Dynamic fluorescence imaging of the free radical products of X-ray absorption in live cells
1 Imaging Physics Lab, Biophysics and Biochemistry Center, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD, 20892, USA
2 Laboratory of Cardiac Energetics, Integrative Physiology Center, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD, 20892, USA
Optical Nanoscopy 2012, 1:5 doi:10.1186/2192-2853-1-5Published: 2 July 2012
The immediate products of x-ray absorption in aqueous biological samples are free radicals including *OH, H2O2, *H and solvated electrons. Because their lifetimes and diffusion ranges are dependent on the local bio-molecular environment, imaging these free radicals in real-time while they are produced by a scanning x-ray nanobeam may provide a biological microscopy method of high resolution. As a first step towards this goal, we investigated the feasibility of imaging the initial free radical products of x-ray absorption in live cells using fluorescent free radical sensors. We selected six commercially available fluorescent sensors for screening tests of their sensitivities towards x-ray radiation in solution form. Two of the six dyes were found to have high sensitivities. One of the two was successfully used for dynamic confocal fluorescence imaging of x-ray generated free radicals in the intracellular space of mouse embryonic fibroblasts. Time series of fluorescence images before and during x-ray radiation were acquired. The rate of increase of cellular fluorescence showed both the initial production of free radicals by the physical ionization events as well as stimulated biological production of reactive oxygen species later on. The implications of the results for future development of microscopy techniques are discussed.