Force spectrum microscopy

Force Spectrum Microscopy (FSM) is an application of active microrheology developed to measure aggregate random forces in the cytoplasm. Large, inert flow tracers are injected into live cells and become lodged inside the cytoskeletal mesh, wherein it is oscillated by repercussions from active motor proteins. The magnitude of these random forces can be inferred from the frequency of oscillation of tracer particles. Tracking the fluctuations of tracer particles using optical microscopy can isolate the contribution of active random forces to intracellular molecular transport from that of Brownian motion.

FSM was developed by Ming Guo and David A. Weitz to probe stochastic intracellular forces generated by motor proteins. Far from a liquid void, the cytoplasm contains a complex meshwork of actin and myosin conferring structural support to the cell, as well as harbouring vesicles and mitochondria among other organelles. Recent research on the macromolecular crowding inside the cytoplasm raises concerns whether diffusive-like motion of large molecules have been mistakenly attributed to Brownian forces. Instead, there are suspicions that myosin motor proteins, which tug randomly on the actin filaments embedded with large molecules, give rise to diffusive-like motion of molecules inside cells. Guo et al. developed an assay to distinguish whether particle motion inside cells are driven by thermal diffusion or by repercussions from active motor proteins like non-muscle myosin II shaking the cellular cytoskeleton.

FSM relies on injecting tracer particles coated with polyethylene glycol (PEG) larger than the cytoskeletal mesh size (>50 nm), settling in between an internetwork of actin filaments and myosin motor proteins. As myosin motor proteins tug on actin filaments to perform cellular work, these actin fluctuations invariably oscillate neighboring PEGylated particles. The magnitude of tracer fluctuation is proportional to the magnitude of aggregate active motor forces. Thus, by recording the displacement of tracer oscillations, FSM can gauge and derive the magnitude of forces exerted by active motor proteins.

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