BPS2026 – Label-free high-precision single-molecule imaging system for probing nanoscale motion of enzymes

The crowded intracellular environment is a non-equilibrium system where metabolic activity is thought to generate non-thermal fluctuations that fluidize the cytoplasm. Theoretical models predict that enzyme molecules in the cytoplasm undergo transient directional movements (ballistic motion of enzymes) during catalytic cycles by converting reaction energy into mechanical motion, thereby generating such non-thermal fluctuations. While indirect evidence, such as enhanced diffusion (increased diffusion coefficient in the presence of substrate) and chemotaxis of enzymes (enzyme motion following substrate concentration gradients), supports this hypothesis, direct observation of this nanoscale motion has remained technically challenging. To estimate the required microscope performance, we developed a physical model of enzyme ballistic motion. Our numerical simulations indicated that direct observation requires a temporal resolution of a few microseconds and a spatial precision of approximately 10 nm. Conventional single-molecule methods, like fluorescence or dark-field microscopy, lack this required spatiotemporal resolution or interfere with intrinsic enzyme dynamics due to labelling. To overcome these limitations, we are developing a label-free interferometric scattering microscopy (iSCAT) system capable of detecting single enzymes with microsecond temporal resolution and nanometer spatial precision. Here, we report our progress in constructing this microscopy system and demonstrate its capability to track nanometer-sized particles. Our study contributes to deepening our understanding of how non-thermal fluctuations are generated and revealing the physical properties of the living cytoplasm.