Ion Beam and Neutron Core Facility
A significant proportion of user-initiated experiments at RARAF are service oriented. Service is generally characterized by routine operation of our current technologies, in which we provide support, but not necessarily significant intellectual input. For such studies, users can choose to come to RARAF for their experiment, or samples can be sent to us for irradiation, and we will irradiate and return the samples. When needed, RARAF staff will provide appropriate training. RARAF guidelines defining a service-based experiment are:
- The experiment is fully designed by the user with minimal input from RARAF staff;
- The experiment uses existing facilities and requires at most minor modifications;
- RARAF is acknowledged in all resulting publications but RARAF personnel are not necessarily co-authors;
- A fee will normally be charged on a per-hour or per-day basis.
In short, collaborative projects involve scientists from RARAF working closely with external investigators who have expertise in other disciplines that can potentially benefit from microbeam technology. Our collaborative projects have typically been the motivators for extensions to our current resources, and we welcome such collaborations. Such collaborations typically drive our technological R&D, and likewise, the technology should significantly advance the scientific frontiers of the collaborative research. RARAF guidelines defining a collaborative project are:
- Interactions that strongly synergize with the existing core research programs or generate new ones that significantly enhance the capabilities of RARAF;
- Collaborative efforts will typically result in a major upgrade to RARAF facilities and will subsequently be provided to service-based users;
- One or more RARAF personnel are closely involved in the project design from start to finish and will be co-authors on joint publications and co-investigators in grant applications;
- No fee will normally be charged for beam time, but the collaborator may be expected to provide support for extra equipment required for the work.
The microbeam facility was designed to deliver defined numbers of helium or hydrogen ions produced by a 5 MV Singletron accelerator, covering a range of LET from 10 to 200 keV/µm, into an area smaller than the nuclei of human cells growing in culture on thin plastic films (0.8 µm diameter beam). The current overall irradiation throughput for our microbeam is about 10,000 cells/h, which may be compared with earlier microbeam system throughputs of about 120 cells/h. At present, the beam is focused by a pair of electrostatic triplet lenses (the initial beam was collimated by a pair of laser-drilled apertures that formed the beamline exit). An integrated computer control program locates the cells, attached in a monolayer to the thin polypropylene base of a cell culture dish, and positions them for irradiation. We are, as always, in the process of developing new technologies to extend the use of our facility for biological experimentation. Current development focuses on adding and improving imaging techniques, increasing throughput, and adding irradiation facilities such as a neutron beam.
Permanent Magnet Microbeam
The permanent magnet microbeam (PMM), under development at RARAF, presents an alternative approach to microbeam design. Instead of focusing the ion beam using electromagnetic or electrostatic lenses, this system uses permanent magnets, which require no power supplies.
4He ions from the accelerator are focused using a compound magnetic lens consisting of two quadrupole triplets. The first triplet is placed 2 m above the object aperture, with a second (identical) triplet placed 2 m above the focal plane of the first. Since each triplet does not have identical demagnifications in the x- and y-axes, the two lenses are rotated by 90º in the x-y plane so that a circular beam spot is obtained (Russian Symmetry). The object aperture was initially covered with a 1.8-μm thick Al scattering foil (since replaced by a phase space sweeper), used to eliminate any correspondence between angle and position for the particles in the beam. A limiting aperture is placed before the first triplet and inside the second triplet to reject ions which have very large aberrations.
The cells to be irradiated are placed at the image plane of the compound lens. The PMM is mounted on the original microbeam endstation consisting of a microscope with a particle detector mounted on one of the objective lenses and an x-y stage positioned by stepping motors.
Just prior to the decommissioning of the RARAF Van de Graaff accelerator in June 2005, we had attained a beam spot size of 20 µm. As a first step, the beam was imaged at the focal plane of the first quadrupole triplet, using a commercial CCD chip. The observed spot size of 50 x 150 µm was in good agreement with that expected from simulations.
As a second step, the beam diameter was measured at the endstation using the knife-edge technique. The spot size was tuned by adjusting all magnets while maintaining Russian symmetry - in particular, we tried to keep quadrupoles 1, 3, 4 and 6 at the same strength and quadrupoles 2 and 5 at the same strength. While the general trends are very similar in both cases, the smallest spot size obtained experimentally was only 20 µm in diameter, two times larger than the theoretical prediction. Simulations have shown that this is probably due to residual high-order fields in the quadrupoles or due to misalignment.
