Definite focus 3

For the determination of single‐cell dry mass, cell length, and width, time‐lapse microscopy was performed using an inverted live‐cell microscope from Zeiss (Axiocam 503, Carl Zeiss Microscopy GmbH, Germany), including a temperature incubator (incubator, XLmulti S2 Dark, Pecon., Germany) mounted on the microscope stage. The system was equipped with the camera wavefront sensor c‐mounted (SID‐4‐sC8 sCMOS, Phaesics, France). The microscope set up included a 100× oil immersion objective, 60× optovar (magnification 160×, 0.045 µm px⁻¹), and an automated defined focus system (Definite Focus 3, Carl Zeiss Microscopy GmbH, Germany) to compensate thermal drifts during long term imaging time‐lapse microscopy. Time‐lapse images were recorded in parallel with ZEN blue (v 3.3, Carl Zeiss Microscopy GmbH, Germany) and PHAST (v 2.4.1, Phaesics, France) every 5 min for 24 h. Before acquisition, a modified Köhler illumination was conducted as described in SID4BIO user's manual for quantitative phase imaging. The image acquisition process was performed as described before [40 (link)].

Confocal microscopic image acquisition was performed on a Zeiss LSM980 laser scanning confocal microscope. Standard (Smart Setup) settings were used for DAPI and EdU, and SA-β-GAL was recorded using the transmitted light detector and the 639nm laser. Koehler illumination and tiling were set up as described above for wide-field microscopy. Here a Plan-Apochromat 10× NA=0.45 lens was used and 3.78 s frame time. Optical zoom of 1.0 resulted in 0.83 μm/pixel resolution in 1024×1024 tile frames. For autofocusing the Zeiss Definite Focus 3 was used. Microplate-based acquisition was set up using the AI sample finder feature of the controller software Zen 2.3, placing one tile region into the center of each well, and data were saved and analyzed as native *.czi files.