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Mitochondria functions as a major power source in diverse cell physiological processes. Mitochondria dysfunction is associated with many diseases, such as neurodegenerative diseases and various cancers. It is crucial to understand mitochondrial metabolism in live cells and related dysfunction in diseases. Currently, three-dimensional (3D) cultured tumor spheroids have been widely used in drug development in cancer therapy fields, due to their similarities with the animal model compared with 2D cultured cells. However, characterization methods to monitor metabolism status in tumor spheroids are limited by the lack of techniques to measure mitochondrial metabolic fluxes in a noninvasive and real-time way. Here, we proposed a Fluorescence Lifetime Imaging Microscopy (FLIM) based method to monitor the mitochondria status in 3D cultured cells. MCF-7 tumor spheroids or 2D cultured cells were labelled with a mitochondria specific dye Rhodamine123 (R123). Images of cultured cells were captured with a FLIM system and analyzed with software SPCImage, providing the size and shape of each tumor spheroid as well as the fluorescence lifetime values of mitochondria. In 2D cultured cells, the fluorescence lifetime values of each dye had little fluctuation, indicating similar physiological status. In tumor spheroids labelled with R123, the fluorescence lifetime values of the inner part of spheroids were uneven compared with those of the outer edge. The reason for this uneven lifetime span of mitochondria may be associated with the difference of status between the inner and outer layers of tumor spheroids. In conclusion, this FLIM based method has shown great advantages to characterize the mitochondria status in 3D cultured tumor spheroids, providing a powerful quantitative method in cellular physiology research fields.
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