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Airborne-mediated microbial diseases such as influenza and tuberculosis represent major public health challenges. A direct approach to prevent airborne transmission is inactivation of airborne pathogens, and the airborne antimicrobial potential of UVC ultraviolet light has long been established; however, its widespread use in public settings is limited because conventional UVC light sources are both carcinogenic and cataractogenic. By contrast, we have previously shown that far-UVC light (207-222 nm) efficiently inactivates bacteria without harm to exposed mammalian skin. This is because, due to its strong absorbance in biological materials, far-UVC light cannot penetrate even the outer (non living) layers of human skin or eye; however, because bacteria and viruses are of micrometer or smaller dimensions, far-UVC can penetrate and inactivate them. We show for the first time that far-UVC efficiently inactivates airborne aerosolized viruses, with a very low dose of 2 mJ/cm

Measurement of ultraviolet (UV) radiation is important for human health, especially with the expanded usage of short wavelength UV for sterilization purposes. This work examines unlaminated Gafchromic EBT3 film for UV radiation monitoring. The authors exposed the film to select wavelengths in the UV spectrum, ranging from 207 to 328 nm, and measured the change in optical density. The response of the film is wavelength dependent, and of the wavelengths tested, the film was most sensitive to 254 nm light, with measurable values as low as 10 µJ/cm2. The film shows a dose-dependent response that extends over more than four orders of magnitude. The response of the film to short wavelength UV is comparable to the daily safe exposure limits for humans, thus making it valuable as a tool for passive UV radiation monitoring.

A horizontal multi-purpose microbeam system with a single electrostatic quadruplet focusing lens has been developed at the Columbia University Radiological Research Accelerator Facility (RARAF). It is coupled with the RARAF 5.5 MV Singleton accelerator (High Voltage Engineering Europa, the Netherlands) and provides micrometer-size beam for single cell irradiation experiments. It is also used as the primary beam for a neutron microbeam and microPIXE (particle induced x-ray emission) experiment because of its high particle fluence. The optimization of this microbeam has been investigated with ray tracing simulations and the beam spot size has been verified by different measurements.

Airborne-mediated microbial diseases such as influenza and tuberculosis represent major public health challenges. A direct approach to prevent airborne transmission is inactivation of airborne pathogens, and the airborne antimicrobial potential of UVC ultraviolet light has long been established; however, its widespread use in public settings is limited because conventional UVC light sources are both carcinogenic and cataractogenic. By contrast, we have previously shown that far-UVC light (207–222 nm) efficiently inactivates bacteria without harm to exposed mammalian skin. This is because, due to its strong absorbance in biological materials, far-UVC light cannot penetrate even the outer (non living) layers of human skin or eye; however, because bacteria and viruses are of micrometer or smaller dimensions, far-UVC can penetrate and inactivate them. We show for the first time that far-UVC efficiently inactivates airborne aerosolized viruses, with a very low dose of 2 mJ/cm2 of 222-nm light inactivating >95% of aerosolized H1N1 influenza virus. Continuous very low dose-rate far-UVC light in indoor public locations is a promising, safe and inexpensive tool to reduce the spread of airborne-mediated microbial diseases.

Methicillin-resistant Staphylococcus aureus (MRSA) and influenza A virus are two of the major targets for new antimicrobial technologies. In contrast to conventional germicidal lamps emitting primarily at 254 nm, which are both carcinogenic and cataractogenic, recent work has shown the potential of far-UVC technology, mainly between 207 and 222 nm, to be an effective means of sterilization of pathogens without apparent harm to mammalian cells. This is because, due to its strong absorbance in biological materials, far-UVC light cannot penetrate even the outer (non living) layers of human skin or eye; however, because bacteria and viruses are of micrometer or smaller dimensions, far-UVC can penetrate and inactivate them. With this report, we present progress on in vitro tests to inactivate MRSA on a surface using far-UVC light from a laser delivered using an optical diffuser. Qualitative and quantitative results show that this means of far-UVC exposure is adequate to inactivate MRSA with a dose comparable to that which would be required using a conventional germicidal lamp. Also included is a report on progress on inactivation of aerosolized influenza A virus. A custom benchtop aerosol exposure chamber was constructed and used to determine the effectiveness of far- UVC. Results indicate that far-UVC efficiently inactivates airborne aerosolized viruses, with a very low dose of 2 mJ/cm 2 of 222-nm light inactivating >95p of aerosolized H1N1 influenza virus. Together these studies help to further establish far-UVC technology as a promising, safe and inexpensive tool for sterilization in many environments.

