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

这项小型企业创新研究（SBIR）一期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，使其达到常规超分辨率（SR）显微镜技术所需的成像精度。由于某些荧光探针的特殊光物理特性，这些新方法绕过了通常与光学显微镜相关的衍射极限，并将其推进到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要求很高。传统的显微镜无法满足这些要求，并且如果不补偿这种漂移，样品相对于图像检测器会发生漂移，从而破坏成像系统的SR能力。该项目将集成一个三轴压电驱动的纳米定位显微镜平台和一个闭环反馈系统，该系统使用EMCCD相机生成的数据，该相机也将作为系统的图像探测器。在样品中稀疏分布的荧光基准将作为评估样品漂移的锚点，并使用纳米定位阶段产生补偿运动，以在所有3个维度上实时稳定样品相对于图像检测器。该项目更广泛的影响/商业潜力在于使基于定位的SR成像方法对工作中的生物学家日常有用。这意味着要使它们在技术上易于实现，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。希望实现这些广泛应用的技术的科学家们面临着一项艰巨的任务：将许多单独的组件组装成一个工作系统，然后使这些组件按照要求无缝地协同工作。我们的目标是开发一种完全集成的显微镜稳定和成像系统，并将其商业化，该系统基于纳米定位平台、EMCCD相机和软件，可以对样品进行实时图像反馈控制。年代的位置。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想要利用SR显微镜作为实验工具所面临的技术挑战，使他们能够将当前的倒置显微镜转换和扩展到潜在的大量具有SR能力的成像系统。

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.