Project Summary/Abstract Fluorescence has significant potential for biomedical imaging applications because of the relatively low cost of imaging equipment, the nominal toxicity of non-ionizing radiation (i.e., light), the potential for molecular imaging using target-specific contrast agents, and the prospect of multiplexed imaging using discretely colored fluorophores. Molecules common in biological tissues including lipids, water, and hemoglobin scatter and absorb light, rendering tissue opaque to visible wavelengths, but longer, near infrared (NIR) wavelengths penetrate deeper, giving us an optical window into the body. To see inside a tissue, we require bright, photostable, highly absorbing, NIR fluorophores and effective imaging equipment. Despite exceptional results in vitro, we can improve on the in vivo performance of organic dyes, fluorescent proteins, and traditional semiconductor quantum dots (QDs), which are typically dim, toxic, not red enough, or all of the above. We have created a material that literally flips a quantum dot inside out to make a quantum shell (QS) comprised of non-toxic elements (In, P, Se, Zn, S) that is tunable from 500 – 900+ nm. Because InP absorbs more efficiently than CdSe, these materials are brighter than previous materials with a smaller size, while emitting in the NIR and reducing toxicity. Our funded technology development plan involves refining the structural and optical properties of these particles to generate a brightness-matched palette of fluorophores to enable multiplexed fluorescence imaging in deep tissue. Through this equipment supplement, we request funding to replace an aging In Vivo Imaging System with a state-of-the-art animal imager that is optimized for multiplexed NIR imaging. A blue-enhanced InGaAs camera will facilitate imaging from 600 – 1600 nm, encompassing the entire first optical tissue window and capturing red tail emission from the QSs in the second optical tissue window as well. A volume Bragg grating enables hyperspectral imaging; high resolution spectral information from every pixel will be used in demixing algorithms to support simultaneous imaging of multiple fluorophores. Installation of this instrument on our primary campus will promote the development and application of contrast agents and imaging protocols for multiplexed near infrared imaging in mice.