A robust approach in quantifying mitochondria dynamics upon transfer: insights into metabolic changes mitochondrial transfer with FLIM intensity-based image segmentation (FIBIS) algorithm

Mitochondrial transfer to recipient cells triggers increased ATP production and enhanced cellular metabolism, yet its precise impact remains unclear. Our study utilizes the intrinsic fluorescence of NADH with Fluorescence Lifetime Imaging Microscopy (FLIM) and introduces the FLIM Intensity-Based Image Segmentation (FIBIS) algorithm. FIBIS improves mitochondrial recognition precision by 35% without the need for biomarker staining. We found that introducing normal epithelial mitochondria into breast cancer cells increased bound NADH levels and their sensitivity to anti-cancer drugs, promoting oxidative stress. FIBIS proves effective in analyzing single mitochondrial metabolism in low signal-to-noise images, offering valuable insights into metabolic changes from mitochondrial transfer.