Mitochondria are complex organelles involved in cellular metabolism, apoptosis, and intracellular signaling. During mitochondrial fusion, they merge into larger interconnected networks, whereas various factors can trigger mitochondrial fission, causing these networks to break apart into smaller fragments. Shorter mitochondria are associated with elevated oxidative stress and the development of pathophysiological conditions such as metabolic disorders, myopathies, and neurodegenerative diseases. Their morphology therefore serves as a reliable indicator of cellular state, making precise and objective quantification of morphological traits essential for understanding and monitoring disease processes. Approaches for characterizing mitochondrial fusion and fission primarily rely on fluorescence microscopy. Qualitative assessment is a common method in which cells are manually categorized based on visual assessment of mitochondrial morphology. However, this type of analysis is subjective and time-consuming, leading to the development of computer algorithms that enable objective quantification of mitochondrial morphology - although these models are often inaccessible or limited to specific use cases. Consequently, there is a growing need for new algorithms or improved versions of existing ones. To address this, we enhanced the “MiNA” algorithm by developing an ImageJ-based algorithm that enables quantitative analysis of mitochondrial length in fluorescence microscopy images. We evaluated the suitability of our cellular model using three cell lines (LA-4, MH-S, and MLg), exposed to different concentrations of H2O2 (0 mM, 0.3 mM, and 3 mM) and to media containing varying levels of nutrients (LCIS (a nutrient-free buffer), complete cell culture medium, and a 50:50 mixture of LCIS and complete medium). Cells were subjected to oxidative stress, which we hypothesized would promote mitochondrial fission, and to short-term starvation, which we expected to promote mitochondrial fusion. Treated cells were stained with the fluorescent dye MitoTracker Green and imaged using a confocal fluorescence microscope. Image analysis performed with the upgraded algorithm revealed, in most cases, a consistent shortening of mitochondrial length with increasing oxidative stress. Short-term starvation resulted in mitochondrial elongation, but only when cells were simultaneously exposed to an additional stressor (3 mM H2O2). The results of the master’s thesis showed that the algorithm, based on the established cell model, provides reliable quantification of mitochondrial morphology and thus represents a suitable tool for further investigation of the mechanisms underlying mitochondrial dynamics, as well as for potential use in studies of cellular responses to less-explored external factors.
|
