Abstract
Charged-particle microbeams (CPMs) provide a unique opportunity to investigate the effects of ionizing radiation on living biological specimens with a precise control of the delivered dose, i.e. the number of particles per cell. We describe a methodology to manipulate and micro-irradiate early stage C. elegans embryos at a specific phase of the cell division and with a controlled dose using a CPM. To validate this approach, we observe the radiation-induced damage, such as reduced cell mobility, incomplete cell division and the appearance of chromatin bridges during embryo development, in different strains expressing GFP-tagged proteins in situ after irradiation. In addition, as the dosimetry of such experiments cannot be extrapolated from random irradiations of cell populations, realistic three-dimensional models of 2 cell-stage embryo were imported into the Geant4 Monte-Carlo simulation toolkit. Using this method, we investigate the energy deposit in various chromatin condensation states during the cell division phases. The experimental approach coupled to Monte-Carlo simulations provides a way to selectively irradiate a single cell in a rapidly dividing multicellular model with a reproducible dose. This method opens the way to dose-effect investigations following targeted irradiation.
Highlights
Charged-particle microbeams (CPMs) present unique features for radiation biology studies: they allow the targeting of sub-cellular compartments with micrometre accuracy, to set a definite irradiation timing and a precise dose control at the single-cell scale[1,2,3]
As the energy deposited by charged-particles depends significantly on the geometry of the target, we developed a three-dimensional realistic voxelized model of a 2-cell stage embryo
As Phalloidin stains the actin-cytoskeleton which is absent in the nucleus, the nuclear volume is indirectly outlined by manual selection based on contrast rendering and cropping the area outside the region of interest (ROI)
Summary
Charged-particle microbeams (CPMs) present unique features for radiation biology studies: they allow the targeting of sub-cellular compartments with micrometre accuracy, to set a definite irradiation timing and a precise dose control at the single-cell scale[1,2,3]. Data from in vitro/in cellulo experiments on monolayer cell culture are very useful to dissect the molecular mechanisms induced by the exposure to ionizing radiation (IR), they are difficult to extrapolate to the in vivo response For this reason, there is a growing interest in applying CPM irradiation techniques to ex vivo and in vivo biological models. The aim of this study was to develop a methodology to micro-irradiate in a reproducible way the 2-cell stage C. elegans embryos with protons Such a micro-irradiation of a dynamic 3D biological model with a fast cell division and rapidly evolving target volumes raises experimental challenges. The validation of this procedure includes an experimental validation of our ability to reproducibly induce radiation damage in embryos. Monte Carlo simulation on realistic 2-cell stage C. elegans phantoms was performed to characterize the specific energy absorbed in different biological compartments (chromatin, nucleus, cytoplasm and whole embryo), while considering different condensation states of chromatin throughout the cell division
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