Csux1a1 nipkow spinning disc unit

High-resolution, multiscale imaging was performed on an inverse microscope equipped with a Yokogawa CSUX1A1 Nipkow spinning disc unit using a PlanApochromat 63 × /1.4 NA oil-immersion objective. Images were acquired for 30 min repeating the 33 × 2 sinterval cycles (as described in Figure 1) with a pixel resolution of 0.211 μm. The culture medium was replaced with phenol-red free DMEM/F12 medium (Gibco) 1 h prior to imaging, and environmental conditions of 37°C and 5% CO₂ were maintained throughout the process.

For live-cell imaging, 30,000 cells were seeded on laminin-rich coated 3.5 cm glass-bottom dishes (WPI, Sarasota, FL, USA, cat. no. FD35-100). One hour after seeding, the medium was exchanged to FCS-free DMEM (high glucose and L-Glutamine, Gibco, Paisley, United Kingdom) supplemented with Penicillin-Streptomycin. The cells were incubated for an additional 23 h. Prior to imaging, the medium was exchanged for DMEM medium without phenol red and FCS (FluoroBrite DMEM, Gibco, Paisley, United Kingdom). Live-cell imaging was performed using a spinning disc microscope (Zeiss Axio Observer Z1, inverse, fully motorized, with hardware autofocus, Yokogawa CSU-X1-A1 Nipkow spinning disc unit) and a Plan-Apochromat 63×/1.4 DIC Oil objective. For UV irradiation experiments, an EC Plan-Neofluar 100×/1.30 Oil Iris objective was used. The microscope is equipped with full environmental control, and imaging took place at 37 °C. EGFP-tagged HK13 and mScarlet-tagged short EPPK1 were illuminated with a 488 nm laser (10% intensity, exposure time 150 ms) and a 561 nm laser (25% intensity, exposure time 200 ms), respectively. The signal was detected with a sCMOS: 2× pco.edge 4.2 camera system (2048 × 2048 pixel, 6.45 µm pixel size, 16 bit, 100 fps, QE 70%). Images were acquired with VisiView 5.0.0.11 acquisition software (Visitron Systems, Puchheim, Germany).