Additional file 16: Video 2. Time-resolved 3D volume rendering of the formation process of an entire organoid culture observed within one Z1-FEP-cuvette. hCCAOs expressing H2B-eGFP as nuclei marker (red) and LifeAct-mCherry as F-actin cytoskeletal marker (green) were imaged for 5 days. The movie shows an excerpt of the first 10 h of the recorded data set. All organoids within the cuvette were segmented and tracked over these first 10 h of recording. The initial processes of cell cluster contraction, lumen formation and subsequent expansion are shown. Depending on the initial cell-cluster size, organoids differ in the time they need to establish a lumen. The changing colours indicate a newly segmented object at each time point. The segmentation and tracking serves only to visualise the behaviour of the organoids on a single cell level and is neither evaluated nor manually curated. Therefore, a segmented cell can disappear because of a mis-segmentation or the cell loses its signal upon cell death. Microscope: Zeiss Lightsheet Z.1; detection objective: W Plan-Apochromat 20x/1.0, illumination objective: Zeiss LSFM 10x/0.2; laser lines: 488 nm, 561 nm; filters: laser block filter (LBF) 405/488/561; voxel size: 1.02 × 1.02 × 2.00 μm3; recording interval: 30 min; 3D rendering and tracking software: Arivis Vision4D. (MP4 88,754 kb)
Download video file (86.7MB, mp4)