Abstract
The spectroscopic methods commonly used to study mitochondria bioenergetics do not show the diversity of responses within a population of mitochondria (isolated or in a cell), and/or cannot measure individual dynamics. New methodological developments are necessary in order to improve quantitative and kinetic resolutions and eventually gain further insights on individual mitochondrial responses, such as studying activities of the mitochondrial permeability transition pore (mPTP ). The work reported herein is devoted to study responses of single mitochondria within a large population after isolation from cardiomyocytes. Mitochondria were preloaded with a commonly used membrane potential sensitive dye (TMRM), they are then deposited on a plasma-treated glass coverslip and subsequently energized or inhibited by additions of usual bioenergetics effectors. Responses were analyzed by fluorescence microscopy over few thousands of mitochondria simultaneously with a single organelle resolution. We report an automatic method to analyze each image of time-lapse stacks based on the TrackMate-ImageJ plug-in and specially made Python scripts. Images are processed to eliminate defects of illumination inhomogeneity, improving by at least two orders of magnitude the signal/noise ratio. This method enables us to follow the track of each mitochondrion within the observed field and monitor its fluorescence changes, with a time resolution of 400 ms, uninterrupted over the course of the experiment. Such methodological improvement is a prerequisite to further study the role of mPTP in single mitochondria during calcium transient loading.
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Acknowledgments
The project was financially supported by the ANR (“Agence Nationale pour la Recherche,” project MITOCARD n° ANR-17-CE11-0041), the University of Bordeaux, the CNRS (“Centre National de la Recherche Scientifique“) and the INSERM (“Institut National de la Santé Et de la Recherche Médicale”). CC acknowledges the University of Bordeaux (Interdisciplinary-pluridisciplinary PhD funding program 2016) for his PhD fellowship. AS acknowledges IHU-LIRYC (Pessac, France) for her PhD fellowship. This study received financial support from the French Government as part of the “Investments of the Future” program managed by the National Research Agency (ANR), Grant reference ANR-10- IAHU-04.
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Colin, C. et al. (2021). Single-Particle Tracking Method in Fluorescence Microscopy to Monitor Bioenergetic Responses of Individual Mitochondria. In: Weissig, V., Edeas, M. (eds) Mitochondrial Medicine . Methods in Molecular Biology, vol 2276. Springer, New York, NY. https://doi.org/10.1007/978-1-0716-1266-8_11
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DOI: https://doi.org/10.1007/978-1-0716-1266-8_11
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