Analysis of mitochondrial mechanical dynamics using a confocal fluorescence microscope with a bent optical fibre

摘要

The cells in the cardiovascular system are constantly subjected to mechanical forces created by blood flow and the beating heart. The effect of forces on cells has been extensively investigated, but their effect on cellular organelles such as mitochondria remains unclear. We examined the impact of nano-Newton forces on mitochondria using a bent optical fibre (BOF) with a flat-ended tip (diameter exceeding 2 μm) and a confocal fluorescence microscope. By indenting a single mitochondrion with the BOF tip, we found that the mitochondrial elastic modulus was proportional to the (-1/2) power of the mitochondrial radius in the 9.6-115 kPa range. We stained the mitochondria with a potential-metric dye (TMRE) and measured the changes in TMRE fluorescence intensity. We confirmed that more active mitochondria exhibit a higher frequency of repetitive transient depolarization. The same trend was observed at forces lower than 50 nN. We further showed that the depolarization frequency of mitochondria decreases under an extremely large force (nearly 100 nN). We conclude that mitochondrial function is affected by physical environmental factors, such as external forces at the nano-Newton level. Lay Description: The cells in the heart system are constantly influenced by mechanical forces created by blood flow and the beating heart. The effect of forces on cells has been extensively studied, but their effect on cellular organelles such as mitochondria remains unknown. Mitochondrial function is recognized as important for health, disease, and aging in human or animal cells. Therefore, we examined the impact of nano-Newton forces on mitochondria using a bent optical fibre (BOF) with a flat-ended tip (diameter exceeding 2 μm) and a confocal laser scanning microscope. In this method, the flat-ended tip of BOF is utilized to apply forces on a single isolated mitochondrion. Simultaneously, to observe the physiological changes in mitochondria depending on mechanical forces, a membrane potential sensitive dye is used to stain mitochondria. By indenting a single mitochondrion with the BOF tip, we found that the mitochondrial hardness was proportional to the (-1/2) power of the mitochondrial radius in the 9.6-115 kPa range. By observing the physiological changes in mitochondria, we confirmed that more active mitochondria exhibit a higher frequency of repetitive transient depolarization. Interestingly, the same trend was observed when applying forces (lower than 50 nN) on the mitochondrion. We further found that the depolarization frequency of mitochondria decreases under an extremely large force (nearly 100 nN). Finally, we conclude that mitochondrial function is affected by physical environmental factors, such as external forces at the nano-Newton level.