RARAF –
Milestones
Our current single-particle microbeam system features an extensively upgraded spatial resolution of 0.6 μm (diameter) for the beam. Complementing this, point and shoot targeting system allows us to move the beam instead of the sample for faster irradiations.
Our multiphoton microscope is online to image 3D tissue samples and small organisms.
Installation of our laser ion source provides more densely-ionizing radiations, i.e., heavier ion beams, with stopping powers from 10 to 4,500 keV/μm, compared with our current beams of H and He ions, which have stopping powers from 10 to 200 keV/μm. A secondary electron ion microscope is available to assess beam quality.
We have added x rays to the list of microbeams we can produce.
Photograph of the Point and Shoot deflector coil.
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.
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.
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.
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.
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.
The diameter of the beam spot for the electrostatic focusing system has continued to decrease with tuning of the focusing systems and development of new lenses, as can be seen in the table below.
The most recent development has been the installation of a compound quadrupole triplet. Two identical triplets 2.20 meters apart are organized in Russian symmetry. Their combined demagnifications have produced our first sub-micron beam spot! The process of adjusting the alignment of the lenses and tuning of the lens voltages is continuing with the aim of obtaining a beam spot diameter of 0.5 µm.
Years
Type of beam
Diameter (µm)
2001
Focused - single quadrupole quadruplet
11
2002-2003
Focused - single quadrupole quadruplet
3
2004-2005
Focused - single quadrupole triplet
3.5
2006
Focused - single quadrupole triplet
2.0
2007
Focused - single quadrupole triplet
1.3
2008
Focused - compound quadrupole triplet
0.8
Multiphoton microscopy, a laser-based, 3D imaging technique is now integrated into the Microbeam II endstation at RARAF. The multiphoton microscope was custom-designed around the Nikon Eclipse E600-FN research fluorescence microscope at the endstation and is intended for imaging cell dynamics in tissue and cell-culture samples following irradiation. Intended for detecting and observing short-term molecular kinetics of radiation responses in living tissue and in cell-culture samples, this multiphoton microscope is the first of its kind to be assembled onto a microbeam cell-irradiation platform.
Multiphoton excitation is a phenomenon where multiple photons, coincident in time and space, can act like a single photon having a superposition of energies from the incident photons. With two photon excitation, for example, two incident photons at a laser focal point can act like one photon of half the wavelength. A Chameleon Ultra II (Coherent Inc.) with a 680-1080 nm wavelength range is the light source for the multiphoton microscope at RARAF. These infrared wavelengths are less photo-damaging and have greater penetration depth than single-photon excitation. At this laser's focal point, the two-photon process effectively provides 340-540 nm. These characteristics support exciting fluorophores, including RFP, at the laser focal plane, and when the imaging technique is coupled to a precision xyz-stage, optical sectioning in 3D tissue samples is possible.
Figure 1. Multiphoton microscope at RARAF for observing responses in cell cultures and in tissues after particle irradiation.
Pictured in Figure 1, the setup of the multiphoton microscope at RARAF includes a scanning laser source that is built into the endstation microscope. The laser path starts at the Ti:S laser, which is bolted to the optics table. The light path proceeds through a variable attenuator and a fast shutter before being directed vertically along the rotation axis of the microscope's pivot mount, which is used to switch between online and offline positions. A beam expander increases the beam to a size that will over-fill the back aperture of the objective lens. The platform attached to the microscope mount accommodates the scanning optics. These optics components point along an axis into the side of a custom-built extension tube for the CCD camera that is used during fluorescence microscopy. A pivoting mirror inside the extension tube selects between fluorescence microscopy or multiphoton microscopy. For multiphoton microscopy, this mirror guides the laser vertically down through both the microscope tube lens and objective lens.
Fluorophores in a sample absorb the laser light through multiphoton excitation at the laser focal point. As these markers fluoresce, signal light entering the objective is deflected towards photomultiplier detectors (H5783, Hamamatsu). Shown in figure 2 (below), signal detection is accomplished with photomultiplier tubes that are mounted to the side of the filter cube changer on the microscope. Control software monitors the correlation between the laser scan position and signal at the photomultiplier tube to produce the images.
