Summary This Small Business Innovation Research (SBIR) Phase I project is focused on the design, development, and testing of a ground-breaking multidimensional multifunctional quantitative optical microscopy module suitable for live whole cell studies. The module will utilize Light Engineering to achieve fast high-sensitivity, low-noise, high- resolution measurements down to the single-molecule level while providing wide volumetric field of view. In spite of the ongoing revolution in optical microscopy, the availability of state-of-the-art commercial solutions has been slow to market, lacking in flexibility, ease of access and affordability. The proposed instrument is based on an integrated design of the illumination, 3D optical response, data collection, and reconstruction algorithms for fluorescence imaging. Specifically, engineered 3D light excitation limits the background noise while reducing photo-damage and photo-toxicity. The engineering of the 3D point spread function enables multiplex functionality including an extended depth of field, high-sensitivity 3D localization of single-molecules or cellular heterogeneities, multi-color, and 3D imaging. The integrated system will enable reconstruction with superb sectioning capability. As a result, the target performance metrics, supported by recent research demonstrations in academic labs, outperform the state of the art in terms of spatial/temporal resolution, signal-to-noise ratio, field of view, and ease of use. This SBIR project is targeted towards commercialization of a cost-effective solution that can be easily integrated with existing scientific microscopes. The commercial module, to be developed from the Phase I prototype, will include a small footprint architecture, a set of novel optical phase masks for point spread function engineering and excitation shaping, a robust optomechanical design, and real-time experiment control software. Tests of the instrument in significant biomedical problems at partners’ labs will validate end-user acceptance and provide valuable feedback towards commercialization. The implications in biomedical imaging are far-reaching. For instance, the instrument would benefit the study of oncogenesis, owing to its degree of molecular sensitivity for detecting the spatial localization of receptors and other signaling molecules within the tumor/extracellular matrix. It would also empower the study of degenerative diseases where the instrument can help reveal their molecular origin and develop novel therapeutic strategies. The new imaging capabilities could further assist in stem cell and brain research. Double Helix Optics is a startup company with a proven record and exclusive licensing rights to the Light Engineering technology from the University of Colorado, as well as the novel Tetrapod and Multicolor PSF localization developments from Stanford University. The company, headquartered in the BioFrontiers Institute in Boulder, is optimally positioned to successfull...