Application suite x v 3

Long-term live-cell imaging was conducted with a Leica SP8 lightning confocal microscope. Data were collected with Leica Application Suite X v. 3.57. The CO₂ level (5%) and both temperature (37 °C) and humidity (80%) were controlled using a gas incubation system (Ibidi). In order to visualize the nuclei, cells were incubated with 10 μM sirDNA (Spirochrome AG) 3 h prior to the measurements. Labeled nuclei were excited at a wavelength of 633 nm. The resulting signal was collected at an emission maximum of around 674 nm through a HCX PL APO 10×/0.40 CS dry objective. Dependent on the experiment the observation time varied between 12 and 72 h with time steps between each frame ranging from 5 to 30 min.

For single-cell time-lapse imaging, a culture was synchronized and stained with PDMPO (330 µM). About 8 h after the end of light starvation, when most cells had gone through cytokinesis, an aliquot of 100 µl was taken and FM4-64 (ThermoFisher Scientific, USA) was added to a final concentration of 4–8 µM to stain the membranes. After adding the membrane dye, a 20 µl drop was mounted on a microscope slide and covered with a glass coverslip, using dental wax as spacer. The samples were visualized using a Leica TCS SP8-STED confocal microscope equipped with a HCS PL APO 86×/1.20 W motCORR objective. FM4-64 and chlorophyll autofluorescence were acquired by white-light laser using 550 nm laser line (6% laser power) and 650 nm laser line (7% laser power), respectively. PDMPO fluorescence was acquired using a 405 nm laser (5% laser power). HyD-SMD detectors were used for PDMPO and FM4-64 with emission collection width set to 474–530 and 608–640 nm, respectively. Chlorophyll autofluorescence emission was collected using a HyD detector with 741–779 nm detection width and the transmission channel was detected with a PMT detector. The cells were imaged for 2–4 h at intervals of 3–60 min. Images were analyzed using Leica Application Suite X v3.5.7. In total, time lapses from over 50 cells were collected over the entire period.

Live-cell imaging was done using a Leica SP8 lightning confocal microscope and data was collected with the Leica Application Suite X v. 3.57. The microscope was equipped with an ibidi gas incubation system for CO2 and O2. Organoids were labeled with sirDNA (10 µM, Spirochrome AG) 3 h before measurement. Organoid age and observation time was set according to the planned experiment time between each image was kept at 10 min. For nuclei visualization samples were excited with 633 nm and detected at an emission maximum around 674 nm, detected with a HCX PL APO 10x/0.40 CS dry objective. Collagen fibers were either visualized by illuminating the samples with 488 nm and detecting the reflected signal or by imaging fluorescently labeled collagen using a HC PL APO 40x/1.10 water immersion objective. High-resolution images of the collagen cage were taken by using an implemented adaptive deconvolution algorithm and the usage of a HC PL APO 63x/1.40 oil immersion objective.