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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

Vertical Microbeams (VMB) are used to irradiate individual cells with low MeV energy ions. The irradiation of cells using VMBs requires cells to be removed from an incubator; this can cause physiological changes to cells because of the lower CO2 concentration, temperature and relative humidity outside of the incubator. Consequently, for experiments where cells require irradiation and observation for extended time periods, it is important to provide a controlled environment. The highly customised nature of the microscopes used on VMB systems means that there are no commercially available environmentally controlled microscope systems for VMB systems. The Automated Microbeam Observation Environment for Biological Analysis (AMOEBA) is a highly flexible modular environmental control system used to create incubator conditions on the end of a VMB. The AMOEBA takes advantage of the recent “maker” movement to create an open source control system that can be easily configured by the user to fit their control needs even beyond VMB applications. When applied to the task of controlling cell medium temperature, CO2 concentration and relative humidity on VMBs it creates a stable environment that allows cells to multiply on the end of a VMB over a period of 36 h, providing a low-cost (costing less than $2700 to build), customisable alternative to commercial time-lapse microscopy systems. AMOEBA adds the potential of VMBs to explore the long-term effects of radiation on single cells opening up new research areas for VMBs.

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.

We describe here an automated imaging system developed at the Center for High Throughput Minimally Invasive Radiation Biodosimetry. The imaging system is built around a fast, sensitive sCMOS camera and rapid switchable LED light source. It features complete automation of all the steps of the imaging process and contains built-in feedback loops to ensure proper operation. The imaging system is intended as a back end to the RABiT-a robotic platform for radiation biodosimetry. It is intended to automate image acquisition and analysis for four biodosimetry assays for which we have developed automated protocols: The Cytokinesis Blocked Micronucleus assay, the γ-H2AX assay, the Dicentric assay (using PNA or FISH probes) and the RABiT-BAND assay.

The RABiT (Rapid Automated Biodosimetry Tool) is a dedicated Robotic platform for the automation of cytogenetics-based biodosimetry assays. The RABiT was developed to fulfill the critical requirement for triage following a mass radiological or nuclear event. Starting from well-characterized and accepted assays we developed a custom robotic platform to automate them. We present here a brief historical overview of the RABiT program at Columbia University from its inception in 2005 until the RABiT was dismantled at the end of 2015. The main focus of this paper is to demonstrate how the biological assays drove development of the custom robotic systems and in turn new advances in commercial robotic platforms inspired small modifications in the assays to allow replacing customized robotics with 'off the shelf' systems. Currently, a second-generation, RABiT II, system at Columbia University, consisting of a PerkinElmer cell::explorer, was programmed to perform the RABiT assays and is undergoing testing and optimization studies.

To analyze the effect of different radiation qualities on the kinetics of p53 Binding Protein 1 (53BP1) formation and decline in human three-dimensional (3-D) vessel models.Two-dimensional (2-D) and 3-D cultures of human umbilical vein cells were exposed to 80 cGy of Gamma radiation and high-energy protons and Fe ions. 53BP1 antibodies were used for foci visualization via immunocytochemistry. Computer analysis was used to determine the number and the size of foci up to 48 hours after irradiation.DNA foci kinetics in 2-D and 3-D human vessel cultures show that foci formation and removal were the same in each type of culture. After 48 h, the number of foci induced by high-energy protons and gamma rays reduced to almost control levels while high linear energy transfer (LET) Fe particles produced more persistent damage.The kinetics of radiation-induced 53BP1 foci in 3-D vessel models is essentially the same as in 2-D monolayers. Since the basal level of spontaneous foci is low in these differentiated non-proliferating cultures, the persistence of radiation-induced 53BP1 foci is detected longer than previously noted. Furthermore, analysis of foci sizes revealed that abnormal radiation-induced foci can persist even when foci frequencies are close to basal levels. The detection of these latent abnormalities could be useful for a more sensitive dosimetry.

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.

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.

The microbeam facility at Columbia University is expanding current protocols for single-particle, single-cell irradiations, so experimenters can locate and irradiate nuclei and cytoplasm of unstained cells. The ion beamline is located directly under the dish, therefore, any new techniques must use reflection microscopy. Two approaches are being integrated and neither require the removal of the cell growth medium prior to irradiation. (1) A novel immersion-based Mirau interferometry lens which uses low-coherence light sources to inhibit unwanted fringing is under design. The process requires tens of nanometers or better precision of vertical stage motion, which will be accomplished with our custom high-precision z-stage. (2) Quantitative Phase microscopy is under testing, also using the z-stage. Future plans include optimization of software routines to decrease time between irradiations. Both methods will be compared further with the automated location routines which use nuclear and cytoplasm stains.

