- Email: [email protected]
10B concentration evaluation in autoradiography images by optical density measurements
Applied Radiation and Isotopes 69 (2011) 1710–1712
Contents lists available at ScienceDirect
Applied Radiation and Isotopes journal homepage: www.elsevier.com/locate/apradiso
10
B concentration evaluation in autoradiography images by optical density measurements A. Portu a,b,n, G. Saint Martin a, D. Brandizzi a,d, O.A. Bernaola a, R.L. Cabrini a,c,d a
´mico Constituyentes (CAC), Comisio ´n Nacional de Energı´a Ato ´mica (CNEA), Av. Gral. Paz 1499, AC: B1650KNA, San Martı´n, Buenos Aires, Argentina Centro Ato ´noma de Buenos Aires, Argentina Consejo Nacional de Investigaciones Cientı´ficas y Te´cnicas (CONICET), Rivadavia 1917, AC: C1033AAJ, Ciudad Auto c ´noma de Buenos Aires, Argentina Facultad de Odontologı´a, UBA, M.T. De Alvear 2142, Ciudad Auto d Laboratorio de Microespectrofotometrı´a (LANAIS-MEF), CONICET-CNEA, Argentina b
a r t i c l e i n f o
a b s t r a c t
Available online 8 May 2011
The quantification and analysis of the tracks forming the autoradiography image of a tissue section is essential for the measurement of particle emitter concentration and distribution (e.g. 10B) in the sample. Conventional counting techniques cannot be used when track density is high because of track overlapping. A densitometry supported by image analysis method suitable for these cases has been developed. Optical density measurements obtained for boron solutions of known concentrations showed a linear behavior in the range of concentrations under consideration. & 2011 Elsevier Ltd. All rights reserved.
Keywords: Autoradiography image Optical density Nuclear tracks
1. Introduction The application of autoradiography techniques using solid state nuclear track detectors (SSNTD) to BNCT can provide substantial information about the determination of 10B in different regions of a tissue sample. For a quantitative analysis of 10B distribution, track density in the detector has to be measured. Different aspects must be considered to select a counting system to evaluate track density: the type and thickness of the detector material, the density of tracks per unit area, the transparency of the material, the type of information desired, the etching process, etc. (Becker, 1969). Besides, a reference system consisting of standard samples with known boron concentration is needed. Nuclear track detectors are often used for detection of a low number of events and track density is usually small. When the number of tracks in SSNTD foils is considerable, conventional counting techniques present difficulties because of track overlapping. In fact, when overexposed and/or overetched, the track detectors enter a saturated regime where direct track counting is not possible anymore. Thus, an approach that takes advantage of a collective effect produced by tracks in the detector should be assessed.
n Corresponding author at: Departamento de Radiobiologı´a, Lab. TANDAR, Centro Ato´mico Constituyentes (CAC), Comisio´n Nacional de Energı´a Ato´mica (CNEA), Av. Gral. Paz 1499, AC: B1650KNA, San Martı´n, Buenos Aires, Argentina. Tel.: þ54 11 6772 7150. E-mail addresses: [email protected] (A. Portu), [email protected] (G. Saint Martin), [email protected] (D. Brandizzi), [email protected] (O.A. Bernaola), [email protected] (R.L. Cabrini).
0969-8043/$ - see front matter & 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.apradiso.2011.04.029
If track density is high enough, the opacity of the etched foil becomes visible to the naked eye. In these cases, optical density (OD) should be considered. With a light source behind the film, the distribution of the emitter element in the sample can be macroscopically seen as a transmission image (Ceberg et al., 1993). For a given particle and SSNTD, OD depends not only on the track density but also on the etching time (Becker, 1969). At certain etching conditions, the amount of damage can be visualized on the detector’s surface and will be proportional to the optical density of the etched detector (El Hofy et al., 1998). This method has been widely used in biological analysis. Quantitative evaluation of nuclear DNA by cytophotometry has been proven to be a valuable aid in tumor assessment (Porschen et al., 1993). In our laboratory, research has been done in order to study DNA image cytophotometry in human tumors (Brandizzi et al., 2009). In this work, an attempt has been made to develop a densitometry supported by an image analysis method suitable for high track density samples. This method could be used as a complementary tool to the quantitative analysis of 10B concentration performed by measuring track densities (Portu et al., 2010). Finally, boron-doped silicon samples were quantitatively evaluated using this method.
