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Visualisation of mitochondria requires imaging technologies across spatial and temporal scales. Conventional fluorescence microscopy techniques, such as wide\u2010field, confocal, spinning\u2010disk, and light\u2010sheet microscopy, enable the real\u2010time observation of mitochondrial networks and dynamics in live cells. Super\u2010resolution 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\u2010mitochondrial structures, such as cristae, overcoming the diffraction limit. Additionally, proximity\u2010based approaches such as FRET (F\u00f6rster Resonance Energy Transfer), split\u2010fluorescent proteins, and proximity ligation assays allow researchers to probe sub\u2010compartmental interactions and organelle contact sites with nanometre\u2010level sensitivity. Electron microscopy (EM) complements optical techniques by offering near\u2010molecular resolution of mitochondrial ultrastructure, including membranes, cristae, and inter\u2010organelle 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.<\/jats:p>","DOI":"10.1111\/jmi.70050","type":"journal-article","created":{"date-parts":[[2025,12,13]],"date-time":"2025-12-13T01:12:55Z","timestamp":1765588375000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["Integrated approaches for multiscale mitochondrial structure and function analysis"],"prefix":"10.1111","author":[{"given":"Adiba","family":"Patel","sequence":"first","affiliation":[{"name":"Department of Biology Indian Institute of Science Education and Research Tirupati (IISERT)  Tirupati Andhra Pradesh India"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Prasanna","family":"Venkhatesh","sequence":"additional","affiliation":[{"name":"Department of Biology Indian Institute of Science Education and Research Tirupati (IISERT)  Tirupati Andhra Pradesh 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