For induction of DNA damage by laser microirradiation, we used the 405 nm‐laser beam of the Nikon's A1R MP confocal and multiphoton microscope. The laser power was set at 80%, and the laser beam was focused on a selected region of interest (ROI) of the nucleus (15 μm × 6 μm) for a total microirradiation time of 40 seconds. For monitoring the response of PARP‐1 to DNA damage induction, a 65 second‐time‐lapse movie was recorded (256 × 256 pixels, 9.5 μs/pixel, 634 frames). FLIM‐FRET microscopy was performed in parallel using the microscope and acquisition settings described above. FLIM‐FRET acquisitions were recorded immediately after laser microirradiation.
For cells analyzed by immunostaining, the induction of DNA damage was set with a laser power at 100%, on a selected ROI of size 3 μm × 3 μm, for a total microirradiation time of 20 seconds. Microirradiated cells were fixed within ~5 minutes after microirradiation.
For cells analyzed by immunostaining, the induction of DNA damage was set with a laser power at 100%, on a selected ROI of size 3 μm × 3 μm, for a total microirradiation time of 20 seconds. Microirradiated cells were fixed within ~5 minutes after microirradiation.