The curse of the red pearl: a fibroblast specific pearl-necklace mitochondrial phenotype caused by phototoxicity

The dynamic nature of mitochondria makes live cell imaging an important tool in mitochondrial research. Although imaging using fluorescent probes is the golden standard in studying mitochondrial morphology, these probes might introduce a-specific features. In this study, live cell fluorescent imaging was applied to investigate a pearl-necklace shaped mitochondrial phenotype that arises when mitochondrial fission is restricted. In this fibroblast specific pearl-necklace phenotype, constricted and expanded mitochondrial regions alternate. Imaging studies revealed that the formation time of this pearl-necklace phenotype differs between laser scanning confocal, widefield and spinning disk confocal microscopy. We found that the phenotype formation correlates with the excitation of the fluorescent probe and is the result of phototoxicity. Interestingly, the phenotype only arises in cells stained with red mitochondrial dyes. Serial section electron tomography pearl-necklace mitochondria revealed that the mitochondrial membranes remained intact, while the cristae structure was altered. Furthermore, filaments and ER were present at the constricted sites. This study illustrates the importance of considering experimental conditions for live cell imaging to prevent imaging artefacts that can have a major impact on the obtained results.