Nearly all microbeam facilities currently employed for radiobiological applications use charged particles - from protons to heavy ions, with LETs (stopping powers) ranging from a few tenths to several hundred keV/µm. There are however considerable benefits in using soft x-ray microbeams for both mechanistic and risk estimation end-points. The higher spatial resolution achievable with modern state-of-the-art x-ray optics elements combined with the localized damage produced by the absorption of low energy photons (~1 keV) represents a unique tool to investigate the radio-sensitivity of sub-cellular and eventually sub-nuclear targets. Moreover, as low-energy x rays undergo very little scattering, by using x rays with an energy of ~5 keV it will be possible to irradiate with micron precision individual cells and/or parts of cells up to a few hundred microns deep inside a tissue sample in order to investigate the relevance of effects such as the bystander effect in 3-D structured cell systems.
We are upgrading the RARAF microbeam to include soft x rays: characteristic Kα x rays from Ti, 4.5 keV (higher energies are not feasible due to Compton scattering effects). The x-ray microbeam (left) is mounted on the end of a horizontal beam line on the first floor of RARAF. Because the x rays are being produced by reflection instead of transmission, the x-ray beam will be vertical.
The x rays will be generated using an electrostatic quadrupole quadruplet lens system to focus protons onto a thick Ti target (best cross sections is at 4.5 MeV). The target consists of a small plug of Ti pressed into a water-cooled copper block. The x rays generated are demagnified using a zone plate. By using the already focused proton microbeam to generate characteristic x rays, it is possible to obtain a nearly monochromatic x-ray beam (very low bremsstrahlung yield) and a reasonably small x-ray source (~20 µm diameter), reducing the requirements on the zone plate.
Based on these parameters our zone plate specifications were determined. The zone plate has a radius of 120 µm and an outmost zone width of 50 nm. The zone plate has been placed relatively close to the x-ray source (250 mm) and has a focal length of 23 mm (demagnification factor of ~11). Currently we have a 5 µm spot size measured using the knife edge occlusion method. This focusing comes from a proton beam spot (50 µm) on the titanium target which translates into a larger object aperture allowing more proton current on the target for a 10x higher dose rate (10 mGy/sec).
We have begun our biological testing of the x-ray microbeam by looking at γ-H2AX foci formation from DNA strand breaks in AG1522 cells. This cell line is well characterized and has a strong dose response signal. We observe an increase in the foci numbers as measured by fluorescent intensity for a linearly increasing dose.
This preliminary data demonstrates the operation of the x-ray microbeam. We look forward to working with our user on this new platform and irradiation modality.