Background Prevention of superficial surgical wound infections from drug-resistant bacteria such as methicillin resistant Staphylococcus aureus (MRSA) currently present major health care challenges. The majority of surgical site infections (SSI) are believed to be caused by airborne transmission of bacteria alighting onto the wound during surgical procedures. We have previously shown that far-ultraviolet C light in the wavelength range of 207-222 nm is significantly harmful to bacteria, but without damaging mammalian cells and tissues. It is important that the lamp be fitted with a filter to remove light emitted at wavelengths longer than 230 nm which are harmful. Aims Using a hairless mouse model of infection of superficial wounds, here we tested the hypothesis that 222-nm light kills MRSA alighting onto a superficial skin incisions as efficiently as typical germicidal light (254 nm), but without inducing skin damage. Methods To simulate the scenario wherein incisions are infected during surgical procedures as pathogens in the room alight on a wound, MRSA was spread on a defined area of the mouse dorsal skin; the infected skin was then exposed to UVC light (222 nm or 254 nm) followed by a superficial incision within the defined area, which was immediately sutured. Two and seven days post procedure, bactericidal efficacy was measured as MRSA colony formation unit (CFU) per gram of harvested skin whereas fixed samples were used to assess skin damage measured in terms of epidermal thickness and DNA photodamage. Results In the circumstance of superficial incisions infected with bacteria alighting onto the wound, 222-nm light showed the same bactericidal properties of 254-nm light but without the associated skin damage. Conclusions Being safe for patient and hospital staff, our results suggested that far-UVC light (222 nm) might be a convenient approach to prevent transmission of drug-resistant infectious agents in the clinical setting.

A novel neutron irradiation facility at the Radiological Research Accelerator Facility (RARAF) has been developed to mimic the neutron radiation from an Improvised Nuclear Device (IND) at relevant distances (e.g. 1.5 km) from the epicenter. The neutron spectrum of this IND-like neutron irradiator was designed according to estimations of the Hiroshima neutron spectrum at 1.5 km. It is significantly different from a standard reactor fission spectrum, because the spectrum changes as the neutrons are transported through air, and it is dominated by neutron energies from 100 keV up to 9 MeV. To verify such wide energy range neutron spectrum, detailed here is the development of a combined spectroscopy system. Both a liquid scintillator detector and a gas proportional counter were used for the recoil spectra measurements, with the individual response functions estimated from a series of Monte Carlo simulations. These normalized individual response functions were formed into a single response matrix for the unfolding process. Several accelerator-based quasi-monoenergetic neutron source spectra were measured and unfolded to test this spectroscopy system. These reference neutrons were produced from two reactions: T(p,n)3He and D(d,n)3He, generating neutron energies in the range between 0.2 and 8 MeV. The unfolded quasi-monoenergetic neutron spectra indicated that the detection system can provide good neutron spectroscopy results in this energy range. A broad-energy neutron spectrum from the 9Be(d,n) reaction using a 5 MeV deuteron beam, measured at 60 degrees to the incident beam was measured and unfolded with the evaluated response matrix. The unfolded broad neutron spectrum is comparable with published time-of-flight results. Finally, the pair of detectors were used to measure the neutron spectrum generated at the RARAF IND-like neutron facility and a comparison is made to the neutron spectrum of Hiroshima.

Radiation-induced bystander effects have been observed in vitro and in cell and tissue culture models, however, there are few reported studies showing these effects in vivo. To our knowledge, this is the first reported study on bystander effects induced by microbeam irradiation in an intact living mammal. The mouse ear was used to investigate radiation-induced bystander effects in keratinocytes, utilizing a 3 MeV proton microbeam (LET 13.1 keV/μm) with a range in skin of about 135 μm. Using a custom-designed holder, the ear of an anesthetized C57BL/6J mouse was flattened by gentle suction and placed over the microbeam port to irradiate cells along a 35 μm wide, 6 mm long path. Immunohistochemical analysis of γ-H2AX foci formation in tissue sections revealed, compared to control tissue, proton-induced γ-H2AX foci formation in one of the two epidermal layers of the mouse ear. Strikingly, a higher number of cells than expected showed foci from direct irradiation effects. Although the proton-irradiated line was ~35 μm wide, the average width spanned by γ-H2AX-positive cells exceeded 150 μm. Cells adjacent to or in the epidermal layer opposite the γ-H2AX-positive region did not exhibit foci. These findings validate this mammalian model as a viable system for investigating radiation-induced bystander effects in an intact living organism.

We present the complete construction methodology for an anatomically accurate mouse phantom made using materials which mimic the characteristics of tissue, lung, and bone for radiation dosimetry studies. Phantoms were constructed using 2 mm thick slices of tissue equivalent material which was precision machined to clear regions for insertion of lung and bone equivalent material where appropriate. Images obtained using a 3D computed tomography (CT) scan clearly indicate regions of tissue, lung, and bone that match their position within the original mouse CT scan. Additionally, radiographic films are used with the phantom to demonstrate dose mapping capabilities. The construction methodology presented here can be quickly and easily adapted to create a phantom of any specific small animal given a segmented CT scan of the animal. These physical phantoms are a useful tool to examine individual organ dose and dosimetry within mouse systems that are complicated by density inhomogeneity due to bone and lung regions.

We have previously shown that 207-nm ultraviolet (UV) light has similar antimicrobial properties as typical germicidal UV light (254 nm), but without inducing mammalian skin damage. The biophysical rationale is based on the limited penetration distance of 207-nm light in biological samples (e.g. stratum corneum) compared with that of 254-nm light. Here we extended our previous studies to 222-nm light and tested the hypothesis that there exists a narrow wavelength window in the far-UVC region, from around 200–222 nm, which is significantly harmful to bacteria, but without damaging cells in tissues. We used a krypton-chlorine (Kr-Cl) excimer lamp that produces 222-nm UV light with a bandpass filter to remove the lower- and higher-wavelength components. Relative to respective controls, we measured: 1. in vitro killing of methicillin-resistant Staphylococcus aureus (MRSA) as a function of UV fluence; 2. yields of the main UV-associated premutagenic DNA lesions (cyclobutane pyrimidine dimers and 6-4 photoproducts) in a 3D human skin tissue model in vitro; 3. eight cellular and molecular skin damage endpoints in exposed hairless mice in vivo. Comparisons were made with results from a conventional 254-nm UV germicidal lamp used as positive control. We found that 222-nm light kills MRSA efficiently but, unlike conventional germicidal UV lamps (254 nm), it produces almost no premutagenic UV-associated DNA lesions in a 3D human skin model and it is not cytotoxic to exposed mammalian skin. As predicted by biophysical considerations and in agreement with our previous findings, far-UVC light in the range of 200–222 nm kills bacteria efficiently regardless of their drug-resistant proficiency, but without the skin damaging effects associated with conventional germicidal UV exposure.

