Project Summary The main objective of this proposal is to develop a new multi-spectral light source with exceptional illumination uniformity we call “Effective Uniform Color-Light Integration Device (EUCLID)” for biological imaging applications. Our project's primary goal is to show how EUCLID impacts multispectral imaging applications and validate in mesoscale brain imaging, a popular neurophotonics application. We anticipate that EUCLID can be integrated into a wide range of biological microscopic and mesoscopic imaging applications, including super-resolution microscopy and birefringence microscopy. In biological widefield imaging application, illumination homogeneity is a crucial factor for excitation performance and resulting data quality. Yet, due to spatial and spectral non- uniformity of conventional imaging systems caused by imperfect optical components, illumination corrections require sophisticated solutions. Illumination design for multi-spectral imaging (MSI) is particularly challenging as field uniformity across a wide range of wavelength is essential. Recent advancements in light emitting and laser diode technology enables creation of multi-spectral light sources with previously unattainable compactness, power, and controllability. Traditionally, achieving homogenous illumination at several wavelengths requires combining collimated beams using dichroic mirrors or beam-splitters and precise alignment. Cost, complexity, and absolute size of the illumination solution require careful consideration when developing a MSI application. The significant novelty of EUCLID is the introduction of a conical geometry allowing for light integration, design optimization and uniformity adjustments. The diffuse-reflective adjustable hollow cavity used in EUCLID alters the source field distribution to compensate spatial non-uniformity caused by the imaging system's optical components while allowing for uniform mixing of light from discrete sources with great efficiency. With a maximum spatial deviation of 1% over a large field of view, preliminary experiments show significantly improved illumination for multispectral imaging in both Nelsonian (critical) and Koehler configuration (FOV). EUCLID is also shown to provide speckle-free laser illumination over a wide field-of-view with a plateau uniformity around 2% for a broad spectrum. This uniformity introduced by the EUCLID is 3 times better than current state-of-the art flat-top illumination technique. In this proposal, we propose to show EUCLIDs performance in multispectral mesoscale brain imaging where neuronal activity (calcium signals), hemodynamic activity (hemoglobin oxygenation), and neuromodulation (acetylcholine levels) are simultaneously monitored across the dorsal cortex of mice with implanted ‘crystal skull’ windows. For that, we will develop two different EUCLID designs. The first one will accept two different commercial LED bulb to provide oblique excitation light to monitor neuronal activ...