The shortwave infrared (SWIR) region of the electromagnetic spectrum has become an exciting avenue for imaging in mammals due to the increased penetration of light through tissue, decreased light scattering, and minimal autofluorescence. In the previous granting period, we showcased an additional advantage of the SWIR region– the enhanced spectral real estate which facilitates non-invasive multicolor imaging in mice. Taken together, all these advantages set the stage for: 1) fundamental biological studies in animal models analogous to those which have been enormously successful in cells and transparent organisms, 2) the ability to evaluate controls in same animal as the experimental group, and 3) advanced clinical diagnostics for both intraoperative and non-invasive use. While the SWIR region has great potential, it cannot be realized without biocompatible contrast agents. This proposal focuses on contrast agent development for high resolution multicolor imaging in the SWIR region, building on the expertise we have gained in polymethine fluorophores over the last granting period. We have prepared over 100 polymethine fluorophores and gained predictive metrics on the most classical photophysical parameters of lmax.abs, lmax,em and FF. We have also learned some key lessons regarding utility of the fluorophores including how to solubilize them in water and have gained appreciation for the different approaches to formulating fluorophores for imaging. In collaboration with the Bruns laboratory, we demonstrated excitation-based multicolor imaging with fluorophores well-matched to common laser lines. In the next granting period, we specifically focus on optimizing fluorophores for use in excitation-based multiplexing with detection in the high-resolution region of the SWIR (above 1400 nm) such that mm resolution can be achieved. Aim 1 explores the chromophore scaffold itself optimizing the photophysical properties for maximum absorption at commercial laser lines and emission >1400 nm. We take a physical organic chemistry approach to fluorophore optimization and capitalize on the continually growing, fully characterized set of systematically modified fluorophores our laboratory has prepared. Our work is aided by computation and theory collaborations with the Lopez and Caram Laboratories. Aim 2 transforms the lead fluorophores from aim 1 into water-soluble, targeted contrast agents. Finally in Aim 3, we aim image the lymphatic system, demonstrating the need for mm resolution imaging and then using excitation-based multicolor imaging to evaluate sentinel lymph node mapping and cancer metastasis.