Ld lci plan apochromat 25x 0.8 na

Here, we refer to single time-points acquisitions and not to time-series commonly known as 4D imaging. For live cell imaging, cultures in HPF carriers were washed with PBS once, transferred to a 35 mm or 10 mm diameter cell culture dish. For samples devoid of genetic fluorescent tags, the HPF carriers were incubated for 20 min with the desired live dyes diluted in the growth medium to the following final concentrations: SiR-actin 100 μM, Hoechst-33342 10 μM, FM4-64 2 μM and BODIPY 493/503 1 μM. The culture dish was connected to the microscope objective by a drop of deionized water, and HPF carriers were flipped with the recess facing the microscope objective lens. Live cell imaging was performed at 37°C and 5% CO₂ with a Zeiss LSM 780 NLO (Carl Zeiss AG, Jena, Germany) equipped with a LD LCI Plan-Apochromat 25x 0.8 NA water immersion objective. To image the whole HPF carrier volume and locate single organoids, we first detected cell nuclei marked with Hoechst-33342 and acquired z-stacks with 6x6 tiles of 512² pixels with 10% overlap, and 10 μm z-step. The final montage was stitched using ZEN-black software. For single organoids, we acquired z-stacks of 1024² pixels at different z-steps ranging from 1.0 to 1.8 μm.

Images were collected on a Zeiss LSM 780 inverted point scanning confocal microscope with a Zeiss LD LCI Plan-Apochromat 25x/0.8 N.A. multi-immersion objective for overview images and a Zeiss Plan Apochromat 63x/1.4 N.A. oil-immersion objective for higher magnification images. Laser settings were adjusted for each sample, but kept constant throughout image collection within the same areas between Pet1-Di and Control-Di sections. The images were imported to and processed with ImageJ (Fiji distribution) for brightness and contrast adjustment, which were also kept constant between Pet1-Di and Control-Di sections.