RARAF –
Milestones
Our current single-particle microbeam system features an extensively upgraded spatial resolution of 0.8 μm (diameter) for the beam. Spatial resolution has been improved by use of our focused lens system and with the aid of our new accelerator.
Our multiphoton microscope is online to image 3D tissue samples and small organisms.
Installation of our laser ion source will provide 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. New state-of-the-art facilities are available for biology.
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 now fully operational. As an example of the use of our multiphoton microscope...
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
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 reliably under 5µm. The PMM is now available as an alternative to the electrostatic microbeam.
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
Immersion Mirau Interferometry (IMI) is a non-stain imaging technique that is based on the principles of phase-shifting interferometry. Several interference images are acquired at different heights with quarter-wavelength phase shifts between their interference patterns. As an interferometric technique, IMI does not require fluorescent staining of the cells, which eliminates potential damage induced by fluorescent stains. It operates at 540 nm (green light), therefore does not induce UV-exposure of the cells. Lastly, immersion mode allows the experimenter to keep the cells in cell medium during the irradiation experiment and minimize cell death due to dehydration.
To facilitate the immersion operation mode, a custom Mirau interferometric attachment is required. Such attachment has been designed and built at RARAF to function as an immersion lens with standard interferometric techniques using a short coherence length.
Custom-built interferometric attachment. The shape is designed to fit the microbeam dish. Two adjustment screws regulate the inclination of the optics with respect to the sample. Aluminum surface is anodized to protect corrosion by the medium. The optics holders are interchangeable. Also attached to the objective is the holder for capacitive sensor.
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.
IMI image of unstained living fibroblasts.
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.
In case of interferometric imaging, even a fraction-of-a-wavelength shift in the vertical direction lowers the image quality. Thus, despite the fact that the microbeam is mounted on the 27" thick concrete floor, the IMI system is still sensitive to unpredictable low-amplitude vibrations.
A recent development is Simultaneous Mirau Interferometry (SIMI). SIMI is a vibration-independent version of IMI and has shorter image acquisition time. SIMI is an integration of Immersion Mirau Interferometry and polarization microscopy.
Quantitative Phase Imaging
In some of the tests, some cells have been missed and there also have been false positives. In general, the quality of the images can be improved by careful tuning of the parameters for the approximate solution to the transport equation. However, to optimize the system for our regular, automated use, there is still work to be done in eliminating false cells and reducing missed cells. Several variables have been isolated and eliminated as causes: plating time, cell-type, cell phase, light color, cell growth surface, amount of medium (depth), percent of medium vs. buffer, and use of a cover glass.
In the picture the QPI algorithm has been applied to normal human dermal fibroblasts under conditions conducive to rapid automatic cell location.
QPI and IMI have been evaluated in comparison to each other. Under consideration were: processing time, reliability, maintenance and ease of use for the experimenter. QPI is a software solution and does not require introduction of fluids or cleaning, whereas IMI does. QPI does not require any additional equipment, while the immersion-Mirau interferometer requires the end-user to use a custom objective. IMI is sensitive to vibrations unlike QPI. At the same time, QPI is more sensitive to the algorithm parameters, which makes Immersion-Mirau interferometry more reliable and ready to automate than QPI. Work is currently being done on vibration-independent approach for IMI.
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 new biology lab as viewed from the entrance to the third floor laboratory space (upper right in layout figure). In view are the two islands that have work stations for up to 12 people.
In 2006, the Trustees of Columbia University contributed the funds ($1.8M) required to build over 2000 square feet of new laboratory and office space on the third floor of the facility. When RARAF was built in the early 1980s, this space had been intended for offices and a meeting room. Since there were never funds available to build these, the space had been used for storage. Construction began in early April 2006 and is now completed. Over half the area is a biology laboratory with a class II laminar flow hood (and provision for two additional laminar flow hoods), several incubators, refrigerators and freezers, and two islands comprising a dozen work stations. There are also six desks for technicians and postdocs. In addition there is a physics laboratory and a dedicated microscopy laboratory with three workstations.
The layout of the third floor of RARAF. Microbeam lab III (permanent magnet lens system) is directly above the original microbeam laboratory. The wall on the left is ~58 feet long. Microbeam II, which houses the electrostatic lens system, is shown in the bottom, right of the center and is half a floor below the new construction, shown with emphasized walls.
Also included in the construction is a new laboratory for the permanent magnet microbeam facility. While this microbeam system was being developed, it was housed in a makeshift room. The magnetic quadrupole triplets and endstation for the system were removed before construction began. Now that construction is complete, the beam line has been reconstructed with the two triplet lenses, and the endstation with the microscope and electronics has been installed. The lab is fitted with a laminar flow hood and an incubator. The magnet system is being adjusted to provide the best focusing using proton and helium ion beams from the accelerator. When the focusing studies are complete, the system will be available again for biological irradiations.
A 5 MV Singletron (right) purchased from High Voltage Engineering Europa (HVEE) in the Netherlands has replaced our Van de Graaff. Installation of the Singletron was completed at the end of 2005. The main technical advantage of this new accelerator is the amount of voltage fluctuation. Energy fluctuations cause an effect similar to wavelength changes in optical lenses because of “chromatic” aberrations in the lenses. The lower the energy fluctuation, the better the electrostatic lens system is able to focus the beam. Our Van de Graaff used a cloth belt, and charge was carried mechanically to the terminal. The Singletron has no moving parts; the high voltage is generated electronically by a Cockcroft-Walton type power supply and therefore is more stable. It is easier to compensate for electronic fluctuations than mechanical fluctuations. A voltage ripple of 200 V peak-to-peak at 3.75 MV is guaranteed with a design aim of 50 V.
A terminal potential of 5 MV also means that using the laser ion source we can obtain heavy ions with usable ranges using lower charge states or longer ranges (higher energies) at the same charge state. It also allows us to obtain higher-energy proton beams so that LETs below 10 keV/μm are available for low-LET experiments on both the microbeam and the track segment (broad beam) facilities.
The new accelerator has been running biological experiments for over two years. The low fluctuation in terminal voltage has helped in the reduction of the beam spot sizes for the PMM and, especially, the electrostatically focused microbeam. The higher terminal voltage has provided more penetrating beams of He ions and protons, which have been used for irradiation of tissue samples using the Track Segment Facility.
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
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.
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.
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.
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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