An emission filter placed before a photomultiplier tube designates the wavelength range (window) of detected signal. With multiple fluorophores, color images can be produced using appropriate dichroic mirrors and emission filters. In figure 3 (below), the image of bovine pulmonary artery endothelial cells (FluoCells prepared slide #1, Molecular Probes) is an example of a color image produced by wavelength-specific detection. The cells in this image were stained with BODIPY FL phallacidin to label the filamentous actin (F-actin) and were counterstained with DAPI to label the nucleus. The objective lens used for imaging these cells was a Nikon CFI LU Epi Plan Fluor 50X objective, with 0.80 numerical aperture and 1.0 mm working distance. To develop this image, 20 to 30 single frames were acquired per color at a rate of 2 seconds/frame. A routine written in MATLAB® subsequently summed and combined the images into a 3D matrix for color display.
Figure 2. Fluorescent signal emitted by the sample is deflected by a dichroic mirror and collected by photomultiplier tubes
Figure 3. Multiphoton microscopy image of bovine pulmonary artery endothelial cells. (FluoCells prepared slide #1, Molecular Probes). Cell nuclei diameter ~10 microns.
With the attachment housing for two photomultiplier tubes, experiments using fluorescence resonance energy transfer (FRET) are possible. FRET optics sets are on hand for the following transitions: BFP > GFP, CFP > YFP, and Alexa 488 > Alexa 594. FRET experiments concurrent with cell irradiation are examples of monitoring post cell-irradiation dynamics using the multiphoton microscope at RARAF.
Particle-Induced Foci Formation
One of our collaborators (David Chen, University of Texas Southwestern, Dallas, TX is using the microbeam to examine particle-induced single strand breaks in DNA within single cells. They are using human HT1080 fibrosarcoma cells that have been transfected with a GFP-tagged XRCC1 DNA single-strand break repair protein. Figure 2 shows multiphoton images of one cell a) before irradiation and b) 4 minutes after exposure to 400 3-MeV alpha particles at a predetermined position within a cell nucleus. Four minutes after irradiation, the region of enhanced GFP signal corresponds to the XRCC1 focus and the irradiation position. In this example, ion-beam targeting was deliberate to avoid areas deficient in GFP (likely nucleoli). This clear response demonstrates that the multiphoton microscope was successful at recording high-resolution, time-lapse images of particle-induced focus formation within a single cell in real time. As an additional capability, projections of 3D z-stacks can quantify focus track volume to correlate with the amount of radiation (number of particles) delivered to the cell nucleus.
The system is fully operational.
For more detail see:
Microbeam-integrated multiphoton imaging system. Bigelow AW, Geard CR, Randers-Pehrson G, and Brenner DJ. Rev. Sci. Instruments 79:123707 (2008).
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 90° 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.
Magnetostatic Lens
The design of the compound lens, used to focus the beam, is based on the one used for our electrostaticly 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.
Beam Tests
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 (A) and quadrupoles 2 and 5 at the same strength (B). The figure shows the theoretical and measured spot size and shape at the end station. 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.
For more details see:
Testing the stand-alone microbeam at Columbia University. Garty G., Ross G.J., Bigelow A., Randers-Pehrson G. and Brenner D.J. Radiat. Prot. Dosim. 122:292-296, 2006.
A single-particle / single-cell microbeam based on an isotopic alpha source. Ross G.J., et al. Nucl. Instrum. Meth.B. 231:207-211, 2005.
A microbeam irradiator without an accelerator. Garty G., et al. Nucl. Instrum. Meth. B 241:392-396, 2005.
Immersion Mirau Interferometry
An immersion-based Mirau interferometric (IMI) objective has been designed and built to function as an immersion lens with standard interferometric techniques using a short coherence length and to otherwise accommodate the endstation requirements for the Columbia University microbeam at RARAF.
To enable interferometry, we used high precision glass for the beamsplitter and the mirror. The surfaces interacting with the medium have a protective layer on top of the coating.
