Integrated approaches for multiscale mitochondrial structure and function analysis

Abstract Mitochondria are double‐membrane organelles whose architecture enables ATP (Adenosine Triphosphate) production, redox signalling, calcium homeostasis, and apoptosis. Visualisation of mitochondria requires imaging technologies across spatial and temporal scales. Conventional fluorescence microscopy techniques, such as wide‐field, confocal, spinning‐disk, and light‐sheet microscopy, enable the real‐time observation of mitochondrial networks and dynamics in live cells. Super‐resolution methods, including structured illumination microscopy (SIM), stimulated emission depletion microscopy (STED), photoactivated localisation microscopy (PALM), stochastic optical reconstruction microscopy (STORM), and expansion microscopy, provide access to fine sub‐mitochondrial structures, such as cristae, overcoming the diffraction limit. Additionally, proximity‐based approaches such as FRET (Förster Resonance Energy Transfer), split‐fluorescent proteins, and proximity ligation assays allow researchers to probe sub‐compartmental interactions and organelle contact sites with nanometre‐level sensitivity. Electron microscopy (EM) complements optical techniques by offering near‐molecular resolution of mitochondrial ultrastructure, including membranes, cristae, and inter‐organelle interfaces. In this review, we comprehensively examined the principles, capabilities, and limitations of these diverse imaging modalities, with a focus on recent advances. We highlight the development of novel fluorescent probes, integrated correlative techniques, and computational analysis pipelines to expand the utility of mitochondrial imaging. By placing these innovations in historical and theoretical contexts, we aim to clarify how each method works and why it is suited to biological questions. Finally, we explore how mitochondrial imaging has revolutionised our understanding of physiology and pathology.