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，以将传统高端倒置光学显微镜稳定到常规实现新兴超分辨率(SR)显微镜技术的成像能力所需的精度。这些最新的方法，通过某些荧光探针的特殊光物理性质，已经绕过了通常与光学显微镜相关的衍射限制，并将其推向了到目前为止只能通过电子显微镜才能实现的领域。这些方法对显微系统的稳定性提出了很高的要求。传统显微镜无法满足这些要求，样品相对于图像探测器会发生漂移，如果这种漂移得不到补偿，可能会破坏成像系统的清晰度能力。该项目将利用EMCCD摄像机产生的数据，将一个三轴压电驱动的纳米定位显微镜工作台与一个闭环反馈系统集成在一起，该摄像机也将作为该系统的图像探测器。稀疏分布在样品中的荧光基准将作为锚点来评估样品漂移，并使用纳米定位阶段产生补偿性运动，以实时稳定样品相对于图像探测器的所有3个维度。该项目的更广泛的影响/商业潜力在于使基于定位的SR成像方法对工作的生物学家常规有用。这意味着使它们在技术上易于实施，并且在经济上是可获得的。目前，稳定传统显微镜并使其成为常规SR显微镜所需的部件并不是单一来源。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：将许多单独的组件组装成一个工作系统，然后根据需要让这些组件无缝地工作在一起。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工作台、EMCCD机和能够对样本S位置进行实时基于图像的反馈控制的软件。它的开发将考虑到基于定位的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都是有用的，例如扩展的活细胞成像。该系统将极大地简化希望将SR显微镜用作实验工具的生物学家所面临的技术挑战，使他们能够将目前的倒置显微镜转换并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.