Single α-particle irradiation permits real-time visualization of RNF8 accumulation at DNA damaged sites

Abstract

As well as being a significant source of environmental radiation exposure, α-particles are increasingly considered for use in targeted radiation therapy. A better understanding of α-particle induced damage at the DNA scale can be achieved by following their tracks in real-time in targeted living cells. Focused α-particle microbeams can facilitate this but, due to their low energy (up to a few MeV) and limited range, α-particles detection, delivery, and follow-up observations of radiation-induced damage remain difficult. In this study, we developed a thin Boron-doped Nano-Crystalline Diamond membrane that allows reliable single α-particles detection and single cell irradiation with negligible beam scattering. The radiation-induced responses of single 3 MeV α-particles delivered with focused microbeam are visualized in situ over thirty minutes after irradiation by the accumulation of the GFP-tagged RNF8 protein at DNA damaged sites.

Figure 1. Characterization of the BNCD membranes.

(a) Experimental set-up. The BNCD membranes were tested under vacuum for electron emission and thickness measurements. Electrical pulses induced by α-particle hits were acquired simultaneously from both CEM (channel electron multiplier) and thick silicon detectors. (b) Impact of the BNCD membranes on the transmitted energy. The red spectrum shows the energy transmitted through a 150 nm thick commercially available Si₃N₄ window. The two BNCD membranes (blue and green spectra) induce the same energy loss, thus the overlapped spectra. The black curve shows the beam energy without any material in its path. When passing through the BNCD membranes, an average energy loss of 200 keV is measured. (c) Channeltron pulse height spectra. The channeltron output signal is clearly separated from background for both BNCD membranes. The red curve shows the spectrum obtained on a Si₃N₄ window without any coating.

Figure 2. CR39 track detectors irradiated with counted α-particles.

(a) The BNCD membranes were validated as vacuum windows by irradiating track detectors or cells with single α-particles in air. Pulses from the CEM were used to trigger the beam shutter when the required number of hits was reached. (b) Single particle irradiation in a regular pattern every 10 μm. Black dots correspond to etched tracks. (c) 10 particles delivered every 20 μm. All particles are delivered in a 5-μm diameter circle.

Figure 3. Induction GFP-RNF8 and γH2AX IRIF in cells irradiated with random α-particles.

(a) Hoechst³³³⁴² staining reveals the nuclear chromatin. (b) IRIF are visualized with γH2AX immuno-detection in fixed cells exposed to ²³⁹Pu source. (c) GFP-RNF8 is re-localized to the DNA damaged areas, and (d) the merged image shows the overlap of GFP-RNF8 and γH2AX 30 minutes after irradiation.

Figure 4. Heterogeneity of GFP-RNF8 response in cell nuclei irradiated with different patterns.

Cells are irradiated with (a) one α-particle, (b) 5 α-particles distributed on a cross pattern. The targeted positions are separated by 4 μm. (c) 10 α-particles focalized on one position, and (d) regular pattern scanned over the whole microscope field of view. 8 α-particles induced-foci are distributed in the nucleus. (e) Fluorescence intensities measured along the two lines drawn in the inset plotted against their length. The blue line shows the constant nuclear fluorescence background; the orange line shows three peaks corresponding to the IRIF. Three different fluorescence intensities are observed suggesting an inhomogeneous chromatin density.

Figure 5. Time-lapse imaging of GFP-RNF8 in a cell nucleus irradiated with single α-particles.

(a) The cell nucleus is targeted and irradiated at time 0 with a single α-particle per point on a cross pattern, each point separated by 4 μm. The re-localization of GFP-RNF8 is observed over 30 min following irradiation and selected time points are shown. (b) Kinetics curves of GFP-RNF8 corresponding to each IRIF obtained from experimetal data. Experimental curves (green points) are normalized and fit (black line) with a model for the first-order response. The intensity fluorescence of GFP-RNF8 protein in the damaged areas is higly variable, as described in each inset by the A parameter. The recruitment time (T) varies independently from the intensity fluorescence reached.