Single-Proton Irradiation of Living Cells - Development of New Tools for Low-Dose Radiation Research

A Single-Ion Hit Facility (SIHF) consists of a custom-build facility based in particle accelerators which offers irradiation controlling the number of delivered particles with a precise targeting localization. The irradiation spot can be confined down to the nanometre scale allowing the irradiation of subcellular compartments with a single particle. Therefore, these facilities have become a very powerful tool for biological applications specifically to study low-dose radiation effects on living cells. A SIHF has been created at the Lund Nuclear Microprobe (LNM-SIHF) and, in order to make it operational, several tools were fabricated. These tools included the necessary software for cell recognition, custom-designed Petri-type dishes suitable for cell culture and irradiation, and other tools which allow the evaluation of the system. Additionally, the importance of reactive oxygen species (ROS) in bystander cells after non-targeted proton irradiation was investigated on the human hepatoma cell line (HepG2). In-house implemented software, SeACell, provides on-line cell recognition and localization in a short time and high efficiency without the use of cell-staining dyes. The program was developed using IDL 6.2 language, and includes automated and manual targeting selection through a user-friendly interface. In addition, table colour display and filter drop-down menus were added to improve the quality of the input image if required. Custom-designed irradiation dishes permit controlling the cells growth position by confining them through limiting structures on the floor of the dish and therefore, facilitating repeated access to the cell position. The epoxy-based photopolymer SU-8 was patterned by UV lithography technique producing irradiation dishes, with a supporting layer of approximately 5 microns thick where 5 microns height walls were used to form the limiting structure. The entire structure contains 400 squares that can be located by a row letter and column number printed outside the grid. Other tools were manufactured by UV large exposure and the SU-8 photoresist: an artificial cell sample, which offered a semi-realistic scenario to test the system's capability, and a calibration sample used to establish the coordinates of the irradiation point in all the microscopes in which the cells were inspected. Also, two Ni dot arrays were fabricated using electron beam lithography to test the targeting accuracy of the system