The development of, and results from an image analysis system are presented for automated detection and scoring of micronuclei in human peripheral blood lymphocytes. The system is part of the Rapid Automated Biodosimetry Tool, which was developed at the Center for High-Throughput Minimally Invasive Radiation Biodosimetry for rapid radiation dose assessment of many individuals based on single fingerstick samples of blood. Blood lymphocytes were subjected to the cytokinesis-block micronucleus assay and the images of cell cytoplasm and nuclei are analyzed to estimate the frequency of micronuclei in binucleated cells. We describe an algorithm that is based on dual fluorescent labeling of lymphocytes with separate analysis of images of cytoplasm and nuclei. To evaluate the performance of the system, blood samples of seven healthy donors were irradiated in vitro with doses from 0-10 Gy and dose-response curves of micronuclei frequencies were generated. To establish the applicability of the system to the detection of high doses, the ratios of mononucleated cells to binucleated cells were determined for three of the donors. All of the dose-response curves generated automatically showed clear dose dependence and good correlation (R(2) from 0.914-0.998) with the results of manual scoring.

Anatomically accurate phantoms are useful tools for radiation dosimetry studies. In this work, we demonstrate the construction of a new generation of life-like mouse phantoms in which the methods have been generalized to be applicable to the fabrication of any small animal. The mouse phantoms, with built-in density inhomogeneity, exhibit different scattering behavior dependent on where the radiation is delivered. Computer models of the mouse phantoms and a small animal irradiation platform were devised in Monte Carlo N-Particle code (MCNP). A baseline test replicating the irradiation system in a computational model shows minimal differences from experimental results from 50 Gy down to 0.1 Gy. We observe excellent agreement between scattered dose measurements and simulation results from X-ray irradiations focused at either the lung or the abdomen within our phantoms. This study demonstrates the utility of our mouse phantoms as measurement tools with the goal of using our phantoms to verify complex computational models.

The immunofluorescence-based detection of γ-H2AX is a reliable and sensitive method for quantitatively measuring DNA double-strand breaks (DSBs) in irradiated samples. Since H2AX phosphorylation is highly linear with radiation dose, this well-established biomarker is in current use in radiation biodosimetry. At the Center for High-Throughput Minimally Invasive Radiation Biodosimetry, we have developed a fully automated high-throughput system, the RABIT (Rapid Automated Biodosimetry Tool), that can be used to measure γ-H2AX yields from fingerstick-derived samples of blood. The RABIT workstation has been designed to fully automate the γ-H2AX immunocytochemical protocol, from the isolation of human blood lymphocytes in heparin-coated PVC capillaries to the immunolabeling of γ-H2AX protein and image acquisition to determine fluorescence yield. High throughput is achieved through the use of purpose-built robotics, lymphocyte handling in 96-well filter-bottomed plates, and high-speed imaging. The goal of the present study was to optimize and validate the performance of the RABIT system for the reproducible and quantitative detection of γ-H2AX total fluorescence in lymphocytes in a multiwell format. Validation of our biodosimetry platform was achieved by the linear detection of a dose-dependent increase in γ-H2AX fluorescence in peripheral blood samples irradiated ex vivo with γ rays over the range 0 to 8 Gy. This study demonstrates for the first time the optimization and use of our robotically based biodosimetry workstation to successfully quantify γ-H2AX total fluorescence in irradiated peripheral lymphocytes.

Background UVC light generated by conventional germicidal lamps is a well-established anti-microbial modality, effective against both bacteria and viruses. However, it is a human health hazard, being both carcinogenic and cataractogenic. Earlier studies showed that single-wavelength far-UVC light (207 nm) generated by excimer lamps kills bacteria without apparent harm to human skin tissue in vitro. The biophysical explanation is that, due to its extremely short range in biological material, 207 nm UV light cannot penetrate the human stratum corneum (the outer dead-cell skin layer, thickness 5–20 μm) nor even the cytoplasm of individual human cells. By contrast, 207 nm UV light can penetrate bacteria and viruses because these cells are physically much smaller. Aims To test the biophysically-based hypothesis that 207 nm UV light is not cytotoxic to exposed mammalian skin in vivo. Methods Hairless mice were exposed to a bactericidal UV fluence of 157 mJ/cm2 delivered by a filtered Kr-Br excimer lamp producing monoenergetic 207-nm UV light, or delivered by a conventional 254-nm UV germicidal lamp. Sham irradiations constituted the negative control. Eight relevant cellular and molecular damage endpoints including epidermal hyperplasia, pre-mutagenic UV-associated DNA lesions, skin inflammation, and normal cell proliferation and differentiation were evaluated in mice dorsal skin harvested 48 h after UV exposure. Results While conventional germicidal UV (254 nm) exposure produced significant effects for all the studied skin damage endpoints, the same fluence of 207 nm UV light produced results that were not statistically distinguishable from the zero exposure controls. Conclusions As predicted by biophysical considerations and in agreement with earlier in vitro studies, 207-nm light does not appear to be significantly cytotoxic to mouse skin. These results suggest that excimer-based far-UVC light could potentially be used for its anti-microbial properties, but without the associated hazards to skin of conventional germicidal UV lamps.

