This Small Business Innovation Research (SBIR) Phase II project is focused on the design, development, and testing of a ground-breaking three-dimensional (3D) multifunctional quantitative optical microscopy module with single-molecule sensitivity suitable for live cell studies and nanometer scale sensing. In spite of the current revolution in optical microscopy, commercial solutions to achieve resolution beyond the diffraction limit have been slow to market, lacking in flexibility, and costly. Furthermore, most commercial products are too tailored to the systems being investigated and are difficult to generalize for widespread use. The instrument to be developed is based on an integrated design of the illumination, optics, data collection, and reconstruction algorithms for fluorescence imaging. These techniques enable the determination of the 3D position of emitters with great precision leading to 3D imaging with better resolution and depth of field than competing methods. Experimental results have consistently shown 3D capability with resolutions below 20 nm, representing one order of magnitude improvement over optical microscopes currently in use at most research institutions.<br/><br/>The broader impact/commercial potential of this project addresses a major opportunity in the expanding microscopy market. The module's unique characteristics and accessible price point will offer multifunctional 3D superresolution imaging capability to thousands of laboratories in the US and worldwide. In an effort to accelerate commercialization, the project seeks to implement a cost-effective, flexible modular subsystem that can be integrated with existing commercial scientific microscopes. With its widespread accessibility, the instrument will impact multiple fields of science and engineering including biophysics, cellular biology, and biomedical science and engineering. From a broader perspective, this project will increase scientific and technical capabilities by providing nanoscale optical imaging for everyday biological research, thereby strengthening the US presence in the worldwide optical microscopy industry.