Fig. 2

General workflow of automated image processing and data analysis pipeline. A Isolated protoplasts are immobilized into 96-well plates and subjected to automated microscopy with daily recordings in bright field mode for 4 days after immobilization (DAIs). Chronological image stacks are generated and subjected to positional correction of slight cell shifts occurring during repeated recordings. B Bright field images are cropped into 9 equal tile sections in order to accelerate further image processing. After preprocessing cell segmentation is done by U-net. This generates result files listing all the identified cells and their corresponding features such as cell position coordinates, area and circularity (C). D After removal of cell clusters and contacting cells, the result file of each well at each time point is used to track individual single cells using Euclidean distance of cell centroid between two time points. E Using this result file, statistical data analysis is carried out to build cell area distribution of cells tracked between two time points. The results of rigorous statistical analysis is used to infer the effect of different conditions on cell behavior, such as expansion and proliferation, and guide future experiments