Depleted uranium (DU) is a dense heavy metal used primarily in military applications. Published data from our laboratory have demonstrated that DU exposure in vitro to immortalized human osteoblast cells (HOS) is both neoplastically transforming and genotoxic. Recent animal studies have also shown that DU is leukemogenic and genotoxic. DU possesses both a radiological (alpha particle) and chemical (metal) component. Since DU has a low specific activity in comparison to natural uranium, it is not considered to be a significant radiological hazard. The potential contribution of radiation to DU-induced biological effects is unknown, and the involvement of radiation in DU-induced biological effects could have significant implications for current risk estimates for internalized DU exposure. The purpose of the current study was to measure the induction of mutagenic damage in V79 cells and to determine if radiation plays a role in the induction of that damage. Mutagenicity at the hypoxanthine (guanine) phosphoribosyltransferase (hprt) locus was measured by selection with 6-thioguanine. There was a dose-dependent increase in mutagenic response following DU exposure ( 10 – 50 μ m ) ; the average increase in mutagenicity above background ranged from 2.54 ± 1.19 to 8.75 ± 1.8 ( P 0.05 ) . Using the same concentration ( 25 μ M ) of two uranyl nitrate compounds that have different uranium isotopic concentrations and, therefore, different specific activities, we examined the effect on hprt mutant frequency in vitro. V79 cells were exposed to either U 238 -uranyl nitrate, specific activity 0.33 μ Ci / g , or DU-uranyl nitrate, specific activity 0.44 μ Ci / g , delivered at a concentration of 25 μ M for 24 h. Results showed, that at equal uranium concentration, a 1.33-fold increase in specific activity resulted in a 1.27 ± 0.11 -fold ( P 0.05 ) increase in hprt mutant frequency. Taken together these data support earlier results showing that radiation can play a role in DU-induced biological effects in vitro.

We describe here an accelerator-based neutron irradiation facility, intended to expose blood or small animals to neutron fields mimicking those from an improvised nuclear device at relevant distances from the epicenter. Neutrons are generated by a mixed proton/deuteron beam on a thick beryllium target, generating a broad spectrum of neutron energies that match those estimated for the Hiroshima bomb at 1.5 km from ground zero. This spectrum, dominated by neutron energies between 0.2 and 9 MeV, is significantly different from the standard reactor fission spectrum, as the initial bomb spectrum changes when the neutrons are transported through air. The neutron and gamma dose rates were measured using a custom tissue-equivalent gas ionization chamber and a compensated Geiger-Mueller dosimeter, respectively. Neutron spectra were evaluated by unfolding measurements using a proton-recoil proportional counter and a liquid scintillator detector. As an illustration of the potential use of this facility we present micronucleus yields in single divided, cytokinesis-blocked human peripheral lymphocytes up to 1.5 Gy demonstrating 3- to 5-fold enhancement over equivalent X-ray doses. This facility is currently in routine use, irradiating both mice and human blood samples for evaluation of neutron-specific biodosimetry assays. Future studies will focus on dose reconstruction in realistic mixed neutron/photon fields.

A laser ion source that will be installed on the new High Voltage Engineering (HVE) 5 MV Singletron accelerator at the Columbia University Radiological Research Accelerator Facility (RARAF) will expand the linear energy transfer (LET) range available for irradiation experiments with mammalian cells. Through laser ablation the laser ion source can produce heavy ions with high charge states from a solid target; after acceleration, these ions will have sufficient energy to irradiate cells on a thin surface at atmospheric pressure. A high-power 100 Hz pulsed Nd:YAG laser used with the laser ion source has produced aluminum ions with charge states greater than nine. Proper power management issues are important in obtaining the high charge states while protecting sensitive laser optics. We expect that the laser ion source will enable us to use ions from hydrogen to iron, providing an LET range of about 10–4500 keV/lm for cell targets.

The stand-alone microbeam, under development at RARAF, presents a novel approach to biological microbeam irradiation studies. Foregoing a conventional accelerator as a source of energetic ions, we propose to use a small, high-specific-activity, alpha emitter. Alpha particles emitted from this source are focused using a compound magnetic lens consisting of 24 permanent magnets arranged in two quadrupole triplets. Using a ‘‘home made’’ 6.5 mCi Polonium source, a 1 alpha particle/s, 10 lm diameter microbeam can be realized. As the alpha source energy is constant, once the microbeam has been set up, no further adjustments are necessary apart from a periodic replacement of the source. The use of permanent magnets eliminates the need for bulky power supplies and cooling systems required by other types of ion lenses and greatly simplifies operation. It also makes the microbeam simple and cheap enough to be realized in any large lab.

