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个集成系统，以稳定传统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，通过某些荧光探针的特殊光物理特性，已经绕过了通常与光学显微镜相关的衍射极限，并将其推向了迄今为止只有电子显微镜才能实现的领域。 这些方法对显微镜系统的稳定性提出了非常高的要求。 传统显微镜无法满足这些要求，并且样品相对于图像检测器会发生漂移，如果这种漂移得不到补偿，可能会破坏成像系统的 SR 功能。 该项目将使用 EMCCD 相机生成的数据将 3 轴压电驱动纳米定位显微镜平台与闭环反馈系统集成，该相机也将充当系统的图像检测器。 样本内稀疏分布的荧光基准参考将用作评估样本漂移的锚点，并使用纳米定位平台产生补偿运动，以在所有 3 个维度上实时稳定样本相对于图像检测器的稳定性。该项目更广泛的影响/商业潜力在于使基于定位的 SR 成像方法对工作生物学家来说通常有用。 这意味着它们在技术上易于实施，并且在经济上易于实现。 目前，稳定传统显微镜并使其常用于超分辨率显微镜所需的组件还没有单一来源。 希望实施这些广泛适用的技术的科学家们面临着一项艰巨的任务，即用许多单独的组件组装一个工作系统，然后让这些组件根据需要无缝地协同工作。 我们的目标是开发并商业化基于纳米定位平台、EMCCD 相机和软件的完全集成的显微镜稳定和成像系统，该系统能够对样品位置进行基于图像的实时反馈控制。 它将在开发时考虑到基于定位的 SR 显微镜，但对于任何需要长期样品稳定性和图像采集的成像实验（例如扩展活细胞成像）非常有用。 该系统将极大地简化想要利用 SR 显微镜作为实验工具的生物学家所面临的技术挑战，使他们能够将当前的倒置显微镜转换和扩展为潜在的大量具有 SR 功能的成像系统。

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.