Phasor plot analysis of mitochondrial polarization in organoids

Phasor plot analysis of mitochondrial polarization in organoids. We analyzed TMRM phasor plot data to investigate NP influence on mitochondrial polarization in intestinal organoids. The PTU files were exported from LAS X lifetime module and processed using the napari-flim-phasor-plotter[HZ1] plugin (Supplementary Code 1). For each PTU file from control (no NP[BD2] ) and NPD[BD3] groups (days 1 and 3), we selected the TMRM channel and manually cropped the organoid region to remove artifacts. Phasor analysis was performed (bin size=1, threshold=10, median filter=10) at 80 MHz laser frequency. G-S coordinates and ROI measurements were exported as CSV files. Then we calculated the mean G and S from G and S coordinates in each CSV file, with data filtering (2.5-97.5 percentile outlier removal of G and S respectively) to remove extreme value, and then converted to mean using the equation: with ω = 80 MHz and draw the boxplot in control (noNP) and NP D groups (days 1 and 3) (Supplementary Code 2).