A central tenet in understanding the biological effects of ionizing radiation has been that the initially affected cells were directly damaged by the radiation. By contrast, evidence has emerged concerning “bystander” responses involving damage to nearby cells that were not themselves directly traversed by the radiation. These long-range effects are of interest both mechanistically and for assessing risks from low-dose exposures, where only a small proportion of cells are directly hit. Bystander effects have been observed largely by using single-cell in vitro systems that do not have realistic multicellular morphology; no studies have as yet been reported in three-dimensional, normal human tissue. Given that the bystander phenomenon must involve cell-to-cell interactions, the relevance of such single-cell in vitro studies is questionable, and thus the significance of bystander responses for human health has remained unclear. Here, we describe bystander responses in a three-dimensional, normal human-tissue system. Endpoints were induction of micronucleated and apoptotic cells. A charged-particle microbeam was used, allowing irradiation of cells in defined locations in the tissue yet guaranteeing that no cells located more than a few micrometers away receive any radiation exposure. Unirradiated cells up to 1 mm distant from irradiated cells showed a significant enhancement in effect over background, with an average increase in effect of 1.7-fold for micronuclei and 2.8-fold for apoptosis. The surprisingly long range of bystander signals in human tissue suggests that bystander responses may be important in extrapolating radiation risk estimates from epidemiologically accessible doses down to very low doses where nonhit bystander cells will predominate.

A single-cell/single-particle microbeam has been designed in which the radiation source is a high specific-activity alpha-particle emitting radioisotope, rather than a particle accelerator. The source is imaged at the focal point of a compound magnetic lens array. A novel precision microscope stage with an extremely compact design is included with the endstations locating, targeting and imaging system. This arrangement allows a 4 m long vertical beamline without any bending magnets. The permanent magnet lens design is derived from an electrostatic lens used in our laboratory in an accelerator-based microbeam. The stand-alone microbeam is expected to produce a 10 lm diameter beam, and will serve as a high-end instrument in a biology laboratory, to precisely deposit ionizing radiation damage within single cells.

The Columbia University Microbeam II has been built to provide a focused ion beam for irradiating designated mammalian cells with single particles. With the interest in irradiating non-stained cells and cells in three-dimensional tissue samples, the endstation was designed to accommodate a variety of imaging techniques, in addition to fluorescent microscopy. Non-stained cells are imaged either by quantitative phase microscopy (QPm) [IATIA, Box Hill North, Victoria, 3129, Australia [1]] or by Mirau interferometry. In conjunction with the focused microbeam, a proposed multiphoton microscope will offer the opportunity to probe live cells at selected depths in tissue samples; dynamic events in such tissue samples are more representative of biological system response than those in cell cultures.

We describe a novel secondary-electron ion microscope (SEIM), designed for diagnostics of the upcoming submicron Columbia University charged-particle microbeam. This secondary-electron ion microscope allows much higher resolutions, at higher single particle detection efficiencies, than previously available, for rapid and accurate diagnostics of sub-micron charged-particle beams. Based on ion electron-emission microscopy (IEEM) and photo-electron microscopy (PEM), the SEIM involves conversion of the incident projectiles on a secondary-electron emitting film. The ejected electrons are focused using a unipolar electrostatic lens and conical electrostatic mirror to form a magnified image on a microchannel plate (MCP). The flight path of the electrons includes two 45� bends; this ‘‘folded’’ geometry results in lower aberrations than a ‘‘straight’’ design, and enables efficient beam imaging down to 100 nm resolution with >50% single electron transfer efficiency. � 2005 Elsevier B.V. All rights reserved.

Normal human fibroblasts in plateau phase ( congruent with 95% G(1) phase) were stained with the vital nuclear dye Hoechst 33342 (blue fluorescence) or the vital cytoplasmic dye Cell Tracker Orange (orange fluorescence) and plated at a ratio of 1:1. Only the blue-fluorescing nuclei were microbeam-irradiated with a defined number of 90 keV/microm alpha particles. The orange-fluorescing cells were then "bystanders", i.e. not themselves hit but adjacent to cells that were. Hit cells showed a fluence-dependent induction of micronuclei as well as delays in progression from G(1) to S phase. Known bystander cells also showed enhanced frequencies of micronuclei (intermediate between those seen in irradiated and control cells) and transient cell cycle delays. However, the induction of micronuclei in bystander cells did not appear to be dependent on the fluence of the particles delivered to the neighboring hit cells. These are the first studies in which the bystander effect has been visualized directly rather than inferred. They indicate that the phenomenon has a quantitative basis and imply that the target for radiation effects cannot be considered to be the individual cell.

