To investigate nanoplastics impact on mitochondrial dynamics and morphology in intestinal organoids, AO-organoids (Porgj-2) were cultured for 24 hours before NPD treatment, followed by additional 1- or 3-days culture. At each timepoint, organoids were stained with WGA (1 hour), washed three times with imaging medium, and then treated with TMRM for mitochondrial visualization. Live imaging was performed using a Leica Stellaris Falcon 8 confocal microscope (1024 × 1024 pixels, 400 Hz scan speed, excitation: 510 nm, emission: 555-650 nm for TMRM) with 2-minute time-lapse acquisition yielding 47 sequential timeframes (t1 to t47). Images were exported as OME-TIFF format using LAS X software and preprocessed to separate WGA, TMRM, and NPD channels using custom Python scripts using in Napari (Supplementary Code 3). Mitochondrial features (TMRM images) were tested using the Nellie plugin in Napari for single timeframes, and multiple timeframes were processed through custom batch-processing scripts based on Nellie code from GitHub (Supplementary Code 4) and exported as CSV files. Ten key parameters were selected: linear velocity, angular velocity, tortuosity, aspect ratio, length, solidity, extent, minor axis length, major axis length, and area, and data analysis was performed at organoid level (mean values of all mitochondria per organoid, visualized with box plots) comparing NP and no NP groups on day 1 and day 3 using custom analysis scripts (Supplementary Code 5).