a 25 mg/cmz kapton foil the protons traversed 2 cm of air and a thin support foil on which the envelopes containing the samples were lying. The proton energy of 24 MeV is high enough so that each proton traverses these foils and the sample without appreciable energy loss. The range of the protons (9 mm) is much larger than the thickness of the samples (1 mm) so the plateau region of the Bragg curve is responsible for the radiation damage done to the molecules. A dosimetry system [3] allows to control the flux of emerging protons with an accuracy of better than 7%. The flux was measured to be constant (<5%) over a beam width of 4 cm diameter. The proton flux was controlled by a calibrated monitor whose units were accumulated to deliver 0, 0.5, 5, and 50 Gy. After irradiation the samples were cooled to 2 °C (batch No. I) or shock-frozen in liquid nitrogen and then transferred into dry-ice (batch No. II) for transportation from Munich to Prague in thermos bottles.
3. Raman Spectroscopy Raman scattering is an inelastic light scattering discovered by C.V. Raman in 1928 [1]. The intensity of the Raman scattering represents about 10.7 of the incident light intensity. The scattered radiation produced by the Raman effect contains information about the energies of molecular vibrations and rotations, and these depend on the particular atoms or ions that comprise the molecule, the chemical bonds connecting them, the symmetry of their molecular structure, and the physico-chemical environment where they reside. To measure Raman spectra, we use the same procedure as in the case of the study of irradiated mice samples [4]. Slices were cut from sample pieces, dried on a filter paper and placed on a sample glass and evenly pressed by a cover glass. Raman spectra of the samples were recorded by a Dilor-Jobin Yvon-Spex Raman spectrometer equipped with an Olympus BX 40 system microscope with a 10Ox objective. This objective is capable to provide a laser spot with a diameter of approximately 1012 #m. An argon-ion laser system with an excitation line at 487 nm and an excitation power of 2.5 mW at the sample was used in the measurements of the tissues. Exposure time was 10-600 s. Raman spectra were taken at 25-30 °C in 10 randomly chosen parts. Sum spectra were corrected by a polynomial baseline using LabSpec software.
4. Results and Conclusions For batch No. I (swine), the Raman spectra for different applied doses were accumulated for the wave number interval 700 cm-11800 cm4. Only the spectra of the samples sealed without any solution (see Fig. 1) or with the physiological solution were suitable for the analysis, the others suffered from severe bacterial contamination. For the batch No. II (human), the Raman spectra were accumulated for the wave number interval 500 cm4- 3500 cm4 (see Figs. 2 and 3). The normal skin sample was contaminated by blood during the surgery (see the region of important blood Raman
$103
lines, 600 cm4- 1700 cm4). Thus the region 2800 cm-~ - 3100 cm4 (CHx bonds) seems to be more suitable for comparison of irradiated normal and tumor skin. Especially in the tumor tissue the difference in sensitivity between unsaturated (2879 cm~, 2935 cm4) and saturated (2854 cm-~) CHx bonds is remarkable: the unsaturated bonds are more sensitive to the applied radiation (lines decreasing with increasing dose). Our investigations have shown that Raman spectroscopy is well-suited to assess the radiation damage done to biological samples by protons, Effects depending on the delivered dose as well as on the type of tissue (normal or tumor) were found, In the future we plan to investigate also single organic compounds and cell nuclei to trace their damages induced by proton irradiation.
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1 A standard presentation of Raman spectra of irradiated normal swine skin (batch No. I) as a function of the wave number for different applied doses. Important intensity changes occurring with increasing dose are indicated by arrows, the wave number shift of the composed 6(CH2) + 6~,(CH3) peak at 1453 cm-~ by a line.
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Figure 2: Intensity of the Raman spectra of irradiated human normal skin (batch No. II) as a function of the wave number for different applied doses. Wave numbers of important peaks are indicated. 30000~
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Wavenumber [cm 4] Figure 3: Intensity of the Raman spectra of irradiated human tumour skin (batch No. II) as a function of the wave number for different applied doses. The Raman spectrum of non-irradiated blood is also displayed. Wave numbers of important peaks are indicated.
Acknowledgments Work supported by the Bavarian Ministry of Environmental Affairs, Grant No. 96e-8801.3-1999/3, and by the Ministry of Education, Youth and Sports of the Czech Republic, Grant CEZ: MSM 223400008.
References [1]
Atkins PW. Spectroscopy 1: rotational and vibrational spectra. In: Atkins PW. Physical Chemistry. 5th Edition, Oxford: Oxford University Press. 1995;545.565-568.576.582-585.
