Far-UVC light: A new tool to control the spread of airborne-mediated microbial diseases.

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.

Automated microbeam observation environment for biological analysis-Custom portable environmental control applied to a vertical microbeam system.

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.

207-nm UV Light-A Promising Tool for Safe Low-Cost Reduction of Surgical Site Infections. II: In-Vivo Safety Studies.

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.

An automated imaging system for radiation biodosimetry.

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.

207-nm UV light - a promising tool for safe low-cost reduction of surgical site infections. I: in vitro studies.

BACKGROUND: 0.5% to 10% of clean surgeries result in surgical-site infections, and attempts to reduce this rate have had limited success. Germicidal UV lamps, with a broad wavelength spectrum from 200 to 400 nm are an effective bactericidal option against drug-resistant and drug-sensitive bacteria, but represent a health hazard to patient and staff. By contrast, because of its limited penetration, ~200 nm far-UVC light is predicted to be effective in killing bacteria, but without the human health hazards to skin and eyes associated with conventional germicidal UV exposure. AIMS: The aim of this work was to test the biophysically-based hypothesis that ~200 nm UV light is significantly cytotoxic to bacteria, but minimally cytotoxic or mutagenic to human cells either isolated or within tissues. METHODS: A Kr-Br excimer lamp was used, which produces 207-nm UV light, with a filter to remove higher-wavelength components. Comparisons were made with results from a conventional broad spectrum 254-nm UV germicidal lamp. First, cell inactivation vs. UV fluence data were generated for methicillin-resistant S. aureus (MRSA) bacteria and also for normal human fibroblasts. Second, yields of the main UV-associated pre-mutagenic DNA lesions (cyclobutane pyrimidine dimers and 6-4 photoproducts) were measured, for both UV radiations incident on 3-D human skin tissue. RESULTS: We found that 207-nm UV light kills MRSA efficiently but, unlike conventional germicidal UV lamps, produces little cell killing in human cells. In a 3-D human skin model, 207-nm UV light produced almost no pre-mutagenic UV-associated DNA lesions, in contrast to significant yields induced by a conventional germicidal UV lamp. CONCLUSIONS: As predicted based on biophysical considerations, 207-nm light kills bacteria efficiently but does not appear to be significantly cytotoxic or mutagenic to human cells. Used appropriately, 207-nm light may have the potential for safely and inexpensively reducing surgical-site infection rates, including those of drug-resistant origin.

UV microspot irradiator at Columbia University.

The Radiological Research Accelerator Facility 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 a live zebrafish embryo.

Optofluidic cell manipulation for a biological microbeam.

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.

An accelerator-based neutron microbeam system for studies of radiation effects.

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 <20 µm in diameter for cells attached to a 3.8-µm thick propylene-bottomed cell dish in contact with the target backing. The neutron flux will roughly be 2000 per second based on the current beam setup at the RARAF singleton accelerator. The dose rate will be about 200 mGy min⁻¹. The principle of this neutron microbeam system has been preliminarily tested at the RARAF using a collimated proton beam. The imaging of the neutron beam was performed using novel fluorescent nuclear track detector technology based on Mg-doped luminescent aluminum oxide single crystals and confocal laser scanning fluorescent microscopy.

Microbeam-integrated multiphoton imaging system.

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.