RARAF – Microbeam

The use of micro-irradiation techniques in radiation biology dates back to the 1950’s to the work of Zirkle (1957) and Munro (1961). However, we are now able to take advantage of recent developments in particle delivery, focusing and detection, image processing and recognition and computer control, coupled with the benefits of new assays of individual cellular response.

In recent years, several groups have developed charged-particle microbeams, in which cells on a dish are individually irradiated by a predefined exact number of alpha particles, allowing the effects of exactly one (or more) alpha particle traversals to be investigated. Biological interest in the microbeam stems from the potential to define the ionizing energy absorbed by a cell, in terms of space, time, and number:

• The microbeam allows irradiation of many cells, each in a highly localized spatial region, such as part of the nucleus, the cytoplasm, or through the immediate neighbor cells of a given cell. Learn more here.

• The microbeam also allows particles to be passed through a cell with a known temporal separation, to investigate, for example, the dynamics of cellular repair. Learn more here.

• Microbeam techniques can deliver exactly one or more particles per cell. Learn more here.

Microbeam Specifications

	A schematic of the microbeam system.

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 an x-ray microbeam.

• Learn more about ongoing upgrades to our microbeam facility here.

• Read select peer-reviewed articles about microbeam irradiation available online here.

Details about the microbeam facility including descriptions of the hardware, control program, and the various protocols available are presented by Randers-Pehrson et al. Click here to read.

Electrostatic Lens Focusing System

There are several basic reasons we are using electrostatic lenses to focus our microbeam:

• We need a small diameter beam, with no halo of scattered particles.

• Electrostatic lenses do not have the hysteresis inherent in magnetic lenses, allowing easy change between differing LET beams.

• Stable voltage is more readily achieved than stable current can be in magnetic systems.

• The focal properties of electrostatic lenses depend only on the accelerating potential.

In terms of electrostatic lens design, we have adopted “Russian symmetry” which insures a circular beam spot. Specifically, the lens strengths are +A,-B,+B,-A for a quadruplet and (+A,-B,+C), (-C,+B,-A) for a double triplet that will focus the particle beam to a spot 0.5 µm in diameter (presently 0.8 µm). One of the main features of the multiplet lens design being used is that part of the alignment of the electrodes is accomplished by using four 1-cm diameter ceramic rods 30 cm long for the entire set of quadrupoles. Evaporating a thin layer of gold onto the entire cylindrical surface in bands creates the positive and negative electrodes. The pole lengths were selected such that the operating voltage on each electrode would be roughly equal - to ensure no “weak links”. Alignment of electrodes relative to each other is “guaranteed” by their one-piece construction.

We first designed and built an electrostatic quadrupole quadruplet lens. Our goal was to optimize the design of the electrodes, and to assess and understand any differences between the results of our optics calculations and measurements on the lens.

As illustrated in the Figure (right), there has been a considerable evolution in the design of the insulated sections of the electrodes to achieve our target voltage of 15 kV:

1st:   Plain insulators were used, with electrode defining gaps
2nd: We added grooves to increase the leakage path
3rd:  We added ion implantation to better define the resistance gradient

In our final design, we have adopted a double triplet system, whereby the lens consists of two electrostatic quadrupole triplets, each 0.3 m long, separated by 1.9 m. One element is below the floor of the microbeam laboratory, and the other is at the table level. A Mathematica model of focusing in the final design is shown here (below).

The top triplet lens was installed, tested, and in use for more than a year. It produced a beam spot with a diameter of ~2 µm. The single lens has been replaced by a pair of lenses identical to the single lens. While adjustments in the alignment of the two lenses and the voltages on their elements are still being made, we have achieved a sub-micron beam with a beam spot of ±0.4µm for 6.0 MeV He ions.

 

Irradiation Protocols

Mouse fibroblast cells, attached to the polypropylene surface of the mini-well, as detected by the automated microbeam image analysis system. The scale bar represents 7 µm.

Individual nuclei are identified and located with an optical image analysis system, which detects the fluorescent staining pattern with 366 nm UV light. For each dish, a computer/microscope-based image analysis system first automatically locates the positions of all the cell nuclei on the dish. A typical example of some imaged cells is shown left.