The new attachment has been tested as a water immersion lens. We confirmed that the two equal-arm light pathways are indeed restored and then provide interference fringes in the environment with sufficient contrast to perform the biological experiments. The acquired images are comparable to those that can be obtained using the commercial attachment that we used for preliminary testing.
Diagram of the immersion-based Mirau lens
The new objective will support rapidly and automatically locating the cell nuclei without stain. The "banding" in some of the examples shows the presence of both a slow drift in z-position and some very slight vibrational interference, due to the requirement that interference patterns needed to reproduce the image are acquired at different times. Even very low amplitude vibrations, if they exist, may prevent maintaining the desirable phase shifts between consecutive frames. One solution is to acquire all interference images at the same time, presently being developed at RARAF.
Custom-built interferometric attachment with an additional window unit
Left: IMI image of normal human dermal fibroblasts taken with the interim immersion-Mirau lens and a neutral density microscope cube. Right: Same sample with a UV cube to reveal the nuclear stain.
For more details see:
Phase-based cell imaging techniques for microbeam irradiations. G.J. Ross , et al. Nucl Instr Meth B 241: 387-391 (2005)
The standard RF ion source that is used on our 5 MV HVE Singletron particle accelerator ionizes atoms from the gas phase and is suitable for experiments involving the irradiation of cells with protons, deuterons and alpha particles having linear energy transfers (LETs) from 10 to 200 keV/μm. To extend the upper LET range of our experiments, ions as heavy as iron (Table 1) are required with high enough energies to have usable particle ranges ≥ 20 μm. Because we are limited to a terminal voltage of 5 MV, highly charged initial heavy ions are needed to obtain the required energies. Laser ion sources, in which a high-power, pulsed light beam is focused to micron dimensions, resulting in power densities on the order of 1012 W/cm2, can produce high initial charge-states of heavy ions in sufficient fluxes for microbeam experiments.
Table 1. Potential Ion Beams for Microbeam Irradiations
Ion Atomic Number Mass
(amu)Ion Energy
(MeV)Min. Charge State for Acceleration Terminal Potential
(MV)Stopping Power
(keV/μm)Range
(μm)p 1 1 5.00 1 5.00 8.4 340 d 1 2 1.20 1 1.20 40 20 He 2 4 10.00 2 5.00 59 105 Li 3 7 15.00 3 5.00 148 68 Be 4 9 15.00 3 5.00 310 37 B 5 11 15.00 3 5.00 515 25 C 6 12 20.00 4 5.00 605 28 N 7 14 20.00 4 5.00 845 22 O 8 16 25.00 5 5.00 955 24 Al 13 27 40.00 8 5.00 1960 22 Cl 17 35 55.00 11 5.00 2760 22 Ti 22 48 70.00 14 5.00 3925 23 Fe 26 56 75.00 15 5.00 4780 22
Our laser ion source (LIS) is based on laser ablation. High-power Nd:YAG laser pulses can enter the accelerator tank through a window, pass through the SF6 insulating gas, and enter a vacuum region through an optical port to deliver a focused, high-power-density pulse onto a solid target. Ions created during an ablation event’s energy transfer are ejected in the form of a plasma plume in a direction that is preferentially normal to the solid target surface. These ions are emitted with a distribution in charge state, kinetic energy, and angle. Following a drift distance for plasma expansion, a spherical electrostatic analyzer (ESA) selects ions with a particular energy per charge and focuses them to a point coinciding with the 3.18-mm entrance aperture of the accelerator tube. The LIS is pictured in a table-top configuration left.
To maximize the ion flux, a Quanta-Ray LAB-190-100 Nd:YAG pulsed laser system from Spectra Physics was chosen for its high repetition rate (100 Hz) and high power (325 mJ/pulse, 10 ns Q-switch pulse duration). A focusing lens (f = 12.2 cm) for the laser is mounted on a holder inside the vacuum system and the focal spot diameter (61 μm) ideally provides a focused power density of 2.2 x 1012 W/cm2. A 2 mm thick piece of AR-coated, BK7 glass is used as a lens protector to inhibit target spatter from accumulating on the focusing lens. The ablation target is a cylindrical solid attached to a differential drive manipulator. The assembly drawing for the Columbia University LIS is shown below.
