Near-infrared fluorescent nanoprobes for irreversibility in nonequilibrium actomyosin networks

Abstract: Actomyosin networks operate far from equilibrium, yet detecting the onset of motor-driven irreversible dynamics remains challenging. Here, we embed near-infrared (NIR) fluorescent single-walled carbon nanotubes (SWCNTs) within reconstituted actin networks, and use their nonphotobleaching emission to optically report ATP-powered myosin contractile activity. G-actin-dispersed SWCNTs are incorporated into polymerized F-actin without perturbing network assembly, enabling long-term, single-emitter fluorescence monitoring. Upon myosin addition, the NIR fluorescence levels of individual SWCNTs exhibit enhanced temporal fluctuations, and population-level statistics reveal deviations from equilibrium behaviour. The index of dispersion (IOD) distributions shift and broaden relative to equilibrium baselines, and the Kullback-Leibler divergence between IOD distributions systematically increases with increasing motor activity. Stationarity analysis further shows a dose-dependent increase in the fraction of nonstationary fluorescence traces, indicating the emergence of irreversible, time-evolving dynamics. These results establish SWCNTs as minimally invasive optical probes of irreversibility in nonequilibrium actomyosin assemblies, with broad applicability to other active biopolymer systems.