The cells shown were stained with a very low concentration (50 nM) of Hoechst 33342 fluorescent dye, which is preferentially taken up in the cellular nucleus, and with orange fluorescent tetramethylrhodamine, which is preferentially taken up in the cytoplasm. This second stain is used when microbeam irradiation of the cytoplasm only is required.

In the standard irradiation protocol each cell is identified and located using an image analysis system. The coordinates of the cells are stored in a computer, and the cell dish is then moved under computer control such that the centroid of each cell nucleus (marked by the image analysis system as a cross) in the dish is in turn positioned over a shuttered, highly collimated beam of charged particles. The nucleus of each cell is exposed to a predetermined exact number of alpha particles, and a particle detector above the cell signals to close the shutter of the accelerator when the desired number of particles (e.g., 1 or 2) are recorded. After this the next cell is moved over the beam and the process is repeated.

In addition to this standard protocol, we have developed and used several other irradiation protocols. In one of these new protocols, developed to irradiate the cytoplasm of each cell, the image analysis system defines the long axis of each cell, after which the computer system delivers particles at two target positions, 8 µm away from each end of the cell nucleus. In these experiments, an exclusion zone around each fluorescent object (even if it is not identified as a nucleus) is automatically generated to ensure that the target positions from one nucleus are not accidentally within the nucleus of an adjacent cell. Wu et al have reported mutation induction by cytoplasmic irradiation using this technique.

All cells in a dish need not be irradiated, and we are using two such variants of the standard protocol to study the bystander effects. In the first variant, described by Zhou et al, all of the cells are imaged, but the computer randomly irradiates only a chosen fraction of them. A second approach is to have a mixture of cells growing in the irradiation dish, but to have only a fraction of them stained with Hoechst 33342 and therefore visible to the image analysis system. The other cells might be stained with a dye of another color so they can be distinguished during later analysis. This technique is used by Geard et al.
 

Imaging and Ion Detection

	Online microscope with stage.

Prior to irradiation, an area ~ 3 mm in diameter is scanned by taking overlapping frames using a 10x objective on the microscope. Each frame is examined for potential cells or groups of cells and the locations of these are stored.

During irradiation, a 40x objective is used to image the cells again in overlapping frames to improve the resolution of the positions of the cell nuclei and to more readily separate cell nuclei that are close together. Cells to be irradiated are moved to the beam position using a combination of a high-resolution three-axis piezo-electric inner stage (Mad City Labs, Madison, WI) with a limited range and a motor-driven outer stage with a larger range but poorer accuracy. The X-Y motions of both the microscope stages are controlled by the Microbeam computer. The specified number of particles is admitted through the beam shutter. The charged particles are detected after passing through the cell using a gas-filled proportional mounted on the 40x objective. The counter has a transparent end window so that the cells can be observed continuously. The beam shutter is a fast electrostatic deflection system allowing the irradiation of each cell nucleus to be quickly terminated after the specified number of particles has been detected, then the next cell moved to the beam position.

The overall spatial precision of the beam, including positioning and beam spread, is about ±0.5µm. Based on our measurements of the morphometric characteristics of exponentially-growing human fibroblast cells, using Monte-Carlo simulations we estimate that the particle beam would miss the targeted nucleus at a rate of < 0.5%.

	The microbeam microscope objectives and stage. The 10x objective is on the left, the 40x objective with the gas proportional counter mounted on it is on the right. The stage (black) is visible at the bottom.		The interior of the Microbeam gas proportional counter. The collector wire and helical grid are seen just below the center, where the window area is located.

Irradiation of Cells in Precise Spatial or Temporal Locations

Whilst it is generally true that radiation is an excellent and commonly used probe of cellular damage and repair mechanisms, such studies normally lack the ability to target specific components of a cell. This is however achievable with a microbeam. A controlled amount of energy can be placed, via a charged particle, through a sub-cellular component, rather than via spatially random depositions. In this way the effect on gene expression, cell cycle signaling, DNA damage and repair, lesion interaction, chromosomal changes, and on later mutagenic and carcinogenic change can be evaluated.

