Project Summary This project describes a technology development effort that will generate three complementary methods for measuring Protein-Protein Interaction (PPI) kinetic rates between membrane proteins. In Aim 1, we develop a new algorithmic approach for single-particle tracking analysis that generates trajectories from image/movie data. It is based on Bayesian inference strategy. We then extend this to extracting kinetic rates of PPIs from two color single particle tracking data. In Aim 2, we develop a ‘smart’ microscope that is capable of adapting illumination and frame rates when a PPI is imminent (particles diffuse close to each other), thereby reducing photobleaching, allowing long-term tracking with organic fluorophores and gaining a factor of 10 to 50 in imaging speed during an interaction. In Aim 3 we implement and demonstrate a ‘quantum imaging’ approach for making a quantum optimal estimation of the distance between two fluorophores of the same type. This method can estimate the distance between incoherent point sources with an uncertainty in the distance measurement near the limit of any possible measurement and substantially better than classical image-then-estimate approaches. In practice, it gives the benefits of two-color tracking using a simplified one-color labeling scheme. Direct detection of PPIs at the single molecule level in living cells is difficult because labeling must be done at densities low enough for single fluorescent labels to be identified and detected with diffraction limited optics. Since molecules must come into contact with each other to initiate a PPI, often through diffusion limited processes, the observed interactions can be temporally rare. The new methods proposed here will allow for other fluorophores, including organic dyes and fluorescent proteins, to be used to quantify PPIs. Our long-term goal is to provide a set of methods that can report on the dynamics of various types of PPIs. The rationale for the proposed Aims is that by providing these new methods, a wider range of PPIs can be studied on living cells, opening new doors for biomedical research. This technology development project is a collaboration between three investigators with complementary expertise. K. Lidke (PI) is an expert on single molecule imaging and microscopy development. F. Becerra (co-PI) is a world leader in the area of ultra-sensitive measurements of coherent states of light using optimized photon counting measurements with fast feedback. D. Lidke (co-I) has developed and applied innovative single molecule techniques to quantify PPI in living cells.