The number of thyroid cancer cases has more than doubled in the past few decades. Most common treatment for all detected cancers, regardless of their size or invasiveness, is a partial or full thyroidectomy. Current clinical imaging techniques cannot distinguish between benign and malignant nodules or between aggressive and indolent thyroid malignancy. The specificity limitations of the imaging techniques lead to the overwhelmingly high number of nodule biopsies as well as to the patient overtreatment. Photoacoustic imaging, with good depth penetration in combination with high contrast and high resolution has a great potential to be clinically utilized for non-invasive, non-ionizing visualization of a superficial organ such as the thyroid. In our preliminary studies focused on follicular thyroid cancer, we successfully developed and utilized a photoacoustic imaging agent that imparts high tumor specificity by targeting thyroid cancer specific biomarker, matrix metalloproteinases (MMP). In this project we propose to optimize the original design and fully characterize the novel agent to develop a non-invasive, clinically applicable molecular photoacoustic imaging strategy for diagnosis of thyroid cancer. We hypothesize that the addition of molecular information will impart necessary specificity to the already sensitive and widely spread technique of thyroid ultrasound imaging. We will first improve signaling, targeting and delivering mechanism of the agent by: selecting the most suitable small molecule dyes (signaling); enhancing substrate specificity for MMP-9 (targeting); and optimizing a delivering vehicle for maximum cellular uptake (delivery). The in vivo behavior of the optimized agent will then be fully characterized by investigating the whole-body and tumor biodistribution, and performing detailed Absorption, Distribution, Metabolism, Excretion and Toxicity (ADME-Tox) studies. Using the optimized and pharmacokinetically characterized agent we will perform detailed photoacoustic imaging of orthotopic thyroid tumors in mice and will correlate the intensity of signal in vivo with the level of the MMP-9 present in the tumors. Finally, to facilitae the clinical translation process, we will develop an imaging protocol utilizing the agent in conjunction with the in-house-made clinical photoacoustic/ultrasound instrument. The results obtained from this proposal are expected to significantly accelerate the translation of molecular photoacoustic imaging from the bench to the physician's room. The imaging strategy that can detect malignant thyroid lesions and identify patients that need treatment would bring major benefit to patient management and reduce the healthcare costs associated with high number of biopsies and unnecessary surgeries.