Mitchell, S. A., Randers-Pehrson, G., Brenner, D. J. and Hall, E. J. The Bystander Response in C3H 10T‰ Cells: The Influence of Cell-to-Cell Contact. Radiat. Res. 161, 397‐401 (2004). Although radiation-induced heritable damage in mammalian cells was thought to result from the direct interaction of radiation with DNA, it is now accepted that biological effects may occur in cells that were not themselves traversed by ionizing radiation but are close to those that were. However, little is known about the mechanism underlying such a bystander effect, although cell-to-cell communication is thought to be of importance. Previous work using the Columbia microbeam demonstrated a significant bystander effect for clonogenic survival and oncogenic transformation in C3H 10T‰ cells. The present study was undertaken to assess the importance of the degree of cell-to-cell contact at the time of irradiation on the magnitude of this bystander effect by varying the cell density. When 10% of cells were exposed to a range of 2‐12 a particles, a significantly greater number of cells (P , 0.0001) were inactivated when cells were irradiated at high density (.90% in contact with neighbors) than at low density (,10% in contact). In addition, the oncogenic transformation frequency was significantly higher in high-density cultures (P , 0.0004). These results suggest that when a cell is hit by radiation, the transmission of the bystander signal through cell-to-cell contact is an important mediator of the effect, implicating the involvement of intracellular communication through gap junctions. q 2004 by Radiation Research Society

Two conflicting phenomena, bystander effect and adaptive response, are important in determining the biological responses at low doses of radiation and have the potential to impact the shape of the dose-response relationship. Using the Columbia University charged-particle microbeam and the highly sensitive AL cell mutagenic assay, we show here that non-irradiated cells acquire mutagenesis through direct contact with cells whose nuclei have been traversed with a single alpha particle each. Pretreatment of cells with a low dose of X-rays four hours before alpha particle irradiation significantly decreased this bystander mutagenic response. Results from the present study address some of the fundamental issues regarding both the actual target and radiation dose effect and can contribute to our current understanding in radiation risk assessment. c2004 COSPAR. Published by Elsevier Ltd. All rights reserved.

A laser ion source is scheduled as an upgrade for the 4 MV Van de Graaff accelerator at the Columbia Universitys Radiological Research Accelerator Facility (RARAF). The source has been designed for use with the RARAF singleparticle single-cell microbeam, though it will be used also for broad-beam irradiations. The operating principle, laser ablation, can produce heavy-ions with high charge states so that their energies will be high enough to provide sufficient range – at least 20 lm – for irradiating cells on a thin surface at atmospheric pressure. The laser ion source being implemented at RARAF consists of three main components: a high-power 100 Hz Nd:YAG laser, a source vacuum chamber and a 24 spherical electrostatic analyzer and a double focusing element with point-to-point focusing in both the horizontal and vertical planes. We expect that the laser ion source will enable us to use ions of sufficient range from hydrogen to iron, providing a range of linear energy transfer from about 10 to 4500 keV/lm. 2003 Elsevier B.V. All rights reserved.

Two conflicting phenomena, bystander effect and adaptive response, are important in determining the biological responses at low doses of radiation and have the potential to impact on the shape of the dose–response relationship. Using the Columbia University charged-particle microbeam and the highly sensitive A L cell mutagenic assay, we show here that non-irradiated cells acquire mutagenesis through direct contact with cells whose nuclei had been traversed with two alpha particles each. Pre-treatment of cells with a 0.1-Gy dose X-rays 4 h before alpha particle irradiation significantly decreased this bystander mutagenic response. Results from the present study address some of the fundamental issues regarding both the actual target and radiation dose effect and can impart on our current understanding in radiation risk assessment.

It has long been accepted that the radiation-induced heritable effects in mammalian cells are the result of direct DNA damage. Recent evidence, however, suggests that when a cell population is exposed to a low dose of alpha particles, biological effects occur in a larger proportion of cells than are estimated to have been traversed by alpha particles. Experiments involving the Columbia University microbeam, which allows a known fraction of cells to be traversed by a defined number of alpha particles, have demonstrated a bystander effect for clonogenic survival and oncogenic transformation in C3H 10T(1/2) cells. When 1 to 16 alpha particles were passed through the nuclei of 10% of a C3H 10T(1/2) cell population, more cells were unable to form colonies than were actually traversed by alpha particles. Both hit and non-hit cells contributed to the outcome of the experiments. The present work was undertaken to assess the bystander effect of radiation in only non-hit cells. For this purpose, Chinese hamster V79 cells transfected with hygromycin- or neomycin-resistance genes were used. V79 cells stably transfected with a hygromycin resistance gene and stained with a nuclear dye were irradiated with the charged-particle microbeam in the presence of neomycin-resistant cells. The biological effect was studied in the neomycin-resistant V79 cells after selective removal of the hit cells with geneticin treatment.

A noninvasive, self-referencing biosensor/probe system has been integrated into the Columbia University Radiological Research Accelerator Facility Microbeam II end station. A single-cell oxygen consumption measurement has been conducted with this type of oxygen probe in 37° C Krebs–Ringer Bicarbonate buffer immediately before and after a single-cell microbeam irradiation. It is the first such measurement made for a microbeam irradiation, and a six fold increment of oxygen flux induced during a 15-s period of time has been observed following radiation exposure. The experimental procedure and the results are discussed.

Sawant, S. G., Randers-Pehrson, G., Metting, N. and Hall, E. J. Adaptive Response and the Bystander Effect Induced by Radiation in C3H 10T½ Cells in Culture. Radiat. Res. 156, 177–180 (2001). This paper discusses two phenomena of importance at low doses that have an impact on the shape of the dose–response relationship. First, there is the bystander effect, the term used to describe the biological effects observed in cells that are not themselves traversed by a charged particle, but are neighbors of cells that are; this exaggerates the effect of small doses of radiation. Second, there is the adaptive response, whereby exposure to a low level of DNA stress renders cells resistant to a subsequent exposure; this reduces the effect of low doses of radiation. The present work was undertaken to assess the relative importance of the adaptive response and the bystander effect induced by radiation in C3H 10T½ cells in culture. When the single-cell microbeam delivered from 1 to 12 α particles through the n...

