Real-Time Observation of Irradiation Response
The observation of samples in real-time during irradiations and subsequent measurements of radiation responses is important for the determination of short term effects which can affect the long term outcomes for the samples. We have developed our microbeam imaging system to use a multi-color, fast switching LED light source, in combination with our EMCCD camera, to observe multiple fluorophores in samples with a relatively high frame rate. Measurements of fluorescent intensity are made as the acquisition occurs directing the further observations to be made in that experiment.
The multicolor movie shows the response of a calcium reporter (green fluorescence) in several touching cells where one of the cells is irradiated through the nucleus (pink cross) while missing the overlapping mitochondria (red fluorescence). The change in the green fluorescent intensity as well as the time variation of that intensity demonstrates that the calcium concentration in the cells is changing on a rapid basis within the first few minutes following irradiation.
This real-time observation technique has been used to look at the temporal mechanism of DNA break repairs. The Real-Time Focus Formation movie demonstrates the observation of 9 cells being irradiated with the microbeam. These are transgenic cells that have GFP-tagged XRCC1 protein which coalesces around DNA damage sites, as part of the repair processes, which we observe as a fluorescent focus formation in each irradiated cell. The measurements shown in the three plotting panels are the intensity across one of the cells on a frame by frame basis, the maximum focus intensity of each cell through time and the area of the focus that forms in each cell. As we can see, each cell responds slightly differently to the same amount of particles. Observation of the individualized single-cell response is our focus for our microbeam irradiator.