Sp8 x smd

Gingiva samples were imaged using confocal microscopy (SP8-X SMD, Leica, Mannheim, Germany; 1024 × 1024 pixels; magnification: 40×) or light-sheet microscopy (LaVision BioTec, Bielefeld, Germany) with MVX10 zoom and 2× objective (Olympus, Tokyo, Japan). As dibenzyl ether (DBE, Sigma-Aldrich) is generally used as imaging medium for light-sheet microscopy and is less toxic than BABB and the RIs of BABB and DBE are similar^{9 (link)}, BABB was replaced with DBE as imaging medium for some gingiva samples.

The U2OS Flp-In/T-Rex cells stably expressing inducible GFP-tagged ZNF384 were incubated with doxycycline (2 μM) for 2.5 h before being irradiated using the previously described ultrasoft X-ray system^{19 (link)}. To obtain locally concentrated DSBs, a custom-designed irradiation mask with parallel apertures (2.5-μm wide) was placed between the bottom of the cell culture dishes and the X-ray source. Cells were irradiated for 10 s at 40 mA emission current and 6 KeV acceleration voltage, resulting in approximately 1000 DSBs per irradiated area. Images of cells and of the irradiation mask were collected five min after exposure. For experiments involving immunofluorescence imaging, cells were exposed as described above, without doxycycline preincubation. Five min after irradiation, cells were fixed and immunostained. Wide-field 3D images were acquired using a Leica DMi8 microscope (×63/1.4 NA) and deconvolved using Huygens Professional (version 19.10). Confocal images of immunostained cells were captured using a Leica SP8-X SMD (×63/1.4 NA).