New Developments

RARAF – New Developments

Our current single-particle microbeam system features an extensively upgraded spatial resolution of 0.8 μm (diameter) for the beam. Further work is continuing with the aim of obtaining a beam spot diameter of 0.5 μm. Current technology development focuses on adding and improving imaging techniques, the capabilities of our existing system and on developing new functionalities for the future.

Among these new developments, a new particle detector to be placed below the samples, higher throughput via point and shoot technology, and development of x-ray microbeams, to be used as additional irradiation facilities.

Sub-Cellular Targeting

Typical examples of sub-cellular targets for the RARAF microbeam have been: 1) the cell nucleus or 2) the cell cytoplasm. Now that a focused charged-particle beam with a sub-micron diameter is routinely available using the compound electrostatic quadrupole triplet lens on the RARAF microbeam, additional targets within cellular systems are accessible. For instance, preliminary radiation experiments that target mitochondria have been conducted on small airway epithelial cells. In these experiments, the multiphoton microscope was used to image GFP-tagged mitochondria sites and position them over the ion beam for irradiation. The figure shows a multiphoton microscope image of mitochondria (green) targets within small airway epithelial cells. The crosshairs (red) mark the center of the image, which coincides with the position of the ion beam. For this image and for post-irradiation time-lapse images of mitochondria behavior, Hoechst was used to counter stain the nuclei.

Point and Shoot

During microbeam irradiation, cells to be irradiated are moved to the beam position using a high-resolution three-axis piezo-electric stage. When a collimated microbeam was being used, this was a necessary but relatively time-consuming method to position cells for irradiation. Unlike a collimated microbeam, a focused microbeam is not restricted to a single location on the exit window and therefore can be deflected to any position in the field of view of the microscope used to observe the cells during irradiation. Moving the beam to the cell position magnetically or electrostatically can be performed much faster than moving the stage.

Photograph of the Point and Shoot deflector coil.

We have developed a "Point-and-Shoot" targeting system for microbeam irradiation based on the wide-field magnetic split-coil deflector system from Technisches Büro Fischer (Ober Ramstadt, Germany). The deflector consists of four interlaced coils in a set of ferrite rings. The coils are paired to create two axes that are orthogonal to each other. When wired in series, a pair of coils on opposite sides will create a temporary dipole magnetic field, which deflects the beam in the direction of the right hand rule. This system has been used for the microbeam facility at Gesellschat für Schwerionenforschung (GSI), Darmstadt, Germany. The deflector can move the beam to as many as 1000 separate locations per second (more than 5 times the speed of movement of the stage!), which will dramatically reduce the irradiation time.

It is possible to "scan" the beam into a rectangular area in the microscope view by using the two pairs of coils independently. The current needed in each coil to deflect the beam to a specific location is mapped and this mapping used as a look-up table for the target locations. The irradiation then proceeds by rapidly switching through the required sets of coil currents to steer the beam to the targets, irradiating each target with the time limited only by the beam current.

We have installed Point-and-Shoot systems on both the Permanent Magnet Microbeam (PMM) and the electrostatic microbeam.

X-Ray Microbeam

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 K_α 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.

SEM micrograph of our Fresnel 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. Preliminary off-line tests confirmed the focusing characteristics of the zone plate. 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). Testing of the x-ray microbeam system is progressing. The final expected dose rate to the sample for a proton beam with a current of 10 nA will be about 30 cGy/min delivered in a 2 micron spot.

Site developed by CE, page last modified by JL on June 16, 2010 .

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