Abstract LB447: Real-time visualization and quantitative analysis of mitochondrial fission and fusion using label-free 3D holotomography imaging

Abstract The balance between mitochondrial fission and fusion is essential for cellular homeostasis, with imbalances implicated in numerous diseases, making these dynamics essential biomarkers. However, morphological analysis of mitochondria using fluorescence and electron microscopy is limited by phototoxicity, photobleaching, and an inability to capture live cell dynamics. This study utilizes Holotomography (HT) imaging with the HT-X1 system for non-invasive, real-time, and 3D analysis of mitochondrial morphology without the need for fluorescent staining. We specifically investigated structural changes in mitochondria of fusion- and fission-induced Hep3B cells, capturing dose-dependent and temporal mitochondrial dynamics. Fusion and fission dynamics were induced by Mdivi-1 and MPP+, respectively. Through HT, we observed significant morphological changes, which were quantitatively assessed using the TomoAnalysis software enhanced with machine learning for accurate mitochondrial segmentation and length measurement. Additionally, we generated 8 distinct ilastik models optimized for each cell type: MCF-7, Hep3B, HeLa, Fibroblast, NIH-3T3, Huh-7, COS-7, and HepG2. By utilizing these distinct ilastik models, we achieved advanced segmentation and measurement of the morphological features of the mitochondria in each cell type. These sophisticated methodological approaches substantially advance mitochondrial research, providing reliable tools for biomarker identification and a more detailed understanding of the cellular processes involved in disease pathogenesis. Citation Format: Sumin Lee, Hoyeon Lee, Jaehyeok Lee, Minju Kim, Hye-Jin Kim, YongKeun Park. Real-time visualization and quantitative analysis of mitochondrial fission and fusion using label-free 3D holotomography imaging [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_2):Abstract nr LB447.