SBIR Phase I: Widefield 3D Superresolution Microscopy Module

这项小型企业创新研究计划（SBIR）一期项目研究了一种具有单分子灵敏度的开创性三维（3D）光学显微镜的可行性，该显微镜适用于活细胞研究和纳米级传感。最近有几种方法显示出远远超过光学衍射极限的分辨率，这限制了成像在横向和轴向尺寸分别大于200和500纳米的特征。迄今为止，这些超分辨率技术已经在专门的实验室中得到了证明，但要在典型的生物学实验室中定期达到基本极限，还需要新的发展。因此，该项目专注于基础技术的发展，以解决即将到来的对普遍的3D超分辨率单分子能力的需求。该仪器基于照明、光学响应、数据收集方法和荧光成像重建算法的联合设计。该仪器利用定制的点扩展函数，能够以极高的精度确定发射器的3D位置，从而获得比竞争方法更好的分辨率和景深。初步的实验室实验已经显示出分辨率低于20nm的3D能力，这相当于目前使用的大多数光学显微镜提高了一个数量级。该项目的广泛影响/商业潜力为不断扩大的生物仪器市场带来了重大机遇。如果成功，它将使许多目前受过去技术限制的生物实验室具备3D超分辨率成像能力。可行性计划通过实施与现有显微镜兼容的具有成本效益的灵活解决方案，为加速商业化道路开辟了机会。超分辨率成像的广泛应用将影响科学和工程的几个领域，包括三维生物物理和生物医学光学成像。在项目期间，公司将建立协同关系，迅速将技术转化为应用。该公司已经建立了合作伙伴关系，在重大的生物学问题中测试该仪器，预计将有助于更好地了解疾病。从更广泛的角度来看，该项目将通过为日常生物研究提供纳米级光学成像来提高科学技术能力，从而加强美国在全球光学显微镜行业的地位。

This Small Business Innovation Research Program (SBIR) Phase I project investigates the feasibility of a ground-breaking three-dimensional (3D) optical microscope with single-molecule sensitivity suitable for live cell studies and nanometer scale sensing. Several methods have recently shown resolution well beyond the optical diffraction limit, which restricts imaging to features larger than about 200 and 500 nm in the transverse and axial dimensions respectively. To date, these super-resolution techniques have been demonstrated in specialized labs but new developments are required to reach the fundamental limits on a regular basis at the typical biology lab. Therefore, this project focuses on fundamental technological developments to address the upcoming need for pervasive 3D superresolution single-molecule capabitlities. The instrument is based on a joint design of the illumination, optical response, data collection approach, and reconstruction algorithms for fluorescence imaging. The instrument makes use of tailored point spread functions that enable the determination of the 3D position of emitters with great precision, leading to better resolution and depth of field than competing methods. Preliminary laboratory experiments have shown 3D capability with resolutions below 20nm, which corresponds to one order of magnitude enhancement over most optical microscopes presently in use. The broader impact/commercial potential of this project addresses a major opportunity in an expanding biological instrumentation market. If successful, it will enable 3D superresolution imaging capability to many biological laboratories currently limited by past technologies. The feasibility plan opens up opportunities for an accelerated commercialization path by implementing a cost-effective flexible solution compatible with existing microscopes. The extensive availability of superresolution imaging will impact several fields of science and engineering including 3D biophysical and biomedical optical imaging. During the project the company will generate synergistic relationships to rapidly transfer the technology into applications. The company has established partnerships for testing the instrument in significant biological problems that are expected to contribute to a better understanding of diseases. From a broader perspective, this project will increase scientific technical capabilities by providing nanoscale optical imaging for everyday biological research, thereby strengthening US presence in the worldwide optical microscopy industry.