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 (ΔΨ_m) 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 (<i>MFN1</i> or <i>MFN2</i>) yields stiff, low-fluctuation mitochondria with reduced ΔΨ_m, whereas MTCH2 loss produces stiff, high-fluctuation, high-ΔΨ_m mitochondria. These three label-free features provide reproducible single-organelle "fingerprints" that resolve bioenergetic states and molecular defects and complement fluorescence and respirometry.