0.75 na objective

After treatment of cells, the medium was changed and cells were cultivated until the next day to allow for micronucleus formation. On the next day, cells were placed in the plate chamber of a Nikon Ti–S fluorescence microscope equipped with a motorized table and an incubation envelope (Okolab) to maintain temperature, CO₂ and humidity. The system was controlled by the software NIS-Elements Advanced Research version 5.10.01 (Nikon). The positions of the chosen micronucleated cells were saved with the software. Images were taken every 10 min for 96 h, covering up to four to five mitoses (cell cycle durations). The originally selected cells are denominated F0, whereas the following generations are denominated F1–F5. Subsequently, sequences for each position were generated. A 40 × 0.75 NA objective (Nikon) and an Andor Luca S (Andor Technology) camera with ND 64 and exposure time of 100 ms were used without binning. Each experiment was repeated five times. Two types of untreated controls were included, one subtype was micronucleated cells and the other subtype was micronucleus-free cells.

We acquired the data on a custom-built epi-fluorescence microscope, with an Olympus 20 $\times$ 0.4 NA objective with an $f=250$ mm tube lens (or Nikon 40 $\times$ 0.75 NA objective, and $f=100$ mm tube lens for 1 Hz acquisition) air objective, and a FITC emission/excitation filter and dichroic mirror set. For the illumination source, we used HORIBA DeltaDiode laser diode model DD-470L, nominal peak wavelength 470 nm spectral Full Width at Half Maximum (FWHM) 10 nm, nominal pulse width of 47 ps, and a nominal pulse energy of 15 pJ. The repetition rate of the diode was set to 25 MH–7 nsz.

All imaging experiments were conducted on glass bottom plates (Cellvis). After treatment of HeLa-H2B-GFP cells, medium was changed and treated cells were cultivated for 24 h to allow for micronucleus induction. Only after radiation, cells were trypsinised from petri dishes and transferred to a glass bottom plate. On the following day, the cell plate was mounted in the live-imaging chamber of a Nikon Ti-S fluorescence microscope equipped with a motorized table and an incubation envelope (Okolab). Temperature, CO₂ and humidity were maintained. The software NIS-Elements Advanced Research version 5.10.01 (Nikon) was used to control the system. Only cells with micronuclei were selected and positions saved for tracking these cells. Every 10 min images were taken for 96 h. Selected micronucleated cells were termed F0, the following generations were termed F1–F5. Images were taken with an Andor Luca S (Andor Technology) camera with ND 64 and exposure time of 100 ms without binning. 40 × 0.75 NA objective (Nikon) was used. Similar experiments with HeLa-H2B-GFP-Lamin B1-dsRed and HeLa-H2B-GFP-LC3B-dsRed cells were conducted after treatment with etoposide or tBHP at ND 8, 300-ms exposure time and 2 × 2-Binning using z-stacks with 5 levels in 2.5 µm steps around focal plane.

Paraffin-embedded samples were sectioned to 6 μm thickness and Verhoeff-Van Gieson stain was used (Elastic Stain Kit; Sigma-Aldrich, St Louis, Mo). The stained sections were examined on a Nikon Eclipse Ci microscope (DS-Fi2 camera), and observed using a 40× 0.75 NA objective (Nikon, Tokyo, Japan). Images were taken across the tissue cross-section and stitched with NIS Elements software (v4.13.03; Nikon). Four random cross-sectional stitched images of the entire wall per tissue were taken, with 2 tissue sections per patient imaged where possible.