Photoacoustic imaging (PAI) represents a hybrid, nonionizing modality, which has been of particular interest because of its satisfactory spatial resolution and high soft tissue contrast. PAI has the potential to provide both functional and molecular imaging in vivo since optical absorption is sensitive to physiological parameters. In this review we summarize our effort to advance molecular PAI with colloidal gold nanobeacons (GNB). GNB represents a robust nanoparticle platform that entraps multiple copies of tiny gold nanoparticles (2–4 nm) within a larger colloidal particle encapsulated by biocompatible synthetic or natural amphilines. The utilization of numerous small gold particles greatly amplifies the signal without exceeding the renal elimination threshold size. With fibrin-targeted GNB, the robust detection of microthrombus formed over a ruptured atherosclerotic plaque has been achieved, which offers an important opportunity to recognize patients with moderate lumen stenosis but high risk of stroke. With the use of second-generation smaller GNBs, the potential to improve sentinel lymph node assessment and biopsy was advanced with respect to rapidity and sensitivity of detection in mice. Finally, for angiogenesis, an essential microanatomical biomarker of tumor and cardiovascular disease progression, integrin-targeted GNBs allowed visualization of numerous angiogenic sprouts and bridges that were otherwise undetectable from inherent blood signal alone, offering sensitive and specific discrimination and quantification of angiogenesis in vivo. Copyright © 2011 John Wiley & Sons, Ltd.