2. Materials and methods Foils of Lexan in contact with solutions of boric acid 99.99% enriched in 10B were irradiated with thermal neutrons at the RA-3 reactor BNCT facility. The known concentrations of the boric acid solutions ranged between 0 and 100 ppm, in order to obtain
A. Portu et al. / Applied Radiation and Isotopes 69 (2011) 1710–1712
1711
different track densities as a result of the boron neutron capture reaction. Irradiation with high neutron fluence (1013 cm 2) was chosen in order to yield a considerable number of events. The foils were chemically attacked with a PEW solution (30 g KOH þ80 g ethyl alcoholþ90 g distilled water) at 70 1C. Etching times of 2 and 4 min were used and the images obtained in both cases were compared. Autoradiography images were acquired with a Zeiss MPM 400 microscope with 40/0.75 objectives. A 560 nm interferential filter was used in front of a digital camera to capture images. Fifty random microscopic fields of about 2 105 mm2 in area were acquired for each sample. A non-irradiated Lexan foil was used as blank. Images were obtained interposing a control filter, in order to correct possible fluctuations in the incident light. Densitometric measurements were made with image analysis software for each concentration value, in order to construct calibration curves. The software evaluates the optical density over the considered area. Thus, the measurements were normalized per unit area (in mm2) in order to allow the comparison of regions of different sizes. In this way, boron concentration could eventually be evaluated in different zones of a tissue sample. As a first application, autoradiography images of boron-doped silicon wafers were analyzed with this technique. Two pieces (SIMO5 and SIMO10) of silicon wafers doped with different (and unknown) quantities of 10B were used for this purpose. The results obtained by this system were compared to those interpolated from a calibration curve constructed by counting individual tracks in samples irradiated with lower neutron fluence (1012 cm 2).
3. Results and discussion
2 min: ODA 1 ¼ 0.00295þ0.00056[10B], R¼0.989 4 min: ODA 1 ¼ 0.01073þ0.0019[10B], R ¼0.991 where ODA 1 is the optical density per unit area expressed in mm 2, and [10B] is the boron-10 concentration in ppm. The concentration values of the silicon samples assessed with the OD calibration are in accordance with those obtained with the automatic track counting system, as shown in Fig. 3. Both methods determine the 10B concentration of the silicon wafers as if they were water equivalent samples. Actually the 10B atoms were superficially implanted on the silicon scaffold, at a depth of 2420 A˚ (Bortolussi, 2007). Thus, the results could not be expressed in terms of ppm in this case. On the other hand, it has been shown that a reference system should be used to evaluate materials in which the particle ranges (in g cm 2) are almost the same as the standards (Saint Martin et al., 2010), and this is not the case of silicon wafers. However, as the aim of this comparison is to evaluate the equivalence between both measuring methods, results are expressed in arbitrary units. The reported relation between the samples SIMO10/SIMO5 is 4.2. The experimental values obtained in this work for OD and
0.25 OD2min OD4min
0.20 OD.A-1 (µm-2)
The acquired images showed a uniform distribution of alpha and lithium tracks, which were quantified without being discriminated. Considerable track superposition was observed for the chosen etching conditions, as observed in Fig. 1(a) and (b). Track area increased with etching time, thus increasing OD. The results showed a linear correlation between OD and 10B concentration for both etching times. Experimental values and its corresponding calibration curves are shown in Fig. 2. The fitting parameters are
Fig. 1. Images of tracks obtained from a 10B solution of 100 ppm in Lexan at 1013 cm 2. Etching time: (a) 2 min and (b) 4 min.
