Project Summary/Abstract Biomedical imaging is an essential modality used in clinical diagnosis. Common imaging modalities such as magnetic resonance imaging (MRI), X-ray imaging, and positron emission tomography (PET), are constrained by cost, acquisition time, and/or use of ionizing radiation. Fluorescence imaging is an optimal modality for biomedical imaging, as it is non-invasive, inexpensive, and safe for living systems. Presently, fluorescence imaging uses near-infrared light (NIR, 700–1000 nm), but the shortwave infrared region (SWIR, 1000–2000 nm) of the electromagnetic spectrum has emerged as a superior region for fluorescence imaging. Advantages such as the reduced light scattering and increased tissue penetration of these lower energy photons, leads to dramatic increases in contrast compared to the NIR and drives innovation for SWIR fluorophores. Our group recently developed a bright flavylium-based SWIR polymethine dye named Flav7. However, the growing field would benefit from even brighter and deeply red-shifted fluorophores. In order to fine-tune flavylium dyes for effective imaging in living systems, an investigation of structural changes and corresponding photophysical properties is necessary. Through systematic derivatization of the Flav7 scaffold, this work seeks to elucidate design principles for the development of a SWIR Fӧrster resonance energy transfer (FRET) turn- on probe. FRET probes are of great interest for imaging as they can lead to greater signal-to- noise ratios compared to free dyes. Our lab aims to recruit SWIR FRET pairs for improved biomedical imaging applications. Using a precedented protease cleavable linker, we will synthesize a SWIR FRET probe for image guided surgery of small tissue sarcoma (STS). The development of a FRET probe reliant on tissue-penetrating SWIR light will greatly improve clinical diagnosis.