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.

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.

We describe here an automated imaging system developed at the Center for High Throughput Minimally Invasive Radiation Biodosimetry. The imaging system is built around a fast, sensitive sCMOS camera and rapid switchable LED light source. It features complete automation of all the steps of the imaging process and contains built-in feedback loops to ensure proper operation. The imaging system is intended as a back end to the RABiT – a robotic platform for radiation biodosimetry. It is intended to automate image acquisition and analysis for four biodosimetry assays for which we have developed automated protocols: The Cytokinesis Blocked Micronucleus assay, the γ-H2AX assay, the Dicentric assay (using PNA or FISH probes) and the RABiT-BAND assay.

The Radiological Research Accelerator Facility (RARAF) at Columbia University has recently added a UV microspot irradiator to a microbeam irradiation platform. This UV microspot irradiator applies multiphoton excitation at the focal point of an incident laser as the source for cell damage, and with this approach, a single cell within a 3D sample can be targeted and exposed to damaging UV. The UV microspot’s ability to impart cellular damage within 3D is an advantage over all other microbeam techniques, which instead impart damage to numerous cells along microbeam tracks. This short communication is an overview and a description of the UV microspot including the following applications and demonstrations of selective damage to live single cell targets: DNA damage foci formation, patterned irradiation, photoactivation, targeting of mitochondria, and targeting of individual cardiomyocytes in the live zebrafish embryo.

Background: Charged particle radiation is known to be more biologically effective than photon radiation. One example of this is the inhibition of the formation of human blood vessels. This effect is an important factor influencing human health and is relevant to space travel as well as to cancer radiotherapy. We have previously shown that ion particles with a high energy deposition, or linear energy transfer (LET) are more than four times more effective at disrupting mature vessel tissue models than particles with a lower LET. For vasculogenesis however, the relative biological effectiveness between particles is the same. This unexpected result prompted us to investigate whether the inhibition of vasculogenesis was occurring by distinct mechanisms. Methods: Using 3-Dimensional human vessel models, we developed assays that determine at what stage angiogenesis is inhibited. Vessel morphology, the presence of motile tip structures, and changes in the matrix architecture were assessed. To confirm that the mechanisms are distinct, stimulation of Protein Kinase C (PKC) with phorbol ester (PMA) was employed to selectively restore vessel formation in cultures where early motile tip activity was inhibited. Results: Endothelial cells in 3-D culture exposed to low LET protons failed to make connections with other cells but eventually developed a central lumen. Conversely, cells exposed to high LET Fe charged particles extended cellular processes and made connections to other cells but did not develop a central lumen. The microtubule and actin cytoskeletons indicated that motility at the extending tips of endothelial cells is inhibited by low LET but not high LET particles. Actin-rich protrusive structures that contain bundled microtubules showed a 65% decrease when exposed to low LET particles but not high LET particles, with commensurate changes in the matrix architecture. Stimulation of PKC with PMA restored tip motility and capillary formation in low but not high LET particle treated cultures. Conclusion: Low LET charged particles inhibit the early stages of vasculogenesis when tip cells have motile protrusive structures and are creating pioneer guidance tunnels through the matrix. High LET charged particles do not affect the early stages of vasculogenesis but they do affect the later stages when the endothelial cells migrate to form tubes.

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 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

Charged-particle microbeams, developed to provide targeted irradiation of individual cells, and then of sub-cellular components, and then of 3-D tissues and now organisms, have been instrumental in challenging and changing long accepted paradigms of radiation action. However the potential of these valuable tools can be enhanced by integrating additional components with the direct ability to measure biological responses in real time, or to manipulate the cell, tissue or organism of interest under conditions where information gained can be optimized. The RARAF microbeam has recently undergone an accelerator upgrade, and been modified to allow for multiple microbeam irradiation laboratories. Researchers with divergent interests have expressed desires for particular modalities to be made available and ongoing developments reflect these desires. The focus of this review is on the design, incorporation and use of multiphoton and other imaging, micro-manipulation and single cell biosensor capabilities at RARAF. Additionally, an update on the status of the other biology oriented microbeams in the Americas is provided.

Multiphoton microscopy has been added to the array of imaging techniques at the endstation for the Microbeam II cell irradiator at Columbia University’s Radiological Research Accelerator Facility (RARAF). This three-dimensional (3D), laser-scanning microscope functions through multiphoton excitation, providing an enhanced imaging routine during radiation experiments with tissuelike samples, such as small living animals and organisms. Studies at RARAF focus on radiation effects; hence, this multiphoton microscope was designed to observe postirradiation cellular dynamics. This multiphoton microscope was custom designed into an existing Nikon Eclipse E600-FN research fluorescence microscope on the irradiation platform. Design details and biology applications using this enhanced 3D-imaging technique at RARAF are reviewed.

