PROJECT SUMMARY: A Quantum Dot Probe for Nanosecond-Timescale Imaging of Fast Biological Processes A great number of known biological functions ? and undoubtedly a much larger number of as-yet unrecognized processes ? are performed by, gated by, or otherwise linked to the motions of small molecules, ions, and protein residues that change geometry or diffuse over biologically relevant distances in nanoseconds (ns, 10-9 s), a timescale readily accessible by a suite of optical methods. Due to limitations of the optical probe or the detector (or both), however, nearly all measurements of evolving biological systems record events with ms (10-3 s) time resolution. The exciting questions are then: What are we missing? How could the search for pharmacological targets be improved by high-time resolution measurements of evolving biological systems? Many examples of fast conformational changes, binding events, redox events, and ion flows critical for biological functions have at least one thing in common: they are coupled to proton (H+) fluxes, and can, in principle, be monitored via high- time resolution tracking of local H+ concentrations. The proposed research program will develop a fundamentally new class of fluorescent quantum dot (QD)-ligand probes to enable all-optical measurements of fast biological processes in live cells using H+?s as an analyte, with nanosecond time resolution. At the end of the 2-yr project period, we aim to have evaluated the feasibility of our ultrafast H+ probe, by exploring strategies to optimize the brightness, sensitivity, and response time of this probe and evaluating the robustness of these properties in simulated biological environments. The longer-term vision for this technology is that it be used within diffraction-limited, and eventually super-resolution, microscopy setups to image processes in space and time with an unprecedented level of detail, and thereby connect pathologies of a vast array of diseases with their underlying molecular-level mechanisms. Our proposed QD-ligand sensor is a visible light- or near-infrared light- emitting QD, coated in organic ligands that introduce tens to hundreds of acidic sites within angstroms of the QD surface. The pKa values at these sites are tunable within various physiologically relevant ranges of pH. The photo-excited state (or ?exciton?) of the QD is an electric dipole itself, so when it ?sees? electric fields generated by, for instance, charged molecules on the surface, the wavelength of the photons that the QD emits changes on the timescale of travel of the electric field (~10-15 s). The color of the QD?s emission is therefore sensitive to the local concentration of H+?s via reversible protonation and deprotonation of its ligands. Importantly, because of the electric field-based sensing mechanism, the change in emission wavelength of the QD H+ sensor should occur effectively instantaneously with a change in local H+ concentration. In contrast, due to the conformational changes, redox processes, proton transfer, or energy transfer required for emission shifts in state-of-the-art GFP- based pH sensors, these sensors have response times of ~20 ms (with an estimated lower limit of 0.5 ms), at least a factor of 105-106 slower than the targeted response time of our QD sensor.