0.15 0.10 0.05 0.00 0
20
40 10B
60
80
100
Concentration (ppm)
Fig. 2. Calibration curves obtained for solutions of boric acid 99.99% enriched in 10 B. Fluence: 1013 cm 2. Etching times: 2 and 4 min.
number of tracks per unit area (N A 1) are 3.970.3 and 4.0 70.1, respectively. Some difficulties related to the reproducibility of measurement conditions were found. Microscope settings such as incident light, condenser height, and focus are difficult to reproduce. They all determine the characteristics of the acquired image, thus affecting OD measurements performed on them. This limitation in the acquisition system diminishes the ability of the method to
1712
A. Portu et al. / Applied Radiation and Isotopes 69 (2011) 1710–1712
facilitates the correlation between a histological tissue image and the boron concentration distribution in its corresponding autoradiography. Further research will be done in order to overcome problems related with optical setup. Additionally the concentration range in which the method is reliable will be established, as well as the resolution in ppm.
Acknowledgments This work was partially supported by Grant PAE 22393, ANPCyT. The authors are grateful to Saverio Altieri and coworkers from Dipartimento di Fisica Nucleare e Teorica (Universita degli Studi di Pavia), for providing the silicon boron-doped wafers. The authors want to thank Lic. E. Pozzi and Lic. S. Thorp for irradiation of the samples. References Fig. 3. Determination of 10B concentration in two Si wafers (SIMO5 and SIMO10) by track counting and OD methods. Etching time: 2 min. ‘‘Relative 10B Concentration’’ means the concentration measured using the reference system.
distinguish between optical densities corresponding to close values of boron concentration. This problem is not so critical in track density measurements, as the important issue is to observe tracks in a way that makes possible individual counting. The purpose of the OD setup is to complement the analysis of track density in those cases where the superposition of tracks leads to miscounting errors. In those situations, OD can give valuable information that supports qualitative analysis.
4. Conclusions We have assessed a method for quantifying nuclear track density by optical densitometry and image analysis that enables the numerical evaluation of boron concentration and distribution in unknown samples. The use of a magnitude such as optical density
Becker, K., 1969. Alpha particle registration in plastics and its application for radon, thermal, and fast neutron dosimetry. Health Phys. 16, 113–123. Bortolussi, S., 2007. Boron Neutron Capture Therapy of disseminated tumours. Ph.D. Thesis. Dipartimento di Fisica Nucleare e Teorica, Universita degli Studi di Pavia. Brandizzi, D., Lanfranchi, H.E., Cabrini, R.L., 2009. Ploidy study in oral carcinomas: use of improved methodology to assess its clinical prognostic value. Oral Surg. Oral Med. Oral Pathol. Oral Radiol. Endodontol. 108 (3), 406–412. Ceberg, C.P., Salford, L.G., Brun, A., Hemler, R.J., Persson, B.R., 1993. Neutron capture imaging of 10B in tissue specimens. Radiother. Oncol. 26 (2), 139–146. El Hofy, M., Elsamman, H., Arafa, W., 1998. Optical density of the etched a-tracks in CR-39 nuclear track detector. Radiat. Meas. 29 (5), 461–464. Porschen, R., Remy, U., Bever, G., Schauseil, S., Hengels, K., Borchard, F., 1993. Prognostic significance of DNA ploidy in adenocarcinoma of the pancreas. a flow cytometric study of paraffin embedded specimen. Cancer 71, 3846–3850. Portu, A., Carpano, M., Dagrosa, A., Pozzi, E., Thorp, S., Cabrini, R.L., Liberman, S., Saint Martin, G., 2010. Reference systems for the determination of 10B through autoradiography images: set up and application to a melanoma experimental model. In: Proceedings of the 14th International Congress on Neutron Capture Therapy, October 25–29, 2010, Buenos Aires, Argentina, pp. 287–290. Saint Martin, G., Santa Cruz, G.A., Bernaola, O.A., Portu, A., 2010. Stochastic simulation of reference systems for 10B measurements in autoradiography. In: Proceedings of the 14th International Congress on Neutron Capture Therapy, October 25–29, 2010, Buenos Aires, Argentina, pp. 279–282.