In 2006, the magnetic quadrupole system had to be removed for the construction of the laboratories on the third floor and was reassembled in early 2007. Without adjusting the magnets, we measured a beam spot size of 20 microns, demonstrating the robustness of this design. Following the replacement of the scattering foil with a phase space sweeper and smaller aperture we have obtained a beam spot approximately 8 µm in diameter.
The magnetostatic lens is in routine use.
The design of the compound lens, used to focus the beam, is based on the one used for our electrostatically focused microbeam. However, in order to simplify the PMM operation, we have elected to use permanent magnets to construct the lens as opposed to the electrostatic lenses. The use of permanent magnets eliminates the need for bulky power supplies and cooling systems required by other types of ion lenses in addition to allowing a tighter configuration (and therefore better optical properties) than common electromagnetic lenses. Magnet strength is adjusted by moving rare earth magnets in and out of a shaped yoke as seen in the figure.
Two permanent magnet quadrupole triplets have been purchased from STI Optronics. The optimized lens, shown here, consists of two outer 4.25 cm long magnetic quadrupoles and an 8.5 cm long center quadrupole with inter-quadrupole gaps of 1.67 cm and a bore of radius 6.35 mm. It should be noted that such a small bore radius is rather difficult to obtain with standard electromagnets.
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.
A significant number of individuals are occupationally exposed to low doses of neutrons, mostly low-energy neutrons. These low-energy neutrons produce biological damage in a fundamentally different way from most photon or high-energy charged particle irradiations. Both the x rays and the high-energy charged particles that our users study damage DNA primarily through atomic ionization, i.e. production of electron vacancies in DNA, directly or via free radicals. By contrast, the primary DNA damage mechanism for low-energy neutrons is via direct knockout of protons in the DNA. In order to better understand possible damage response mechanisms such as the bystander effect from neutrons on biological systems, we are developing a neutron microbeam in addition to the broad beam neutron irradiator.
Our neutron microbeam design is based on the existing charged particle microbeam technology at RARAF. The principle of the neutron microbeam is to use the proton beam with a micrometer-sized diameter impinging on a very thin lithium fluoride target system. From the kinematics of the7Li(p,n)7Be reaction near the threshold of 1.881 MeV, the neutron beam is confined within a narrow, forward solid angle. Calculations show that the neutron spot using a target with a 17 µm thick gold backing foil will be less than 20 µm in diameter for cells attached to a 3.8 µm thick propylene-bottomed cell dish in contact with the target backing. The neutron flux will roughly be 2000 per second based on the current beam setup at RARAF Singleton accelerator. The dose rate will be about 200 mGy/min. By reducing the target thickness to the minimum necessary, the production of resonance gamma rays in the thin target will be limited. The principle of this neutron microbeam system has been preliminarily tested at RARAF using a collimated proton beam.
We have produced the first-ever neutron microbeam. It is based on a proton microbeam at specific and precise energy (1.886 MeV) impinging on a lithium target. we have now reached a ~30 µm diameter. This is sufficiently small that initial biological testing is now underway.
UV Microspot Irradiator
We have integrated a UV microspot irradiator into our microbeam system. Where traditional UV laser microbeam design involves an elongated laser path of exposure, our system is an improvement over the conventional design because it utilizes multiphoton excitation to produce a micro-volume of effective UV radiation - the defining characteristic behind our term UV microspot. What makes our UV microspot unique is that it is integrated within the Microbeam II charged-particle cell-irradiation platform to provide a cocktail of photon and particle irradiations within one system.