Sawant, S. G., Randers-Pehrson, G., Geard, C. R., Brenner, D. J. and Hall, E. J. The Bystander Effect in Radiation Oncogenesis: I. Transformation in C3H 10T½ Cells In Vitro can be Initiated in the Unirradiated Neighbors of Irradiated Cells. It has long been accepted that radiation-induced genetic effects require that DNA be hit and damaged directly by the radiation. Recently, evidence has accumulated that in cell populations exposed to low doses of α particles, biological effects occur in a larger proportion of cells than are estimated to have been traversed by α particles. The end points observed include chromosome aberrations, mutations and gene expression. The development of a fast single-cell microbeam now makes it possible to expose a precisely known proportion of cells in a population to exactly defined numbers of α particles, and to assay for oncogenic transformation. The single-cell microbeam delivered no, one, two, four or eight α particles through the nuclei of all or just 10% of C3H 10...

A short probe-forming system is developed for the Columbia Microprobe that includes four electrostatic quadrupoles with a Russian quadruplet configuration. The smallest beam spot size and appropriate optimal parameters of the probe-forming systems have been found. These parameters of the system are compared with appropriate parameters of other field configurations including the electrostatic and magnetic fields with dipole, quadrupole, and rotational symmetry. The new original construction of the electrostatic quadruplet has been manufactured. The sensitivity of this quadruplet to some misalignments of the construction is investigated.

Ever since x-rays were shown to induce mutation in Drosophila more than 70 years ago, prevailing dogma considered the genotoxic effects of ionizing radiation, such as mutations and carcinogenesis, as being due mostly to direct damage to the nucleus. Although there was indication that alpha particle traversal through cellular cytoplasm was innocuous, the full impact remained unknown. The availability of the microbeam at the Radiological Research Accelerator Facility of Columbia University made it possible to target and irradiate the cytoplasm of individual cells in a highly localized spatial region. By using dual fluorochrome dyes (Hoechst and Nile Red) to locate nucleus and cellular cytoplasm, respectively, thereby avoiding inadvertent traversal of nuclei, we show here that cytoplasmic irradiation is mutagenic at the CD59 (S1) locus of human–hamster hybrid (A L ) cells, while inflicting minimal cytotoxicity. The principal class of mutations induced are similar to those of spontaneous origin and are entirely different from those of nuclear irradiation. Furthermore, experiments with radical scavenger and inhibitor of intracellular glutathione indicated that the mutagenicity of cytoplasmic irradiation depends on generation of reactive oxygen species. These findings suggest that cytoplasm is an important target for genotoxic effects of ionizing radiation, particularly radon, the second leading cause of lung cancer in the United States. In addition, cytoplasmic traversal by alpha particles may be more dangerous than nuclear traversal, because the mutagenicity is accomplished by little or no killing of the target cells.

An important technique used for the grading of voltage drop along high voltage ceramic insulators is to provide some surface conduction to bleed off accumulated surface charge. We have used metal ion implantation to modify the surface of high voltage ceramic vacuum insulators to provide a uniform surface resistivity of order 10 10 Ω/square. A vacuum arc ion source based implanter was used to implant Pt at an energy of about 125 keV to a dose of order 10 16 ions/cm 2 into the surface of ceramic rods used to support the ion focusing system of the Columbia

A novel technique for label-free imaging of live biological cells in aqueous medium that is insensitive to ambient vibrations is presented. This technique is a spin-off from previously developed immersion Mirau interferometry. Both approaches utilize a modified Mirau interferometric attachment for a microscope objective that can be used both in air and in immersion mode, when the device is submerged in cell medium and has its internal space filled with liquid. While immersion Mirau interferometry involves first capturing a series of images, the resulting images are potentially distorted by ambient vibrations. Overcoming these serial-acquisition challenges, simultaneous immersion Mirau interferometry incorporates polarizing elements into the optics to allow simultaneous acquisition of two interferograms. The system design and production are described and images produced with the developed techniques are presented.

A design is given of a laser ion source for the 4.2 MV Van de Graaff accelerator at the Columbia University Radiological Research Accelerator Facility (RARAF). The source has been designed with application in mind for the RARAF single-particle single-cell microbeam, though it will also be used for broad-beam irradiations. The operating principle, laser ablation, can produce heavy ions with high charge states so that their energies will be high enough to provide sufficient range—at least 20 μm—for irradiating cells on a thin surface at atmospheric pressure. The laser ion source being implemented at RARAF is based on the laser operated ion source used by Hughes at the University of Arkansas and consists of three main components: laser generator, source vacuum chamber, and spherical electrostatic analyzer.

A soft x-ray microbeam using proton-induced x-ray emission (PIXE) of characteristic titanium (K(α) 4.5 keV) as the x-ray source has been developed at the Radiological Research Accelerator Facility (RARAF) at Columbia University. The proton beam is focused to a 120 μm × 50 μm spot on the titanium target using an electrostatic quadrupole quadruplet previously used for the charged particle microbeam studies at RARAF. The proton induced x-rays from this spot project a 50 μm round x-ray generation spot into the vertical direction. The x-rays are focused to a spot size of 5 μm in diameter using a Fresnel zone plate. The x-rays have an attenuation length of (1/e length of ~145 μm) allowing more consistent dose delivery across the depth of a single cell layer and penetration into tissue samples than previous ultra soft x-ray systems. The irradiation end station is based on our previous design to allow quick comparison to charged particle experiments and for mixed irradiation experiments.