[2]
Besserer J, de Boer J, Dellert Jet al. An irradiation facility with a vertical beam for radiobiological studies. Nucl Instr Meth A 1999;430:154-60.
[3]
de Boer J, Besserer J, Moosburger M et al. Dosimetry of low-energy protons on the vertical-beam facility at the Munich accelerator. Phys Med (Suppl. 1) 2001 ;17:143-3.
[4] Synytsya A, Alexa P, de Boer Jet al. Raman spectroscopy of tissue samples after irradiation by 24 MeV protons. In: de Boer J, editor. Proceedings of the Workshop on Radiation Therapy and Dosimetry. Munich: Munich University. 2001 ;5-10.
The effect of proton-irradiation on the Raman spectroscopy of tissue samples J. de Boera, A. Synytsya b, P. Alexab, J. Besserera, S. Froschauera, a a b a M. Loewe , M, Moosburger , K. Volka , M. WQrkner Sektion Physik, Universit~t M0nchen, D-85748 Garching, Germany E-mail: deboer~,physik.uni-muenchen.de b
Institute of Chemical Technology, Technick& 5, 166 28 Prague 6, Czech Republic E-mail: Petr.Alexa~vscht.cz
Summary Tumor and healthy tissue samples were irradiated by 24 MeV protons. The samples were exposed to doses from 0 to 50 Gy and subsequently examined by Raman spectroscopy. The analysis of the intensity of characteristic peaks as a function of radiation dose exhibits different trends for the two types of tissue.
Keywords: tissue samples; proton irradiation; radiation dose; Raman spectroscopy 1. Sample Preparation Normal (healthy) swine skin samples (batch No. I) about 5x5 mm each were extracted from the frozen swine skin provided by the Veterinary Institute of the Munich University. After thermalization up to room temperature the samples were cut into four pieces each of about 1 mm thickness (in controlled atmosphere) and sealed in thin wrappers of plastic foil: without any solution or in the physiological solution (0.9% Sodium Chloride) or in two different formulations of DULBECCO's cell culture medium. Two human skin samples (batch No. II) 5x5 mm each were extracted from the skin of the scalp with the appropriate patient consent at the Clinic of Dermatology, Hospital Kralovsk~ Vinohrady, Prague. They comprise a piece of normal skin and a central region of a skin basalioma. After transportation from Prague to Munich in a thermos bottle at 2 °C, the samples were cut into four pieces each of about 1 mm thickness (in controlled atmosphere) and sealed in thin wrappers of plastic foil without any solution.
2. Irradiation and Transportation Protons were accelerated to an energy of 24 MeV by the Munich tandem accelerator and piped to a vertical-beam facility [2]. After leaving the vacuum through a 25 mg/cmz kapton foil the protons traversed 2 cm of air and a thin support foil on which the envelopes containing the samples were lying. The proton energy of 24 MeV is high enough so that each proton traverses these foils and the sample without appreciable energy loss. The range of the protons (9 mm) is much larger than the thickness of the samples (1 mm) so the plateau region of the Bragg curve is responsible for the radiation damage done to the molecules. A dosimetry system [3] allows to control the flux of emerging protons with an accuracy of better than 7%. The flux was measured to be constant (<5%) over a beam width of 4 cm diameter. The proton flux was controlled by a calibrated monitor whose units were accumulated to deliver 0, 0.5, 5, and 50 Gy. After irradiation the samples were cooled to 2 °C (batch No. I) or shock-frozen in liquid nitrogen and then transferred into dry-ice (batch No. II) for transportation from Munich to Prague in thermos bottles.
3. Raman Spectroscopy Raman scattering is an inelastic light scattering discovered by C.V. Raman in 1928 [1]. The intensity of the Raman scattering represents about 10.7 of the incident light intensity. The scattered radiation produced by the Raman effect contains information about the energies of molecular vibrations and rotations, and these depend on the particular atoms or ions that comprise the molecule, the chemical bonds connecting them, the symmetry of their molecular structure, and the physico-chemical environment where they reside. To measure Raman spectra, we use the same procedure as in the case of the study of irradiated mice samples [4]. Slices were cut from sample pieces, dried on a filter paper and placed on a sample glass and evenly pressed by a cover glass. Raman spectra of the samples were recorded by a Dilor-Jobin Yvon-Spex Raman spectrometer equipped with an Olympus BX 40 system microscope with a 10Ox objective. This objective is capable to provide a laser spot with a diameter of approximately 1012 #m. An argon-ion laser system with an excitation line at 487 nm and an excitation power of 2.5 mW at the sample was used in the measurements of the tissues. Exposure time was 10-600 s. Raman spectra were taken at 25-30 °C in 10 randomly chosen parts. Sum spectra were corrected by a polynomial baseline using LabSpec software.
