Recent technological advances and new radiobiology challenges are behind the great interest in the use of microirradiation techniques for radiobiological studies. Radiobiological microbeams are facilities able to deliver precise doses of radiation to preselected individual cells (or part of them) in vitro and assess their biological consequences on a single cell base. They are therefore uniquely powerful tools to address specific problems where very precise targeting accuracy and dose delivery are required. The majority of radiobiological microbeams are centred on particle accelerators in order to irradiated biological samples with an exact number of ions. Currently there are only three microbeam facilities in routine use which employ focused X-rays: two are based on laboratory bench X-ray sources (Queen's University Belfast and Nagasaki University) and one developed using synchrotron X-ray beams (Photon Factory in Tsukuba, Japan). While low dose rates limit laboratory bench sources to a few keV, micronsize X-ray probes of a few tens of keV are achievable using synchrotron sources. Each facility has however their own benefits and draw back points. Techniques for focusing X-rays are well established and continuously improving with focal spots down to 50 nm achievable for ultrasoft X-rays using circular diffraction gratings known as "zone plates". Reflection X-ray optics such as Kirkpatrick-Baez mirrors and polycapillary systems are also used to produce micron size X-ray spots. Combined with nano-positioning accuracy of the new generation of stages, improved optics and image analysis algorithms, X-ray microbeams are able to address radiobiological issues in an unprecedented way. Microbeams have contributed significantly to the discovery and characterization of important new findings regarding the mechanisms of interaction of ionizing radiation with cells and tissues. In particular, they have played a fundamental role in the investigation of non-targeted effects where radiation response is induced in samples whose DNA has not been directly exposed. The exquisite resolution offered by focused X-ray probes has allowed important questions regarding the locations and mechanisms of subcellular targets to be precisely addressed. Evidences of the critical role played by the cytoplasm have been collected and radio-sensitivity across the cell nucleus itself is attracting considerable interest. Moreover using the microbeam single cell approach, it has been possible to study the mechanisms underpinning the bystander effect where radiation damage is expressed in cells which have not been directly irradiated but were in contact or shared medium with directly exposed samples. As a result, microbeam facilities are regarded as a main tool for the formulation of a new radiobiological paradigm where direct damage to cellular DNA is not a requirement. Additionally, the implications of the non-targeted effects in in vivo systems and ultimately humans have still to be fully understood. The new generation of X-ray microbeams equipped with 3D image stations and higher X-ray energies offers the perfect approach to extend targeted studies to complex biological models. Finally, the single-cell approach and the high spatial resolution offered by the microbeam provide the perfect tool to study and quantify the dynamic processes associated with the production and repair of DNA damage. Using green fluorescent protein (GFP), it is now possible to follow the spatio-temporal development of the DNA damage sites which is currently of great interest in order to monitor the remodelling of chromatin structure that the cell undergoes to deal with DNA damage.
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