**2.1. Microbeam**

For low-dose radiation circumstances which are extremely relevant to the environmental radiation exposure, individual cells hardly experience traversals by radiation particles. The biological effects of such low-dose radiation are unknown, and it is very difficult to study with conventional broad radiation beam exposure due to Poisson distribution of tracks. Also the cell-to-cell communication cannot be addressed directly because of the random radiation of broad beam particles. The microbeam system can overcome these difficulties. Microbeam is an irradiation system which delivers a certain number of particles with a micron-sized diameter spot to a chosen biological target, which allows damage to be precisely deposited within specific locations (e.g., nuclei or cytoplasm of single cells). Charged particle microbeams have been significant contributions to defining the biological targets of ionizing radiations. Also with microbeam, cell nuclear and cytoplasmic responses of targeted cells are established along with the responses of non-targeted bystander cells [1]. There is evidence that radiationinduced bystander signals between cells may originate with a diffusible mediator [2–4].

For more than 15 years, the Radiological Research Accelerator Facility (RARAF) of Columbia University has built and operated a charged particle microbeam facility capable of irradiating the nuclei of individual biological cells with as few as one helium ion or proton [5, 6]. The RARAF microbeam II is an updated system, and it is driven by a 5.5 MV HVEE Singleton Accelerator. The particles are ionized by a radio-frequency (RF) ion source inside the Singleton Accelerator and are accelerated from a DC high-voltage terminal to reach the desired energy. Then, the particle beam passes through a beam transport system to the beam end station where irradiation experiments take place. The beam transport system includes a few beam manipu‐ lation elements (**Figure 1**). The main beam slits and beam stop are used to eliminate unwanted ion beams, to limit the size of the beam entering a 90° bending magnet, and to stop the beam from entering the bending magnet when irradiations are not expected. The magnetic steering magnet is used to make fine adjustments, aiming the ion beam at the entrance of the bending magnet. The 90° bending magnet is used to bend the beam into the vertical direction. The beam deflector/shutter is an electrostatic system that steers the beam rapidly (~1 ms) to end the irradiation of a cell. The object aperture (~30 μm diameter) limits the initial beam size. A custom built electrostatic double-quadrupole triplet system [7] is used to focus the beam at the position of the cells to be irradiated. In front of the first triplet lens, an angular limiting aperture is used to eliminate particles entering at large angles to help reduce the diameter of the beam spot. The beam exit window is a thin silicon nitride foil attached to a stainless steel disk, with thickness of either 500 nm or 100 nm (for a sub‐micron beam spot) to minimize scattering the beam. The cell dish holder is designed to locate cells at the focusing plane of the microbeam and is in the view range of a customized microscope. An imaging/targeting system comprises a precision XYZ stage (MadCity Labs, Inc. WI), a Nikon Eclipse E600 microscope, and an attached PhotonMAX-512B EMCCD camera (Princeton Instruments, NJ). This equipment combination also allows us to image the wide range of fluorescent proteins that have been developed. A computer control program written in Visual Basic locates the cells, plated in a cell culture dish, and positions them for irradiation. While the RARAF microbeam is primarily used for charge particle irradiation, the accelerator can be used as source for neutral particle radiation, for example, neutron radiation [8–10].

**Figure 1.** RARAF microbeam.

degenerative diseases. With the dramatic increase in exposure of the human population to lowdose radiation either from diagnostic procedures, industrial applications, mining, cleanup of contaminated sites, and space travel, there has been a great scientific need for better estimates of the risks to such exposures. One of the driving forces behind the low-dose radiation re‐ search is developing specialized radiation technology and novel, versatile biophysics tools for

For low-dose radiation circumstances which are extremely relevant to the environmental radiation exposure, individual cells hardly experience traversals by radiation particles. The biological effects of such low-dose radiation are unknown, and it is very difficult to study with conventional broad radiation beam exposure due to Poisson distribution of tracks. Also the cell-to-cell communication cannot be addressed directly because of the random radiation of broad beam particles. The microbeam system can overcome these difficulties. Microbeam is an irradiation system which delivers a certain number of particles with a micron-sized diameter spot to a chosen biological target, which allows damage to be precisely deposited within specific locations (e.g., nuclei or cytoplasm of single cells). Charged particle microbeams have been significant contributions to defining the biological targets of ionizing radiations. Also with microbeam, cell nuclear and cytoplasmic responses of targeted cells are established along with the responses of non-targeted bystander cells [1]. There is evidence that radiationinduced bystander signals between cells may originate with a diffusible mediator [2–4].

For more than 15 years, the Radiological Research Accelerator Facility (RARAF) of Columbia University has built and operated a charged particle microbeam facility capable of irradiating the nuclei of individual biological cells with as few as one helium ion or proton [5, 6]. The RARAF microbeam II is an updated system, and it is driven by a 5.5 MV HVEE Singleton Accelerator. The particles are ionized by a radio-frequency (RF) ion source inside the Singleton Accelerator and are accelerated from a DC high-voltage terminal to reach the desired energy. Then, the particle beam passes through a beam transport system to the beam end station where irradiation experiments take place. The beam transport system includes a few beam manipu‐ lation elements (**Figure 1**). The main beam slits and beam stop are used to eliminate unwanted ion beams, to limit the size of the beam entering a 90° bending magnet, and to stop the beam from entering the bending magnet when irradiations are not expected. The magnetic steering magnet is used to make fine adjustments, aiming the ion beam at the entrance of the bending magnet. The 90° bending magnet is used to bend the beam into the vertical direction. The beam deflector/shutter is an electrostatic system that steers the beam rapidly (~1 ms) to end the irradiation of a cell. The object aperture (~30 μm diameter) limits the initial beam size. A custom built electrostatic double-quadrupole triplet system [7] is used to focus the beam at the position of the cells to be irradiated. In front of the first triplet lens, an angular limiting aperture is used to eliminate particles entering at large angles to help reduce the diameter of the beam spot.

such radiation biology and radiation physics studies.

**2. Single-cell microbeam irradiation**

**2.1. Microbeam**

112 Radiation Effects in Materials

#### **2.2. Single-cell microbeam irradiation with oxygen micro-biosensor**

To physically demonstrate the radiation-induced oxygen flux changes outside the irradiated cell, a self-referencing amperometric electrochemical sensor was proposed for use with singlecell microbeam irradiation experiment. This type of micro-biosensor allows sensitive and noninvasive measurement of the flux of radical mediators, such as oxygen (or nitric oxide), in single cells. This is achieved by repeatedly moving the micro-biosensor/probe tip through the extracellular gradient at a known frequency and a known distance apart. This so-called selfreferencing technique minimizes the measurement challenges caused by the random drift of the sensor output. In conjunction with a microbeam, it has the capacity to accurately detect selected ionic and molecular gradient changes surrounding a single cell with high spatial resolution. The first radiobiology experiment was to analyze metabolic oxygen consumption in individual living lung cell after sub-cellular irradiation and to explore the radiation response of such cells. The self-referencing oxygen electrochemical system was developed at the Bio-Currents Shared Resource at the Marine Biological Laboratory (MBL), Woods Hole, MA. It was integrated with the RARAF single-particle, single-cell microbeam to form a single-cell irradiation response detection platform.
