Time-Lapse Monitoring of DNA Damage Colocalized With Particle Tracks in Single Living Cells.

PURPOSE: Understanding the DNA damage and repair induced by hadron therapy (HT) beams is crucial for developing novel strategies to maximize the use of HT beams to treat cancer patients. However, spatiotemporal studies of DNA damage and repair for beam energies relevant to HT have been challenging. We report a technique that enables spatiotemporal measurement of radiation-induced damage in live cells and colocalization of this damage with charged particle tracks over a broad range of clinically relevant beam energies. The technique uses novel fluorescence nuclear track detectors with fluorescence confocal laser scanning microscopy in the beam line to visualize particle track traversals within the subcellular compartments of live cells within seconds after injury. METHODS AND MATERIALS: We designed and built a portable fluorescence confocal laser scanning microscope for use in the beam path, coated fluorescence nuclear track detectors with fluorescent-tagged live cells (HT1080 expressing enhanced green fluorescent protein tagged to XRCC1, a single-strand break repair protein), placed the entire assembly into a proton therapy beam line, and irradiated the cells with a fluence of ∼1 × 10(6) protons/cm(2). RESULTS: We successfully obtained confocal images of proton tracks and foci of DNA single-strand breaks immediately after irradiation. CONCLUSIONS: This technique represents an innovative method for analyzing biological responses in any HT beam line at energies and dose rates relevant to therapy. It allows precise determination of the number of tracks traversing a subcellular compartment and monitoring the cellular damage therein, and has the potential to measure the linear energy transfer of each track from therapeutic beams.

Nanoscale measurements of proton tracks using fluorescent nuclear track detectors.

PURPOSE: The authors describe a method in which fluorescence nuclear track detectors (FNTDs), novel track detectors with nanoscale spatial resolution, are used to determine the linear energy transfer (LET) of individual proton tracks from proton therapy beams by allowing visualization and 3D reconstruction of such tracks. METHODS: FNTDs were exposed to proton therapy beams with nominal energies ranging from 100 to 250 MeV. Proton track images were then recorded by confocal microscopy of the FNTDs. Proton tracks in the FNTD images were fit by using a Gaussian function to extract fluorescence amplitudes. Histograms of fluorescence amplitudes were then compared with LET spectra. RESULTS: The authors successfully used FNTDs to register individual proton tracks from high-energy proton therapy beams, allowing reconstruction of 3D images of proton tracks along with delta rays. The track amplitudes from FNTDs could be used to parameterize LET spectra, allowing the LET of individual proton tracks from therapeutic proton beams to be determined. CONCLUSIONS: FNTDs can be used to directly visualize proton tracks and their delta rays at the nanoscale level. Because the track intensities in the FNTDs correlate with LET, they could be used further to measure LET of individual proton tracks. This method may be useful for measuring nanoscale radiation quantities and for measuring the LET of individual proton tracks in radiation biology experiments.

Co-visualization of DNA damage and ion traversals in live mammalian cells using a fluorescent nuclear track detector.

The geometric locations of ion traversals in mammalian cells constitute important information in the study of heavy ion-induced biological effect. Single ion traversal through a cellular nucleus produces complex and massive DNA damage at a nanometer level, leading to cell inactivation, mutations and transformation. We present a novel approach that uses a fluorescent nuclear track detector (FNTD) for the simultaneous detection of the geometrical images of ion traversals and DNA damage in single cells using confocal microscopy. HT1080 or HT1080-53BP1-GFP cells were cultured on the surface of a FNTD and exposed to 5.1-MeV/n neon ions. The positions of the ion traversals were obtained as fluorescent images of a FNTD. Localized DNA damage in cells was identified as fluorescent spots of γ-H2AX or 53BP1-GFP. These track images and images of damaged DNA were obtained in a short time using a confocal laser scanning microscope. The geometrical distribution of DNA damage indicated by fluorescent γ-H2AX spots in fixed cells or fluorescent 53BP1-GFP spots in living cells was found to correlate well with the distribution of the ion traversals. This method will be useful for evaluating the number of ion hits on individual cells, not only for micro-beam but also for random-beam experiments.

Absorbed dose in ion beams: comparison of ionisation- and fluence-based measurements.

A direct comparison measurement of fluorescent nuclear track detectors (FNTDs) and a thimble ionisation chamber is presented. Irradiations were performed using monoenergetic protons (142.66 MeV, ϕ=3×10(6) cm(-2)) and carbon ions (270.55 MeV u(-1), ϕ=3 × 10(6) cm(-2)). It was found that absorbed dose to water values as determined by fluence measurements using FNTDs are, in case of protons, in good agreement (2.4 %) with ionisation chamber measurements, if slower protons and Helium secondaries were accounted for by an effective stopping power. For carbon, however, a significant discrepancy of 4.5 % was seen, which could not be explained by fragmentation, uncertainties or experimental design. The results rather suggest a W-value of 32.10 eV ± 2.6 %. Additionally, the abundance of secondary protons expected from Monte-Carlo transport simulation was not observed.

Subcellular spatial correlation of particle traversal and biological response in clinical ion beams.