Contents lists available at ScienceDirect
Applied Radiation and Isotopes journal homepage: www.elsevier.com/locate/apradiso
10
B concentration evaluation in autoradiography images by optical density measurements A. Portu a,b,n, G. Saint Martin a, D. Brandizzi a,d, O.A. Bernaola a, R.L. Cabrini a,c,d a
´mico Constituyentes (CAC), Comisio ´n Nacional de Energı´a Ato ´mica (CNEA), Av. Gral. Paz 1499, AC: B1650KNA, San Martı´n, Buenos Aires, Argentina Centro Ato ´noma de Buenos Aires, Argentina Consejo Nacional de Investigaciones Cientı´ficas y Te´cnicas (CONICET), Rivadavia 1917, AC: C1033AAJ, Ciudad Auto c ´noma de Buenos Aires, Argentina Facultad de Odontologı´a, UBA, M.T. De Alvear 2142, Ciudad Auto d Laboratorio de Microespectrofotometrı´a (LANAIS-MEF), CONICET-CNEA, Argentina b
a r t i c l e i n f o
a b s t r a c t
Available online 8 May 2011
The quantification and analysis of the tracks forming the autoradiography image of a tissue section is essential for the measurement of particle emitter concentration and distribution (e.g. 10B) in the sample. Conventional counting techniques cannot be used when track density is high because of track overlapping. A densitometry supported by image analysis method suitable for these cases has been developed. Optical density measurements obtained for boron solutions of known concentrations showed a linear behavior in the range of concentrations under consideration. & 2011 Elsevier Ltd. All rights reserved.
Keywords: Autoradiography image Optical density Nuclear tracks
1. Introduction The application of autoradiography techniques using solid state nuclear track detectors (SSNTD) to BNCT can provide substantial information about the determination of 10B in different regions of a tissue sample. For a quantitative analysis of 10B distribution, track density in the detector has to be measured. Different aspects must be considered to select a counting system to evaluate track density: the type and thickness of the detector material, the density of tracks per unit area, the transparency of the material, the type of information desired, the etching process, etc. (Becker, 1969). Besides, a reference system consisting of standard samples with known boron concentration is needed. Nuclear track detectors are often used for detection of a low number of events and track density is usually small. When the number of tracks in SSNTD foils is considerable, conventional counting techniques present difficulties because of track overlapping. In fact, when overexposed and/or overetched, the track detectors enter a saturated regime where direct track counting is not possible anymore. Thus, an approach that takes advantage of a collective effect produced by tracks in the detector should be assessed.
n Corresponding author at: Departamento de Radiobiologı´a, Lab. TANDAR, Centro Ato´mico Constituyentes (CAC), Comisio´n Nacional de Energı´a Ato´mica (CNEA), Av. Gral. Paz 1499, AC: B1650KNA, San Martı´n, Buenos Aires, Argentina. Tel.: þ54 11 6772 7150. E-mail addresses: [email protected] (A. Portu), [email protected] (G. Saint Martin), [email protected] (D. Brandizzi), [email protected] (O.A. Bernaola), [email protected] (R.L. Cabrini).
0969-8043/$ - see front matter & 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.apradiso.2011.04.029
If track density is high enough, the opacity of the etched foil becomes visible to the naked eye. In these cases, optical density (OD) should be considered. With a light source behind the film, the distribution of the emitter element in the sample can be macroscopically seen as a transmission image (Ceberg et al., 1993). For a given particle and SSNTD, OD depends not only on the track density but also on the etching time (Becker, 1969). At certain etching conditions, the amount of damage can be visualized on the detector’s surface and will be proportional to the optical density of the etched detector (El Hofy et al., 1998). This method has been widely used in biological analysis. Quantitative evaluation of nuclear DNA by cytophotometry has been proven to be a valuable aid in tumor assessment (Porschen et al., 1993). In our laboratory, research has been done in order to study DNA image cytophotometry in human tumors (Brandizzi et al., 2009). In this work, an attempt has been made to develop a densitometry supported by an image analysis method suitable for high track density samples. This method could be used as a complementary tool to the quantitative analysis of 10B concentration performed by measuring track densities (Portu et al., 2010). Finally, boron-doped silicon samples were quantitatively evaluated using this method.
