Zorikova, E. O.; Chourasia, S.; Rosenhek-Goldian, I.; Cohen, S. R.; Nesterov, S. V.; Gross, A.

Mitochondria play a pivotal role in energy production, signaling, and apoptosis. Yet, probing their functional state at the single-organelle level without invasive labels remains a major challenge. Here, we introduce a novel, label-free approach that leverages Atomic Force Microscopy (AFM) beyond its traditional imaging role, transforming it into a powerful tool for functional analysis of individual, isolated mitochondria. By immobilizing mouse liver mitochondria on polylysine-coated mica, we achieved nanoscale resolution of mitochondrial mechanical properties including height, height fluctuation power spectra, and Young\'s modulus, under different respiratory states. Strikingly, fluctuations in mitochondrial height fluctuations below 20 Hz showed robust correlation with the mitochondria membrane potential ({Delta}{psi}), a cornerstone of mitochondrial function. This relationship allows AFM to sensitively detect changes in the mitochondria bioenergetic status. Applying this method to mitochondria from liver-specific MTCH2 liver-conditional knockout mice, a model of mitochondrial malfunction, we confirmed AFM\'s diagnostic potential. The technique reliably distinguished malfunctional mitochondria, mirroring and adding new insights beyond conventional fluorescence assays. By bridging nanomechanics and mitochondrial bioenergetics, this approach paves the way for non-invasive, high-resolution diagnostics at the single-organelle level, holding promise to monitor the actual functional state of mitochondria in clinical settings.