This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The rapid development of optical imaging systems for diagnostic and therapeutic use has been greatly enhanced by the development of highly fluorescent probes. In particular, imaging probes such as exogenous fluorophores that are tagged to proteins or nanoparticles (e.g. polymers and other metals) have demonstrated cancer-specific imaging in small animals. Agents are typically developed to have specificity to a cancer-specific enzyme, receptor, or metabolic by-product, and thus enable functional and molecular imaging within the whole body. Optical imaging agents that emit in the near-infrared (NIR) are favorable for deep tissue imaging where background autofluorescence is low. To aid in the design of these agents, in vitro diagnostics are necessary in order to characterize lifetime and spectral changes upon binding prior to small animal injection and clinical translation. Flow cytometry is an ideal method for these characterization studies since it allows measurement of free dye, dye bound in solution to other molecules and dye bound to cells and beads. The capabilities in lifetime measurement and full spectral flow cytometry are of particular interest in characterizing the new polymer dyes due to the complex molecular interactions that can occur upon binding of the dye with the polymer as well as binding of the polymer with cellular receptors. Upon development of the full-spectral phase-sensitive flow cytometer, cells tagged with exogenous fluorophores, polymer-bound fluorophores, or nanoparticle-enhanced fluorophores, will be provided by Dr. Chun Li's group for the measurement of lifetime and discrimination of intrinsic fluorescence signatures from cells. A number of approaches to analyze fluorophore-protein and polymer constructs for diagnostic imaging will be planned. First, the possibility of multi-exponential decay is present when a fluorophore is bound to different sites on a polymer. Therefore multi-frequency measurements on a high-throughput phase system will be used to resolve heterogeneous fluorescence decay and fit the phase data to multi-exponential decay models to more accurately obtain the fluorophore concentration. Secondly, lifetime changes may result depending on proximity of the fluorophore to different polymer residues. Fluorophores that are more intercalated at one location on the polymer may not be available to quenching molecules as are fluorophores that are bound to alternate monomers. Thus, lifetime measurements will be acquired to identify the binding sites of the fluorophore to the polymer. Thirdly, lifetime changes in exogenous dyes bound to cell surfaces may occur due to self-quenching because the number of fluorophores bound to the cell varies, and the number of binding sites on the cell surface will vary. Lifetime measurements will be performed to accurately measure the quenching effects and optimize cell-surface binding. Lastly, the lifetime of a dye upon internalization into a cancer cell will be measured. Here, lifetime measurements will be useful when combined with sorting in order to separate cells that have internalized dye-ligand constructs or cells with dyes bound to receptors over-expressed on the surface of the cell.