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Electrostatic Microbeam

Specifications

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.

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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 focuses the particle beam to a spot 0.5 µm in diameter (almost to the theoretic limit for such
lenses).

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.

img10As 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).

img11The 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

microbeam

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, 8µm 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

microbeam

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%.


microbeam microbeam

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.

 

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

 

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tel: (914) 591-9244
fax: (914) 591-9405
Radiological Research Accelerator Facility Nevis Laboratories
P.O. Box 21, 136 S. Broadway, Irvington, N.Y. 10533