From Microscopy to Nanoscopy: Contemporary Physical Methods in Mitochondrial Structural Biology.

Mitochondria play a crucial role in cellular bioenergetics, signaling, and metabolism; yet, many fundamental mechanisms such as the proton transfer along the membranes, the link between membrane curvature and oxidative phosphorylation, and the nanoscale organization of enzyme supercomplexes remain poorly understood due to the limitations of classical biochemical approaches. This review addresses this gap by systematically analyzing the contemporary physical methods used to investigate the mitochondrial structure and function from the micro to nano scale. It covers advanced fluorescence and super-resolution microscopy, electron and volume electron microscopy, and scanning probe techniques, as well as cryo-electron tomography for resolving supramolecular assemblies in near-native conditions. The review highlights the applications of the modern fluorescent probes, expansion and phase microscopy, and machine-learning-based image analysis for a quantitative assessment of the mitochondrial morphology, membrane potential, and dynamics in living cells and tissues. Complementary spectroscopic and scattering methods, including Raman spectroscopy, NMR, and X-ray and neutron scattering, are discussed as tools for probing the redox state, metabolite composition, and membrane organization. Emphasis is placed on integrating high-resolution experimental data with advanced computational frameworks to test competing models of mitochondrial function and pathology, and to guide the development of biomimetic and biomedical technologies.