This grant supports research by Chunte (Sam) Peng, an Assistant Professor of Chemistry at Massachusetts Institute of Technology (MIT) and a member of the MIT-Harvard Broad Institute, which will focus on creating novel probes for single particle tracking to quantitatively study the nonequilibrium dynamics of molecular motors in vivo, using these dynamics as a window into the functioning of living systems and far-from-equilibrium physics. Professor Peng proposes to learn about intracellular dynamics by making movies of molecular motors ‘tagged’ with fluorescent probes. The probes—called Up Conversion NanoParticles (UCNPs)—have three features that make them superior to existing fluorescent probes and which suit them for intracellular single particle tracking. First, UCNPs fluoresce at different colors than the light emitted by other cellular components, thus offering superior contrast relative to nearby objects. Second, UCNP fluorescence is spectrally much narrower than that of commonly used fluorescent probes, allowing a larger number of distinct cellular components—each tagged with a different color—to be simultaneously tracked. Third, UCNPs can be used to track a cellular object for much longer (hours or days) than is typical using existing fluorescent probes (a few minutes). Peng will use UCNPs to study the nonequilibrium dynamics of two molecular motors, dynein and kinesin, as they transport ‘cargo’ from place to place within a cell along intracellular protein polymers. The research team will attach probes to cargo and/or motors and then record movies that track probe position as revealed by the UCNP fluorescence. The plan is to begin by quantifying motor dynamics at various points in a motor’s traversal of a cell and then explore why motor behavior varies by gaining an ever-increasing level of detail about the local cellular environment. Particular experimental attention will be paid to how motor efficiency varies in relation to varying cellular conditions, efficiency-affecting interactions between motors and between motors and cargo, and the relation of observed motor efficiency to efficiency constraints predicted by thermodynamic theory.