Plasma Membrane Staining and Live-Cell Confocal Imaging

After post-irradiation incubation of 1, 6, 24, and 72 h, the cells were labeled with a 1.5X CellMask Orange plasma membrane stain solution (Thermo Fisher Scientific) for 10 min at 37°C, 100% humidity, and 5% CO₂, resulting in a stable, homogeneous fluorescence labeling of the plasma membrane in living cells.

For live-cell confocal imaging, a custom-made live-cell imaging container and a confocal microscope (Leica TCS SP8 3X) were used. Sample, microscope stage, and microscope were kept at a constant temperature of 37°C by a climate chamber. The excitation laser wavelength for CellMask Orange was 554 nm, with a detection range of 567–635 nm. Laser power for the excitation laser was in the range of 5 mW. In order to record a large area with best resolution, mosaic images were acquired using a 100× oil objective (Leica HCX PL APO 100x/1.4 Oil), resulting in a lateral resolution of 250 nm and an axial resolution of about 600 nm. Per sample, 100 partial images with an overlap of 20% were acquired and collected together to create one final merged image. This image has a size of about 670 μm × 670 μm. Each final merged image contains between 30 and 200 cells per sample. All samples were acquired in z-stacks with a step size of 400 nm and a pixel size of 40 nm. Live-cell imaging was preferred to cell fixation in order to avoid TNT breakage and distortion (18). The cells were scanned bidirectionally and with a scanning speed of 600 Hz to ensure a fast image acquisition, which reduces movement artifacts and stressing of the cells caused by long light exposures. Additionally, the complete image acquisition duration was kept under 1 h to ensure as few network changes as possible during image capture.