To demonstrate the capabilities of RARAF’s UV microspot irradiator, the Columbia University crown logo was irradiated onto a live single cell nucleus. The cells for this demonstration were HT1080 human Fibro Sarcoma cells with nuclei containing GFP-tagged XRCC1 (repair protein for single-strand DNA damage). Provided by our user David Chen, UT Southwestern, Dallas, Texas, these cells were plated on Petri dishes and kept under physiological conditions during the irradiation and imaging phases. Of the two cell nuclei visible in the 3D multiphoton microscopy image above, the lower of the two cell nuclei was irradiated using a Titanium Sapphire laser tuned to 976 nm, which “acts as” 488 nm and 325 nm in the two- and three-photon modes, respectively. The crown pattern was drawn by precision stage motions to 59 irradiation locations. Each location received 16 mW of laser power for 1 second in an elliptical volume, 0.65 µm radial by 2.8 µm axial full width half maximum (FWHM), corresponding to the point spread function of the laser through our 60X NA 1.0 water-dipping objective. Typical spacing between points was 1 µm. As DNA damage occurred in the cell nucleus, XRCC1 repair protein formed foci at the damage sites. Following irradiation, multiphoton microscope z-stack imaging of the GFP concentration in the nucleus revealed the crown pattern in the irradiated cell nucleus. This 3D image was processed with the deblurring program AutoQuant and the image size is 58.1 µm wide by 55.8 µm high.
Track Segment Charged-Particle Irradiator
Because the beam is vertical, the track-segment facility can irradiate attached cells growing in dishes filled with culture medium. The dishes are 3.5 cm i.d. stainless steel rings with 6 micron thick Mylar epoxied onto them. The vertical beam passes through a thin metal foil into the atmosphere, through the Mylar dish bottom, and irradiates the sample attached to the Mylar. A slot-shaped aperture approximately 6 mm wide defines the beam irradiating the samples. A stepping motor rotates a wheel containing up to 20 dishes at a rate defined by the desired dose and the instantaneous beam current striking the beam-defining aperture. Each point on a dish passes through the beam in about 25 steps. A heated enclosed wheel to which humidified gas can be supplied is available for oxygen enhancement ratio (OER) or fractionation experiments.
In track-segment irradiation, an initially monoenergetic beam of charged particles passes through the thin sample (~10mm) so that the same segments of the particle tracks are deposited in all the material of interest. Particles with linear energy transfer (LET) between 10 and 200 keV/mm are available utilizing beams of protons, deuterons, helium-3, and helium-4 ions.
Neutrons are generated at RARAF using nuclear reactions in thin targets and thus are essentially monoenergetic, in contrast to neutrons generated by reactors or by high-energy deuterons bombarding beryllium targets. RARAF's neutron production targets are hydrogen isotopes absorbed into thin titanium coatings on water-cooled copper backings. Monoenergetic neutrons with energies from 15 MeV down to 220 keV are available. Also currently available are low-dose rate lower-energy spectra. As discussed in the section on slow neutrons, even lower-energy neutron beams (<40 keV) are available.
Large numbers of 14 MeV neutrons can be produced using the T(d,n)4He reaction. The neutron energy, fluence, and dose rate are nearly independent of the angle so that planning irradiations and designing fixtures to hold samples are relatively easy. A significant fraction of the energy deposited in tissue by 14 MeV neutrons is from alpha particles and heavy-ion recoils. Approximately 70% of the energy deposition is from proton recoils.
Facilities Available while Using the Beams
- Cell Culture
- Skin Models
- C. elegans
- Zebrafish and Medaka
- Mouse Ear
- Equipment for Biological Assays
- Microbeam Online Imaging
- Offline Imaging
- Multi-Photon Imaging
- Irradiation Response
- Capillary Electrophoresis
- Optoelectronic Manipulation
- Cell Sorter
- Cell Dispenser
Application Process for Service-Based Beam Time
RARAF invite prospective service-based users to submit proposed experiments. We ask that prospective users discuss the proposed experiment with the RARAF Chief Physicist, Gerhard Randers-Pehrson, at 914-591-9244 or at firstname.lastname@example.org.
All applicants for service-based beam time should fill out a Service Experiment Request Form, which is available online: Service Request Form. Service-based applications are promptly evaluated, generally by the RARAF staff. When approved, beam time will be conveniently scheduled on a month-to-month basis.
Initiating Collaborative-Based Research
Collaboration requests should be initiated through direct discussions between the principal investigators. Appropriate contacts are David Brenner, RARAF Director (212-305-5660, email@example.com), or Gerhard Randers-Pehrson, RARAF chief physicist (914-591-9244, firstname.lastname@example.org), though initial contact with any member of the RARAF team is welcome.
After discussions, we will ask you to document your request, which will be reviewed by the RARAF staff, and also by the RARAF External Advisory Committee.