Development of High-Speed 3D Super-Resolution Microscope

项目总结/摘要 尽管在超分辨率成像方面进行了十年的工作，但大量的亚细胞现象仍然存在于- 由于当前技术的时间分辨率差而不可见。因此，迫切需要一个 具有改进的时间和空间分辨率的灵活、可访问和定量的超分辨率成像系统 lution.例如，DNA的特征长度在5 nm和150 nm之间， 在小至1 ms的时间尺度上的变化，这些时间尺度和长度不在能力范围内 当前的超分辨率方法。我们的长期目标是开发和应用测量工具， 进一步了解亚细胞现象的机制和功能障碍。的总体目标 这个应用和实现这个长期目标的下一步是大幅提高时间分辨率， 通过单次采集超分辨率显微镜以及3D定位实现超分辨率， 荧光团密度大一个数量级。这项研究的基本原理是，目前的超级- 分辨率仪器不足以使许多小规模和快速的亚细胞过程可视化。达到 这些更小的长度和更快的时间尺度需要一种新的方法来可视化这些现象，如果我们 就是要足够准确地理解它们，以治疗或诊断疾病。为了确保我们实现目标- 本申请的目的，我们已经制定了以下具体目标：1）单收购的发展 超分辨率显微镜，这将导致在衍射极限分辨率提高40%，在每一个im. 年龄，以及2）开发3D密集增强图像分析算法，这将导致能够 位于3D发射器中，距离为60 nm，具有来自单个图像或重建的10 nm各向同性分辨率， 构建较近发射器的3D亚衍射密度。这些新的和改进的功能将使 对获取超分辨率数据的时间分辨率的改进。方法是创新的 在我们看来，因为它利用了一种新的成像系统（贝塞尔光束显微镜）， 空间分辨率的信息，这与目前的超分辨率系统不同， 空间分辨率的解决方案。这项研究之所以重要，是因为它扩展了超分辨率成像 在一个特别迫切需要的领域，提供高速亚衍射极限3D再现， 构建能力将允许实时监测，例如，染色质包装水平和硬度， 响应转录。此外，STORM/PALM等随机超分辨率技术将贝内 从密集标记的精确定位，提高其时间分辨率。

Project Summary/Abstract Despite a decade of work on super-resolution imaging, a large amount of sub-cellular phenomena remains in- visible due to the poor temporal resolution of current techniques. As a result, there is an urgent need for a flexible, accessible, and quantitative super-resolution imaging system with improved temporal and spatial reso- lution. For example, DNA has characteristic lengths between 5 nm and 150 nm that exhibit significant dynamic changes over time scales as small as 1 ms, which are length and time scales that are not within the capabilities of current super-resolution methodologies. Our long-term goal is to develop and apply measurement tools to further our understanding of mechanisms and dysfunction of sub-cellular phenomena. The overall objective of this application and the next step in attaining this long-term goal is to drastically improve the temporal resolu- tion of super-resolution through both single-acquisition super-resolution microscopy as well as 3D localization at an order of magnitude greater fluorophore density. The rationale underlying this research is that current super- resolution instruments are inadequate for visualizing many small-scale and fast sub-cellular processes. Reaching these smaller lengths and faster time scales requires a new approach toward visualizing these phenomena if we are to understand them with sufficient fidelity to treat or diagnose diseases. To ensure that we achieve the ob- jective of this application we have developed the following Specific Aims: 1) Development of Single-Acquisition Super-Resolution Microscopy, which will result in a 40% improvement in diffraction limited resolution in every im- age, and 2) Development of 3D Dense-Emitter Image Analysis Algorithm, which will result in the ability to either locate in 3D emitters as close together as 60 nm with 10 nm isotropic resolution from a single image or recon- struct the 3D sub-diffraction density of closer emitters. Together these new and improved capabilities will enable improvements to the temporal resolution with which super-resolution data is acquired. The approach is innovative in our opinion, because it utilizes a novel imaging system (Bessel Beam Microscopy) that trades low-frequency information for spatial resolution, which differs from current super-resolution systems that trade temporal reso- lution for spatial resolution. The proposed research is significant because it expands super-resolution imaging capability in an area where there is a particularly urgent need. Providing high-speed sub-diffraction limit 3D re- construction capabilities will allow real-time monitoring of, for example, chromatin packing level and stiffness in response to transcription. Additionally, stochastic super-resolution techniques such as STORM/PALM will benefit from the accurate localization at dense labeling, improving their temporal resolution.