Purpose: Over the past five years the Center for Minimally Invasive Radiation Biodosimetry at Columbia University has developed the Rapid Automated Biodosimetry Tool (RABiT), a completely automated, ultra-high throughput biodosimetry workstation. This paper describes recent upgrades and reliability testing of the RABiT. Materials and methods: The RABiT analyses fingerstick-derived blood samples to estimate past radiation exposure or to identify individuals exposed above or below a cut-off dose. Through automated robotics, lymphocytes are extracted from fingerstick blood samples into filter-bottomed multi-well plates. Depending on the time since exposure, the RABiT scores either micronuclei or phosphorylation of the histone H2AX, in an automated robotic system, using filter-bottomed multiwell plates. Following lymphocyte culturing, fixation and staining, the filter bottoms are removed from the multi-well plates and sealed prior to automated high-speed imaging. Image analysis is performed online using dedicated image processing hardware. Both the sealed filters and the images are archived. Results: We have developed a new robotic system for lymphocyte processing, making use of an upgraded laser power and parallel processing of four capillaries at once. This system has allowed acceleration of lymphocyte isolation, the main bottleneck of the RABiT operation, from 12 to 2 sec/sample. Reliability tests have been performed on all robotic subsystems. Conclusions: Parallel handling of multiple samples through the use of dedicated, purpose-built, robotics and high speed imaging allows analysis of up to 30,000 samples per day.

Presented here is a novel microbeam technology--the Flow-And-ShooT (FAST) microbeam--under development at RARAF. In this system, cells undergo controlled fluidic transport along a microfluidic channel intersecting the microbeam path. They are imaged and tracked in real-time, using a high-speed camera and dynamically targeted, using a magnetic Point and Shoot system. With the proposed FAST system, RARAF expects to reach a throughput of 100,000 cells per hour, which will allow increasing the throughput of experiments by at least one order of magnitude. The implementation of FAST will also allow the irradiation of non-adherent cells (e.g. lymphocytes), which is of great interest to many of the RARAF users. This study presents the design of a FAST microbeam and results of first tests of imaging and tracking as well as a discussion of the achievable throughput.

A novel neutron microbeam is being developed at the Radiological Research Accelerator Facility (RARAF) of Columbia University. The RARAF microbeam facility has been used for studies of radiation bystander effects in mammalian cells for many years. Now a prototype neutron microbeam is being developed that can be used for bystander effect studies. The neutron microbeam design here is based on the existing charged particle microbeam technology at the RARAF. The principle of the neutron microbeam is to use the proton beam with a micrometre-sized diameter impinging on a very thin lithium fluoride target system. From the kinematics of the 7Li(p,n)7Be reaction near the threshold of 1.881 MeV, the neutron beam is confined within a narrow, forward solid angle. Calculations show that the neutron spot using a target with a 17-µm thick gold backing foil will be

In cell biology studies it is often important to avoid the damaging effects caused by fluorescent stains or UV-light. Immersion Mirau Interferometry (IMI) is an epi-illumination label-free imaging technique developed at the Columbia University Radiological Research Accelerator Facility. It is based on the principles of phase-shifting interferometry (PSI) and represents a novel approach for interferometric imaging of living cells in medium. To accommodate the use of medium, a custom immersion Mirau interferometric attachment was designed and built in-house. The space between the reference mirror and the beam splitter is filled with liquid to ensure identical optical paths in the test and reference arms. The interferometer is mountable onto a microscope objective. The greatest limitation of standard PSI is the sensitivity to environmental vibrations, because it requires consecutive acquisition of several interferograms. We are developing Simultaneous Immersion Mirau Interferometry (SIMI), which facilitates simultaneous acquisition of all interferograms and eliminates the effects of vibration. Polarization optics, incorporated into the design, introduces a phase delay to one of the components of the test beam. This enables simultaneous creation and spatial separation of two interferograms, which are combined with the background image to reconstruct the intensity map of the specimen. Our results of imaging live and fixed cells with IMI and SIMI show that this system produces images of a quality that is sufficient to perform targeted cellular irradiation experiments.

In response to the recognized need for high throughput biodosimetry methods for use after large-scale radiological events, a logical approach is complete automation of standard biodosimetric assays that are currently performed manually. The authors describe progress to date on the RABIT (Rapid Automated BIodosimetry Tool), designed to score micronuclei or gamma-H2AX fluorescence in lymphocytes derived from a single drop of blood from a fingerstick. The RABIT system is designed to be completely automated, from the input of the capillary blood sample into the machine to the output of a dose estimate. Improvements in throughput are achieved through use of a single drop of blood, optimization of the biological protocols for in situ analysis in multi-well plates, implementation of robotic-plate and liquid handling, and new developments in high-speed imaging. Automating well-established bioassays represents a promising approach to high-throughput radiation biodosimetry, both because high throughputs can be achieved, but also because the time to deployment is potentially much shorter than for a new biological assay. Here the authors describe the development of each of the individual modules of the RABIT system and show preliminary data from key modules. System integration is ongoing, followed by calibration and validation.