1. Neutron autoradiography in nuclear track detectors: simultaneous observation of cells and nuclear tracks from BNC reaction by UV C sensitization of polycarbonate 1 Comisión Nacional de Energía Atómica, Argentina 2 Consejo Nacional de Investigaciones Científicas y Técnicas, Argentina 3 Facultad de Odontología, Universidad de Buenos Aires, Argentina 4 Laboratorio de Microespectrofotometría, CONICET-CNEA Agustina Portu 1,2, Andrés Rossini 1, Mario A. Gadan 2, Omar A. Bernaola 2†, S. I. Thorp 1, P. Curotto 1, E.C.C Pozzi 1, Rómulo L. Cabrini 2,3,4, Gisele Saint Martin 2

2. AUTORADIOGRAPHY APPROACHES QUALITATIVE QUANTITATIVE High density of tracks. Differences in shades of gray. Information about the spatial distribution of boron. Individual counting. Absolute value of boron concetration. Different regions of the tissue can be evaluated.

3. Qualitative autoradiography QTA High resolution Tumor and premalingnat tissue sections of hamster cheek pouchand its corresponding autoradiography images. GB-10 (50mg.kg -1 ) Quantitative autoradiography 5 ppm10 ppm 100 ppm

4. Haematoxylin stained melanoma tumor (NUDE) cryosection and its corresponding qualitative autoradiography image. BPA (350 mg.kg -1 ), 10 13 n.cm -2. Magnification: 10 X. S T S T Conventional autoradiography

5. QTA Portu et al., ARI 69 (2011) 1698–1701 – Portu et al., Biotech Histochem 88(2013): 217–221

6. High resolution autoradiography Simultaneous observation of nuclear tracks and biological material UV C sensibilization (CR-39) K. Amemiya et al., Rad Meas 40 (2005) 283 – 288. T. Konishi et al., J. Radiat Res, 48 (2007)255–261. HRQAR High resolution quantitative autoradiography Solares and Zamenhof, Radiat Res 144 (1995) 50 – 58. Kiger 3rd WS, Micca PL, Morris GM, Coderre JA, Radiat Prot Dosimetry (2002) 99:409 – 12..

7. To develop a methodology to produce an “imprint” of cells cultivated on a polycarbonate detector by exposure of the detector to UV C radiation… …in order to observe cells and tracks simultaneously. To quantify BPA concentration of MEL J cells in vitro. Aim

8. Effect of UV C on polycarbonate Photo-oxidation: polimeric chains clevage Photo-Fries: chains re-arragement UV Spectrum: UV-A: (320-400) nm UV-B: (280-320) nm UV-C: (200-280) nm yellowing

9. UV-C exposure UV-C irradiation of the assembly sample-detector. Wavelenght: 254 nm - Increase of etching velocity - Imprints formation Proposal

10. Cells superpositionMonolayer Detector: circular foils of polycarbonate (Lexan™, 250 μm) on petri dishes (60 mm). Cells: human melanoma line MEL J (≈5.10 5 cells ) Incubation: BPA (10 ppm) for 2 h. Fixation: Glutharaldehide (5%). H&E. Magnification: 100x 1

11. thermal neutron irradiation 2 10 12 n.cm -2 10 13 n.cm -2 BPA incubation 3 0 and 10 ppm UV-C irradiation 4 2, 4 and 6 h Photodegradation of polycarbonate Field uniformity: 15 cm Irradiance (mW.cm -2 ) Build-up and characterization of the irradiation facility H&E and exploration 5 etching 6 2, 3 and 4 min Optimal conditions? Methodology (cont) Irradiance (mW.cm -2 ) distance (cm) Irradiance at central point A 0,0 F UV Lamp 1 2 34 5 6 h = 1 cm h = 14 cm z x y 4 h, 2 min

12. 2 h UV-C2 h UV-C 2 min 3 min 4 min

13. 2 min 3 min 4 min 4 h UV-C

14. UV-C time 4 min 3 min

15. ControlBPA OPTIMAL CONDITIONS

16. Control – UV 6h – 4 minBPA – UV 6h – 4 min SEM images

17. UV-A irradiation (λ=360 nm) Exposition time: 6 h at a rate of 0.9 mW.cm -2 (same irradiance as UV-C)

18. 10 B quantification Mean value: (33±7) ppm Image Proplus TM

19. Conclusions We developed a methodology for the simultaneous visualization of cell imprints and tracks originated from 10 B using polycarbonate as a nuclear track detector. The best conditions were established in order to visualize imprints and tracks. It could be concluded that the photoinduced damage mechanisms of the polymeric detector responsible for the imprint creation, are more effective for photon energies higher than that corresponding to UV A radiation. We were able to quantify boron concentration inside the cells.

20. This methodology will make possible an extensive comparison between cell lines and the evaluation of different boron compounds under development through their distribution in vitro.

21. Tissue imprints a)Haematoxylin-Eosin stained premalignant tissue section mounted on Lexan (BPA, 1.25x). b)Autoradiography image of a sucessive section. 1 h UV-C; 2 min etching

22. 40x Better resolution on epithelium assesment Improvement of the technique!

23. Thank you very much!

24. Mercury lamp TUV G15T8 de 15 W (Philips) Sample positioning

25. Etching kinetics Diameter measurement of fission fragments ( 252 Cf)

26. A 0,0 F UV Lamp 1 2 34 5 6 h = 1 cm h = 14 cm z x y Irradiance (mW.cm -2 ) distance (cm) Irradiance at central point Field uniformity: 15 cm Irradiance (mW.cm -2 )

27. thermal neutrons (10 13 n.cm -2 ) Lexan BPA Photodegradation of polycarbonate UV C Trypsin Etching PEW 70° C 2, 3 and 4 min 2, 4 and 6 h