Ions generated from the laser ablation process have a wide distribution in charge state and energy. To select a particular ion charge state and energy, the plasma first expands through a drift distance of 70 cm and then enters a 24-degree spherical ESA that was designed and built in-house. This ESA is a double-focusing element with fringe field shunts and interchangeable entrance and exit apertures. A photograph of our spherical ESA is shown below. The ESA is a self-contained module mounted on a six-inch flange. An electrostatic field within the ESA selects ions by energy/charge. So for each pulsed, laser-ablation event, the ions that pass through the ESA can be characterized by a time-of-flight (TOF) spectrum. The highest charge-state ions have the highest energy and arrive at the accelerator entrance aperture first, while the lowest charge-state ions arrive last.
Several considerations were made to tailor-fit the LIS footprint into the Singletron machine. Auxiliary power available at the Singletron terminal is limited, but adequate, for a non-evaporable getterer (NEG) pump to maintain proper vacuum conditions within the target ablation area. Additionally, it is crucial to have a match between the ion source emittance and the acceptance of the Singletron accelerator tube. In collaboration with HVE, appropriate modifications were made to the primary accelerator tube sections to optimize the transmission of the LIS-produced ions and also to conserve the function of the primary RF ion source incorporated on the Singletron.
For more details see:
Laser ion source for the Columbia University microbeam. (Nucl. Instrum. Meth. B 241:874-879, 2005)
Secondary Electron Ion Microscope
As we improve the spatial characteristics of the microbeam system, it becomes increasingly important to be able to assess the beam quality, in order to adjust the system to its optimum capabilities. For this purpose we are developing a secondary electron ion microscope (SEIM). This device will enable us to measure the beam profile and position, in real time, with sub-micron resolution.
The SEIM design is inspired by the common technique of photoelectron microscopy (PEM). It is based on conversion of incident projectiles using a secondary electron emitting (SEE) film, generating one (or more) electrons per projectile. The ejected electrons are then imaged, using an electrostatic unipotential lens, forming a 500 times magnified image on an image intensified CCD This high magnification enables 100 nm resolution using a 50 μm resolution CCD.
In order to overcome the chromatic and spherical aberrations inherent in the electrostatic lens, the electrons are bent by a 45ş angle, reflected by an electrostatic mirror and bent by an additional 45ş before reaching the detector. This “folded” design of the SEIM is a novel one, developed at RARAF.
From systematic simulations we have found the “ideal” SEIM configuration (a 150° mirror biased at 1.5 V with respect to the SEE film as well as the required lens voltage as a function of mirror placement), giving a single-electron resolution of 300-500 nm and single electron transport efficiency of 55%-80%, depending on the mirror bias. This should be compared with ~1% for “straight” geometries utilizing an aperture to eliminate energetic electrons). We expect that this resolution will improve as the square root of the electron yield which is expected to be higher than unity for heavy/energetic ions, where the SEIM is needed most.
Preliminary tests, using an “unfolded” SEIM, consisting of the electrostatic lens and the electron detector but without the magnet and mirror agree well with simulations. For these measurements we replaced the SEE foil with a quartz window on which a micron scale pattern of aluminum was evaporated. The pattern was illuminated with low intensity UV light and the resulting photoelectrons were imaged. A comparison of the optical- and electron-based images of the pattern are shown on the right. The width of the spot edge allows us to estimate the SEIM resolution at 4.3 μm RMS. This is in good agreement with the simulated prediction of 4-5 μm. The predicted magnification (16x) is also in good agreement with the measured 20x.
For more details see:
Development of a secondary-electron ion-microscope for microbeam diagnostics. Garty G., Randers-Pehrson G. and Brenner D.J. Nucl. Instr. Meth. B. 231:60-64 (2005).
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