SBIR Phase I: Real-time Active Image Stabilization for Microscopy

该小型企业创新研究（SBIR）第一阶段项目旨在开发一种集成系统，以稳定传统的高端倒置光学显微镜，使其达到常规实现新兴超分辨率（SR）显微镜技术成像能力所需的精度。 这些最近的方法，使特定的光物理特性的某些荧光探针，已经绕过了衍射极限通常与光学显微镜，并将其推到领域迄今为止只有电子显微镜实现。 这些方法对显微系统的稳定性提出了很高的要求。 传统的显微镜不能满足这些要求，并且相对于图像检测器发生样本漂移，如果不补偿该漂移，则会破坏成像系统的SR能力。 该项目将利用EMCCD相机生成的数据将3轴压电驱动纳米定位显微镜载物台与闭环反馈系统集成，EMCCD相机也将作为系统的图像检测器。 稀疏分布在样品中的荧光基准参考将作为锚点，从该点评估样品漂移，并产生补偿运动，使用纳米定位阶段，以稳定样品相对于图像检测器在所有3个维度，在real-time.The更广泛的影响/商业潜力，该项目在于使本地化为基础的SR成像方法经常有用的工作生物学家。 这意味着使它们在技术上易于实现，并且在经济上易于获得。 目前，稳定传统显微镜并使其常规用于SR显微镜所需的组件没有单一来源。 希望实现这些非常广泛适用的技术的科学家们面临着一项艰巨的任务，即从许多单独的组件中组装一个工作系统，然后让这些组件根据需要无缝地协同工作。 我们的目标是开发，然后商业化一个完全集成的显微镜稳定和成像系统的基础上的纳米定位阶段，EMCCD相机，和软件，使实时图像为基础的反馈控制的样品？的位置。 它将与基于定位的SR显微镜一起开发，但对于任何需要长期样品稳定性和图像采集的成像实验都很有用，例如延长的活细胞成像。 该系统将极大地简化生物学家所面临的技术挑战，希望利用SR显微镜作为实验工具，使他们能够转换和扩展他们目前的倒置显微镜到潜在的大量SR成像系统。

This Small Business Innovation Research (SBIR) Phase I project is aimed at developing an integrated system to stabilize a conventional high-end, inverted optical microscope to the precision required to routinely achieve the imaging power of emerging super-resolution (SR) microscopy techniques. These recent methods, enabled by particular photo-physical properties of certain fluorescent probes, have circumvented the diffraction limit normally associated with light microscopy and pushed it into realms thus far only achievable with electron microscopy. These methods put very high demands on the stability of the microscopy system. Conventional microscopes fail to meet these demands, and sample drift occurs relative to the image detector that can destroy the SR capabilities of the imaging system if this drift is not compensated. This project will integrate a 3-axis piezo-driven nanopostioning microscope stage with a closed-loop feedback system using data generated from an EMCCD camera, which will also serve as the image detector for the system. Fluorescent fiduciary references sparsely distributed within the sample will serve as anchor points from which to assess sample drift, and produce compensatory movement using the nanopositioning stage to stabilize the sample relative to the image detector in all 3 dimensions, in real-time.The broader impact/commercial potential of this project lies in making localization-based SR imaging methods routinely useful to working biologists. This means making them technically straightforward to implement, and economically accessible. Presently, there is no single source for the components required to stabilize a conventional microscope and make it routinely useful for SR microscopy. Scientists hoping to implement these very broadly applicable techniques are left with the daunting task of assembling a working system from its many individual components, and then getting these components to work together seamlessly as required. Our goal is to develop and then commercialize a fully-integrated microscope stabilization and imaging system based on a nanopositioning stage, an EMCCD camera, and software enabling real-time image-based feedback control of the sample?s position. It will be developed with localization-based SR microscopy in mind, but will be useful for any imaging experiment that requires long-term sample stability and image acquisition, such as extended live-cell imaging. This system will greatly simplify the technical challenges faced by biologists wanting to utilize SR microscopy as an experimental tool, enabling them to convert and extend their current inverted microscopes into potentially a large number of SR-capable imaging systems.