2. Materials and methods Foils of Lexan in contact with solutions of boric acid 99.99% enriched in 10B were irradiated with thermal neutrons at the RA-3 reactor BNCT facility. The known concentrations of the boric acid solutions ranged between 0 and 100 ppm, in order to obtain
A. Portu et al. / Applied Radiation and Isotopes 69 (2011) 1710–1712
1711
different track densities as a result of the boron neutron capture reaction. Irradiation with high neutron fluence (1013 cm 2) was chosen in order to yield a considerable number of events. The foils were chemically attacked with a PEW solution (30 g KOH þ80 g ethyl alcoholþ90 g distilled water) at 70 1C. Etching times of 2 and 4 min were used and the images obtained in both cases were compared. Autoradiography images were acquired with a Zeiss MPM 400 microscope with 40/0.75 objectives. A 560 nm interferential filter was used in front of a digital camera to capture images. Fifty random microscopic fields of about 2 105 mm2 in area were acquired for each sample. A non-irradiated Lexan foil was used as blank. Images were obtained interposing a control filter, in order to correct possible fluctuations in the incident light. Densitometric measurements were made with image analysis software for each concentration value, in order to construct calibration curves. The software evaluates the optical density over the considered area. Thus, the measurements were normalized per unit area (in mm2) in order to allow the comparison of regions of different sizes. In this way, boron concentration could eventually be evaluated in different zones of a tissue sample. As a first application, autoradiography images of boron-doped silicon wafers were analyzed with this technique. Two pieces (SIMO5 and SIMO10) of silicon wafers doped with different (and unknown) quantities of 10B were used for this purpose. The results obtained by this system were compared to those interpolated from a calibration curve constructed by counting individual tracks in samples irradiated with lower neutron fluence (1012 cm 2).
3. Results and discussion
2 min: ODA 1 ¼ 0.00295þ0.00056[10B], R¼0.989 4 min: ODA 1 ¼ 0.01073þ0.0019[10B], R ¼0.991 where ODA 1 is the optical density per unit area expressed in mm 2, and [10B] is the boron-10 concentration in ppm. The concentration values of the silicon samples assessed with the OD calibration are in accordance with those obtained with the automatic track counting system, as shown in Fig. 3. Both methods determine the 10B concentration of the silicon wafers as if they were water equivalent samples. Actually the 10B atoms were superficially implanted on the silicon scaffold, at a depth of 2420 A˚ (Bortolussi, 2007). Thus, the results could not be expressed in terms of ppm in this case. On the other hand, it has been shown that a reference system should be used to evaluate materials in which the particle ranges (in g cm 2) are almost the same as the standards (Saint Martin et al., 2010), and this is not the case of silicon wafers. However, as the aim of this comparison is to evaluate the equivalence between both measuring methods, results are expressed in arbitrary units. The reported relation between the samples SIMO10/SIMO5 is 4.2. The experimental values obtained in this work for OD and
0.25 OD2min OD4min
0.20 OD.A-1 (µm-2)
The acquired images showed a uniform distribution of alpha and lithium tracks, which were quantified without being discriminated. Considerable track superposition was observed for the chosen etching conditions, as observed in Fig. 1(a) and (b). Track area increased with etching time, thus increasing OD. The results showed a linear correlation between OD and 10B concentration for both etching times. Experimental values and its corresponding calibration curves are shown in Fig. 2. The fitting parameters are
Fig. 1. Images of tracks obtained from a 10B solution of 100 ppm in Lexan at 1013 cm 2. Etching time: (a) 2 min and (b) 4 min.