Spatially, multiple particle traversals can be placed intra-nuclearly, intra-nuclearly at the nuclear margins, perinuclearly in the cytoplasm, and at other cytoplasmic locations. Thus the microbeam provides a probe with a spatial precision lacking from all other sources of ionizing radiation. This is particularly pertinent for examining mechanisms of chromosome aberration formation, and of early gene responses to spatially precise energy deposition.

As an example, the effect of cell-to-cell communication is becoming of increasing interest in understanding damage processing mechanisms. Several investigators (e.g., Nagasawa and Little 1992, Hickman et al 1994, Deshpande et al 1996) have presented convincing evidence of scenarios where more cells exhibited radiation damage than were estimated to have had alpha particle tracks actually pass through them. Their conclusions were that cells are exhibiting the effects of a radiation track passing through a neighbor (or near neighbor) cell, via extracellular signaling responses. If this conclusion is true, it would have wide ranging consequences. Clearly a microbeam can address this central issue directly, for example by irradiating the neighbors of particular cells, but not the particular cells themselves, after which the non-irradiated cells can be assayed. Such an approach is unattainable with any conventional means of cell perturbation.

As well as evaluating the consequences of precise numbers of events with or without precise placement, the timing between energy deposition events can be altered such that the kinetics of interactional repair processes can be probed. Also given the capacity of the microbeam to record and return to individual cellular positions, timing between particle irradiations can range from microseconds to periods within one phase of the cell cycle, between phases of the cell cycle, and even between parent-progeny over cellular generations.
 

Irradiation with an Exact Number of Particles

Microbeam techniques are necessary to elucidate the biological effects of exactly one particle because, due to the random (Poisson) distribution of tracks, this cannot practically be simulated in the laboratory using conventional broad-field exposures. Microbeam techniques can overcome this limitation by delivering exactly one (or more) particle per cell nucleus. This is important, since at the low doses of relevance to environmental radiation exposure, individual cells only rarely experience traversals by an ionizing particle, and almost never experience more than one traversal. For example, in the case of radon, which dominates the radiation exposure of the general public, radon risk estimation involves epidemiological studies of uranium miners. The average lifetime radon exposure of these miners is sufficiently high that risk estimates are driven by data on miners whose target bronchial cells are subject to multiple alpha particle traversals. On the other hand, for an average house occupant, about 1 in 2,500 target bronchial cells will be exposed per year to a single alpha particle, but less than 1 in 10⁷ cells to traversals by more than one particle. Therefore, in order to extrapolate from miner to environmental exposures, it is necessary to be able to extrapolate from the effects of multiple traversals to the effects of single traversals of a particles.

To see how one might predict the effects of single particles consider an experiment designed to measure the effects of single a particles traversing, say, C3H10T½ cells. Assuming an LET of 150 keV/µm, at a low practical dose of about 0.1 Gy, on average each cell nucleus will be traversed by a single alpha particle. However, as the number of traversals of a given cell is Poisson distributed, about 26% of the cells will be traversed by more than one particle, about 8% by more than two, and about 2% by more than three a particles. But we are interested in the effects of exactly one alpha particle.

Approximate number of cells (~10³) exposed to different numbers of a particle traversals in the bronchial epithelium of a) a Colorado uranium miner (averaged), b) an average environmentally-exposed person, and c) in a 0.1 Gy in vitro experiment (from Brenner, 1989).

The most direct solution to this problem is the use of a microbeam which can deliver exactly one particle to a cell. True single-particle irradiations should thus allow measurement of the effects of exactly one alpha particle traversal, relative to multiple traversals. The application of such systems to low frequency processes such as oncogenic transformation depends very much on the technology involved. With an irradiation rate of at least 5,000 cells per hour, experiments with yields of the order of 10^-4 can practically be accomplished.

See also Do Low Dose-Rate Bystander Effects Influence Domestic Radon Risks?

Information:
How To Make Polypropylene µ-Beam Cell Dishes
How to Plate Cells for Microbeam Experiments

Site developed by CE, page last modified by JL on August 19, 2009 .

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