PURPOSE: To report on the spatial correlation of physical track information (fluorescent nuclear track detectors, FNTDs) and cellular DNA damage response by using a novel hybrid detector (Cell-Fit-HD). METHODS AND MATERIALS: The FNTDs were coated with a monolayer of human non-small cell lung carcinoma (A549) cells and irradiated with carbon ions (270.55 MeV u(-1), rising flank of the Bragg peak). Phosphorylated histone variant H2AX accumulating at the irradiation-induced double-strand break site was labeled (RIF). The position and direction of ion tracks in the FNTD were registered with the location of the RIF sequence as an ion track surrogate in the cell layer. RESULTS: All RIF sequences could be related to their corresponding ion tracks, with mean deviations of 1.09 µm and -1.72 µm in position and of 2.38° in slope. The mean perpendicular between ion track and RIF sequence was 1.58 µm. The mean spacing of neighboring RIFs exhibited a regular rather than random spacing. CONCLUSIONS: Cell-Fit-HD allows for unambiguous spatial correlation studies of cell damage with respect to the intracellular ion traversal under therapeutic beam conditions.

Engineering cell-fluorescent ion track hybrid detectors.

BACKGROUND: The lack of sensitive biocompatible particle track detectors has so far limited parallel detection of physical energy deposition and biological response. Fluorescent nuclear track detectors (FNTDs) based on Al2O3:C,Mg single crystals combined with confocal laser scanning microscopy (CLSM) provide 3D information on ion tracks with a resolution limited by light diffraction. Here we report the development of next generation cell-fluorescent ion track hybrid detectors (Cell-Fit-HD). METHODS: The biocompatibility of FNTDs was tested using six different cell lines, i.e. human non-small cell lung carcinoma (A549), glioblastoma (U87), androgen independent prostate cancer (PC3), epidermoid cancer (A431) and murine (VmDk) glioma SMA-560. To evaluate cell adherence, viability and conformal coverage of the crystals different seeding densities and alternative coating with extracellular matrix (fibronectin) was tested. Carbon irradiation was performed in Bragg peak (initial 270.55 MeV u⁻¹). A series of cell compartment specific fluorescence stains including nuclear (HOECHST), membrane (Glut-1), cytoplasm (Calcein AM, CM-DiI) were tested on Cell-Fit-HDs and a single CLSM was employed to co-detect the physical (crystal) as well as the biological (cell layer) information. RESULTS: The FNTD provides a biocompatible surface. Among the cells tested, A549 cells formed the most uniform, viable, tightly packed epithelial like monolayer. The ion track information was not compromised in Cell-Fit-HD as compared to the FNTD alone. Neither cell coating and culturing, nor additional staining procedures affected the properties of the FNTD surface to detect ion tracks. Standard immunofluorescence and live staining procedures could be employed to co-register cell biology and ion track information. CONCLUSIONS: The Cell-Fit-Hybrid Detector system is a promising platform for a multitude of studies linking biological response to energy deposition at high level of optical microscopy resolution.

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.

Novel Al2O3:C,Mg fluorescent nuclear track detectors for passive neutron dosimetry.

The latest advances in the development of a fluorescent nuclear track detector (FNTD) for neutron and heavy charged particle dosimetry are described and compared with CR-39 plastic nuclear etched track detectors (PNTDs). The technique combines a new luminescent aluminium oxide single crystal detector (Al(2)O(3):C,Mg) with an imaging technique based on laser scanning and confocal fluorescence detection. Detection efficiency was obtained after irradiations with monoenergetic neutron and proton beams. Dose dependences were measured for different configurations of the detectors exposed in fast- and thermal-neutron fields. A specially developed image processing technique allows for fast fluorescent track identification and counting. The readout method is non-destructive, and detectors can be reused after thermal annealing.

New Al2O3:C,Mg crystals for radiophotoluminescent dosimetry and optical imaging.

Optical and dosimetric properties of a new radiophotoluminescent material based on aluminum oxide doped with carbon and magnesium (Al2O3:C,Mg) and having aggregate oxygen vacancy defects are presented. The Al2O3:C,Mg crystals are characterized by several new optical absorption and emission bands. It is suggested that the main optical properties of this material are due to the formation of aggregate defects composed of two oxygen vacancies and two Mg-impurity atoms. Radiation-induced optical absorption bands are centered at 335 and 620 nm and produce fluorescent emission at 750 nm with a 75 +/- 5 ns lifetime. The dose measurements are performed by illumination of the Al2O3:C,Mg crystal with 335 nm or 650 nm light and by measuring the intensity of the 750 nm fluorescence. The detector material is insensitive to room light before and after the irradiation and the traps are stable up to 600 degrees C. A dose measurement range between 5 mGy and 200 Gy, suitable for therapeutic radiology applications, was demonstrated. The short luminescent lifetime and nondestructive readout is favorable for imaging applications.

Confocal fluorescent imaging of tracks from heavy charged particles utilising new Al2O3:C,Mg crystals.

A completely optical, non-destructive imaging of tracks in a fluorescent crystal provides a new way to detect and to assess doses from heavy charged particles and neutrons. The technique combines confocal fluorescent microscopy with a new radiation-sensitive, luminescent material based on aluminium oxide single crystals doped with carbon, magnesium and having aggregate oxygen vacancy defects (Al2O3:C,Mg). Radiation-induced colour centres in the new material have an absorption band at 620 nm and produce fluorescence at 750 nm with a high quantum yield and a short, 75 +/- 5 ns, fluorescence lifetime. Three-dimensional spatial distribution of fluorescent intensity allows one to obtain depth-dose distributions and to discriminate between high- and low-linear energy transfer radiations. Images of single tracks produced by different types of radiation have been obtained. Irradiations with a calibrated 241Am alpha source showed high efficiency for track detection. Thermal neutrons were detected using a nuclear reaction with a 6LiF radiator and production of alpha particles and tritium ions. Fast neutrons were detected using recoil protons produced in a polyethylene radiator installed in front of the crystalline detector. Three-dimensional reconstruction of a recoil proton propagating through the crystal was demonstrated.