0.15 0.10 0.05 0.00 0
20
40 10B
60
80
100
Concentration (ppm)
Fig. 2. Calibration curves obtained for solutions of boric acid 99.99% enriched in 10 B. Fluence: 1013 cm 2. Etching times: 2 and 4 min.
number of tracks per unit area (N A 1) are 3.970.3 and 4.0 70.1, respectively. Some difficulties related to the reproducibility of measurement conditions were found. Microscope settings such as incident light, condenser height, and focus are difficult to reproduce. They all determine the characteristics of the acquired image, thus affecting OD measurements performed on them. This limitation in the acquisition system diminishes the ability of the method to
1712
A. Portu et al. / Applied Radiation and Isotopes 69 (2011) 1710–1712
facilitates the correlation between a histological tissue image and the boron concentration distribution in its corresponding autoradiography. Further research will be done in order to overcome problems related with optical setup. Additionally the concentration range in which the method is reliable will be established, as well as the resolution in ppm.
Acknowledgments This work was partially supported by Grant PAE 22393, ANPCyT. The authors are grateful to Saverio Altieri and coworkers from Dipartimento di Fisica Nucleare e Teorica (Universita degli Studi di Pavia), for providing the silicon boron-doped wafers. The authors want to thank Lic. E. Pozzi and Lic. S. Thorp for irradiation of the samples. References Fig. 3. Determination of 10B concentration in two Si wafers (SIMO5 and SIMO10) by track counting and OD methods. Etching time: 2 min. ‘‘Relative 10B Concentration’’ means the concentration measured using the reference system.
distinguish between optical densities corresponding to close values of boron concentration. This problem is not so critical in track density measurements, as the important issue is to observe tracks in a way that makes possible individual counting. The purpose of the OD setup is to complement the analysis of track density in those cases where the superposition of tracks leads to miscounting errors. In those situations, OD can give valuable information that supports qualitative analysis.
4. Conclusions We have assessed a method for quantifying nuclear track density by optical densitometry and image analysis that enables the numerical evaluation of boron concentration and distribution in unknown samples. The use of a magnitude such as optical density
Becker, K., 1969. Alpha particle registration in plastics and its application for radon, thermal, and fast neutron dosimetry. Health Phys. 16, 113–123. Bortolussi, S., 2007. Boron Neutron Capture Therapy of disseminated tumours. Ph.D. Thesis. Dipartimento di Fisica Nucleare e Teorica, Universita degli Studi di Pavia. Brandizzi, D., Lanfranchi, H.E., Cabrini, R.L., 2009. Ploidy study in oral carcinomas: use of improved methodology to assess its clinical prognostic value. Oral Surg. Oral Med. Oral Pathol. Oral Radiol. Endodontol. 108 (3), 406–412. Ceberg, C.P., Salford, L.G., Brun, A., Hemler, R.J., Persson, B.R., 1993. Neutron capture imaging of 10B in tissue specimens. Radiother. Oncol. 26 (2), 139–146. El Hofy, M., Elsamman, H., Arafa, W., 1998. Optical density of the etched a-tracks in CR-39 nuclear track detector. Radiat. Meas. 29 (5), 461–464. Porschen, R., Remy, U., Bever, G., Schauseil, S., Hengels, K., Borchard, F., 1993. Prognostic significance of DNA ploidy in adenocarcinoma of the pancreas. a flow cytometric study of paraffin embedded specimen. Cancer 71, 3846–3850. Portu, A., Carpano, M., Dagrosa, A., Pozzi, E., Thorp, S., Cabrini, R.L., Liberman, S., Saint Martin, G., 2010. Reference systems for the determination of 10B through autoradiography images: set up and application to a melanoma experimental model. In: Proceedings of the 14th International Congress on Neutron Capture Therapy, October 25–29, 2010, Buenos Aires, Argentina, pp. 287–290. Saint Martin, G., Santa Cruz, G.A., Bernaola, O.A., Portu, A., 2010. Stochastic simulation of reference systems for 10B measurements in autoradiography. In: Proceedings of the 14th International Congress on Neutron Capture Therapy, October 25–29, 2010, Buenos Aires, Argentina, pp. 279–282.