The stand-alone microbeam at Columbia University presents a novel approach to biological microbeam irradiation studies. Foregoing a conventional accelerator as a source of energetic ions, a small, high-specific-activity, alpha emitter is used. 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(-1), 10 microm diameter microbeam can, in principle, be realised. 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 realised in any large lab. The Microbeam design as well as first tests of its performance, using an accelerator-based beam are presented here.

There is increasing evidence biological responses to ionizing radiation are not confined to those cells that are directly hit, but may be seen in the progeny at subsequent generations (genomic instability) and in non-irradiated neighbors of irradiated cells (bystander effects). These so called non-targeted phenomena would have significant contributions to radiation-induced carcinogenesis, especially at low doses where only a limited number of cells in a population are directed hit. Here we present data using a co-culturing protocol examining chromosomal instability in alpha-irradiated and bystander human fibroblasts BJ1-htert. At the first cell division following exposure to 0.1 and 1Gy alpha-particles, irradiated populations demonstrated a dose dependent increase in chromosome-type aberrations. At this time bystander BJ1-htert populations demonstrated elevated chromatid-type aberrations when compared to controls. Irradiated and bystander populations were also analyzed for chromosomal aberrations as a function of time post-irradiation. When considered over 25 doublings, all irradiated and bystander populations had significantly higher frequencies of chromatid aberrations when compared to controls (2-3-fold over controls) and were not dependent on dose. The results presented here support the link between the radiation-induced phenomena of genomic instability and the bystander effect.

An apertured microbeam is used for single‐particle single‐cell irradiation to study radiobiological effects at the Radiological Research Accelerator Facility (RARAF), Center for Radiological Research, Columbia University. The present sample targeting system involves imaging techniques and a stepping motor stage to sequentially position a cell nucleus above a vertical ion beam. An interest expressed by the biology research community in targeting subnuclear components has spurred the development of microbeam II, a next‐generation facility to include a focused ion beam and a more precise sample manipulator, a voice coil stage. Sample positioning precision will rely on a feedback circuit incorporating linear variable differential transformer (LVDT) position measurements. In addition, post‐lens electrostatic deflection is a contender for a point‐and‐shoot system that could speed up the cell irradiation process for cells within an image frame. Crucial to this development is that ion beam blow up must be minimal...

The radiation sciences are increasingly interdisciplinary, both from the research and the clinical perspectives. Beyond clinical and research issues, there are very real issues of communication between scientists from different disciplines. It follows that there is an increasing need for interdisciplinary training courses in the radiological sciences. Training courses are common in biomedical academic and clinical environments, but are typically targeted to scientists in specific technical fields. In the era of multidisciplinary biomedical science, there is a need for highly integrated multidisciplinary training courses that are designed for, and are useful to, scientists who are from a mix of very different academic fields and backgrounds. We briefly describe our experiences running such an integrated training course for researchers in the field of biomedical radiation microbeams, and draw some conclusions about how such interdisciplinary training courses can best function. These conclusions should be applicable to many other areas of the radiological sciences. In summary, we found that it is highly beneficial to keep the scientists from the different disciplines together. In practice, this means not segregating the training course into sections specifically for biologists and sections specifically for physicists and engineers, but rather keeping the students together to attend the same lectures and hands-on studies throughout the course. This structure added value to the learning experience not only in terms of the cross fertilization of information and ideas between scientists from the different disciplines, but also in terms of reinforcing some basic concepts for scientists in their own discipline. The two main fields of radiation medicine, radiology and radiation oncology, are both becoming increasingly interdisciplinary, both from the research and the clinical perspectives. Just to consider a few examples, and every reader

This paper describes the fabrication and integration of light-induced dielectrophoresis for cellular manipulation in biological microbeams. An optoelectronic tweezers (OET) cellular manipulation platform was designed, fabricated, and tested at Columbia University's Radiological Research Accelerator Facility (RARAF). The platform involves a light induced dielectrophoretic surface and a microfluidic chamber with channels for easy input and output of cells. The electrical conductivity of the particle-laden medium was optimized to maximize the dielectrophoretic force. To experimentally validate the operation of the OET device, we demonstrate UV-microspot irradiation of cells containing green fluorescent protein (GFP) tagged DNA single-strand break repair protein, targeted in suspension. We demonstrate the optofluidic control of single cells and groups of cells before, during, and after irradiation. The integration of optofluidic cellular manipulation into a biological microbeam enhances the facility's ability to handle non-adherent cells such as lymphocytes. To the best of our knowledge, this is the first time that OET cell handling is successfully implemented in a biological microbeam.