Novel Quantitative Assessment Pipeline of Organoid Growth Dynamics Using Adapted Light Absorption and Surface Area Normalization Models

2D characterization of organoids by light microscopy with live cell imaging systems provides a powerful, rapid approach toward characterizing organoid growth patterns and behavior under different conditions with high temporal resolution. However, current conventional analysis methods display critical flaws in their approximations, including inaccurate assumptions of linear light absorption kinetics and inappropriate normalization of organoid darkness. Organoid darkness represents cellular shedding and debris accumulation in the lumen of organoids and is thus proportional to the surface area, rather than to a 2D organoid projection as is conventionally used. This novel model and image processing pipeline accounts for these shortcomings by incorporating logarithmic light absorption parameters, a noncumulative measure of darkness, and surface area‐normalized darkness values which yield accurate and highly reproducible representation of organoid growth kinetics.