4. Results and Conclusions For batch No. I (swine), the Raman spectra for different applied doses were accumulated for the wave number interval 700 cm-11800 cm4. Only the spectra of the samples sealed without any solution (see Fig. 1) or with the physiological solution were suitable for the analysis, the others suffered from severe bacterial contamination. For the batch No. II (human), the Raman spectra were accumulated for the wave number interval 500 cm4- 3500 cm4 (see Figs. 2 and 3). The normal skin sample was contaminated by blood during the surgery (see the region of important blood Raman
$103
lines, 600 cm4- 1700 cm4). Thus the region 2800 cm-~ - 3100 cm4 (CHx bonds) seems to be more suitable for comparison of irradiated normal and tumor skin. Especially in the tumor tissue the difference in sensitivity between unsaturated (2879 cm~, 2935 cm4) and saturated (2854 cm-~) CHx bonds is remarkable: the unsaturated bonds are more sensitive to the applied radiation (lines decreasing with increasing dose). Our investigations have shown that Raman spectroscopy is well-suited to assess the radiation damage done to biological samples by protons, Effects depending on the delivered dose as well as on the type of tissue (normal or tumor) were found, In the future we plan to investigate also single organic compounds and cell nuclei to trace their damages induced by proton irradiation.
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50 Gy
5 Gy
%,~ 0 5 Gy
-700
0 Gy
800
900
t000
i100
t200
Wavenumber
1300
t400 i 5 0 0
1600
1700
[ c m ~]
1 A standard presentation of Raman spectra of irradiated normal swine skin (batch No. I) as a function of the wave number for different applied doses. Important intensity changes occurring with increasing dose are indicated by arrows, the wave number shift of the composed 6(CH2) + 6~,(CH3) peak at 1453 cm-~ by a line.
l~Ii{
25000 ........................................................................................................... 7 ° ° °
...................................
..................................................................................................................
Is 60o0~ ..................0,5 Gy j ....................... o Gy
.......................
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5000. 15000
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10000
2000, 5000 1000,
0¸ 8~
i200
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280o
3000 3too
34oo
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Figure 2: Intensity of the Raman spectra of irradiated human normal skin (batch No. II) as a function of the wave number for different applied doses. Wave numbers of important peaks are indicated. 30000~
.................................................................
25000.
50 Gy ....................5 Gy 0,5 Gy ........................0 Gy .........................blood
= O~ ~
25000,
--50 Gy .................. 5 Gy ~ 0
, , ~ , ~ , , , - ~ - , , . ~
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........................
Gy blood
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I 15000-
=
10000.
5000-
0-~
0~ 600
800
1000
i200
1400
1600
1800
2800
3000
3200
3400
Wavenumber [cm 4] Figure 3: Intensity of the Raman spectra of irradiated human tumour skin (batch No. II) as a function of the wave number for different applied doses. The Raman spectrum of non-irradiated blood is also displayed. Wave numbers of important peaks are indicated.
Acknowledgments Work supported by the Bavarian Ministry of Environmental Affairs, Grant No. 96e-8801.3-1999/3, and by the Ministry of Education, Youth and Sports of the Czech Republic, Grant CEZ: MSM 223400008.
References [1]
Atkins PW. Spectroscopy 1: rotational and vibrational spectra. In: Atkins PW. Physical Chemistry. 5th Edition, Oxford: Oxford University Press. 1995;545.565-568.576.582-585.
[2]
Besserer J, de Boer J, Dellert Jet al. An irradiation facility with a vertical beam for radiobiological studies. Nucl Instr Meth A 1999;430:154-60.
[3]
de Boer J, Besserer J, Moosburger M et al. Dosimetry of low-energy protons on the vertical-beam facility at the Munich accelerator. Phys Med (Suppl. 1) 2001 ;17:143-3.
[4] Synytsya A, Alexa P, de Boer Jet al. Raman spectroscopy of tissue samples after irradiation by 24 MeV protons. In: de Boer J, editor. Proceedings of the Workshop on Radiation Therapy and Dosimetry. Munich: Munich University. 2001 ;5-10.