Neutron Autoradiography Combined With UV-C Sensitization: Toward the Intracellular Localization of Boron

GADAN M; LLOYD R; SAINT-MARIN G; OLIVERA MS; L. POLICASTRO; PORTU A

Congreso; 18 th International Congress of Neutron Capture Therapy; 2018

Introduction            Ourgroup has reported the imprint formation of biological material onpolycarbonate nuclear track detectors (NTD) by UV-C exposure, as an approach tosimultaneously visualize cell contours and nuclear tracks coming from the boronneutron capture reaction. A reduction in track density (fading) on sensitizedpolycarbonate foils was also reported by our group. In this work we present animprovement of the neutron autoradiography technique combined with UV-Csensitization of polycarbonate NTD with the aim to enhance spatial resolutionof nuclear tracks etched pits respect to main cell structures or compartments,such as nucleus and cytoplasm. We focused our attention on reducing the UV-C irradiationtime, achieving contrast enhancement of cells imprints while preserving nucleartrack etched pits in a single image, thus avoiding the need of using imageco-registration methods. Materials and methods            Consideringthat cell nucleus has a higher UV absorption than cytoplasm and thathaematoxylin has even higher UV absorption and preferentially stains nucleus,we proposed to enhance the contrast between this two main cell structures bymeans of haematoxylin staining before UV-C sensitization. Several experimentswere performed in order to optimize UV-C exposure parameters together withetching conditions for cells imprints formation using the SKBR3 breast cancercell line. Cells were cultured on polycarbonate detectors (LexanTM)and incubated with BPA-f. Cell attachment strategies, toxicity assays and boronincubation times were explored. The assemblies ?cells+NTD? were irradiated withthermal neutrons at the RA-3 reactor. Regarding cell imprints, differentparameters were also explored, such as: UV-C exposure times; haematoxylinstaining before UV-C and staining times; and finally different etching times.  Results            Thus,we established the parameters of UV-C sensitization and chemical etchingconditions that let us achieve clear images of cell imprints together withnuclear track etch pits in a single image. The proposed method improvessignificantly the resolution of the imprints images and allowed us to clearlydifferentiate the cell nucleus from the rest of the cell. Indeed, the optimizedmethod even reproduces the findings in the stained cell culture, such asmultinuclear cells or division processes in SKBR3 cell line. In addition, thismethod reduces the UV-C exposure time from 6 h (according to previous works)down to 5 min, which is an important experimental issue. This remarkablereduction in the exposure time would prevent fading of nuclear tracks.  Conclusion            Itis fair to emphasize that this proposal involves no difficult experimentalsetup or complex image acquisition systems. The contrast enhancement wasachieved just by adding the haematoxylin staining step previous to UV-C lampexposure and, as in the ?conventional? autoradiography technique, only a lightmicroscope equipped with a digital camera is necessary. The proposed methodologycan be applied to study the boron distribution independently of the chosen cellline and/or boron compounds. The obtained images together with details anddiscussion of the performed method will be presented.