Anupam Patgiri (@apatgiri.bsky.social)

Bridging nanomechanics and bioenergetics of single mitochondria by atomic force microscopy Mitochondria orchestrate energy conversion and cell fate, yet label-free approaches that report both functional and physical states at the single-organelle level are nonexistent. Here, we combine atomic force microscopy (AFM) imaging with single-mitochondrion phenotyping by quantifying stiffness, height, and spontaneous low-frequency height fluctuations at the nanoscale. Across respiratory activators, inhibitors, and uncouplers, the integrated 0- to 20-Hz fluctuation power correlates with mitochondrial membrane potential (ΔΨm) and does not covary with changes in mitochondrial height (a proxy for swelling). In liver mitochondria lacking mitochondrial carrier homolog 2 (MTCH2), a regulator of mitochondrial metabolism, dynamics, and apoptosis, AFM reveals a compact, mechanically stiff, high-fluctuation state consistent with hyperpolarization and distinct from inhibited/uncoupled signatures. Extending the assay to mitochondria isolated from mouse embryonic fibroblasts, AFM data can distinguish between genotypes: loss of the mitochondrial pro-fusion proteins mitofusin 1 or 2 ( MFN1 or MFN2 ) yields stiff, low-fluctuation mitochondria with reduced ΔΨm, whereas MTCH2 loss produces stiff, high-fluctuation, high-ΔΨm mitochondria. These three label-free features provide reproducible single-organelle “fingerprints” that resolve bioenergetic states and molecular defects and complement fluorescence and respirometry. This study includes no data deposited in external repositories. All data supporting the findings of this study are available within the article and its supplementary information files. Original data images are available from the corresponding author upon reasonable request. https://www.life-science-alliance.org/content/9/5/e202503602