High Resolution 3D X-ray Diffraction Microscope

我们建议向10 nm分辨率三维成像的目标发展x射线衍射显微镜。 冰冻的水合细胞。这一目标是建立在我们最近成功获得30 nm分辨率的2D图像的基础上的 在先进光源上进行的实验中的冷冻干燥酵母--酿酒酵母 (ALS)在劳伦斯伯克利国家实验室。 X射线衍射显微镜是最近发展起来的一种方法，在这种方法中，相干衍射图是一种 测量非晶态样品，然后通过迭代算法对其进行分相，以产生真实空间图像。 它提供了一种在x射线成像中获得最大可能空间分辨率的方法，方法是避免 辐射剂量增加对光学效率和最大收集角度的限制 一种X射线光学系统。在NIH的支持下，我们建立了一台新的装置来获取冻结的倾斜序列数据 水合标本，并已将其安装在ALS的光束线上。有了这个仪器，我们收集了 获得了冻干酵母的一系列2D衍射图，并在30 nm处获得了重建图像 决议。我们还开始收集冷冻水合酵母的衍射数据，并证实 冷冻水合细胞的辐射耐受性与我们10纳米分辨率的目标是兼容的。 为了达到我们对冷冻水化标本进行高分辨率3D成像的目标，我们计划提供 仪器附近的样品制备能力，并改进我们的仪器，以允许更快 通过结合扫描成像功能对不同制剂进行评价。我们还计划改进 我们的数据采集程序通过开发一种新的波束止动装置，使我们能够更好地捕获低速数据 空间频率。我们将改进数据重建方法和软件，以处理 3D成像带来的挑战。我们将进行标记研究和相关显微镜检查，以期 解读这种新模式提供的酵母菌图像。我们将改进我们目前运营的光束线 以提供在更高的光子能量下工作的能力，以便处理更厚的样品。我们会 还采取步骤建立一个新的设施(包括定制设计的波浪器和 光束线)，这将使该技术常规地可供更广泛的用户使用 社区。该项目在肌萎缩侧索硬化症战略计划中具有首要地位。 发展研究细胞超微结构的改进方法对于提高我们的 对生物结构和功能的理解。软x射线在成像方面具有基本优势。 未切片的、水合的细胞和衍射显微镜可以最大限度地利用 在给定的辐射照射下获得的。

We propose to develop x-ray diffraction microscopy towards the goal of 10 nm resolution 3D imaging of frozen hydrated cells. This goal is built upon our recent success in obtaining 30 nm resolution 2D images of a freeze-dried yeast, Saccharomyces cerevisiae, in experiments carried out at the Advanced Light Source (ALS) at Lawrence Berkeley National Laboratory. X-ray diffraction microscopy is a-recently-developed method in which the coherent diffraction pattern of a non-crystalline sample is measured, and then phased by an iterative algorithm to yield a real-space image. It provides an approach to achieve the maximum possible spatial resolution in x-ray imaging by avoiding the radiation dose increasing limitations of optic efficiency and maximum collection angle otherwise imposed by an x-ray optical system. With NIH support, we have built a new apparatus to acquire tilt series data of frozen hydrated specimens, and have installed it on a beamline at the ALS. With this apparatus, we have collected a series of 2D diffraction patterns of freeze-dried yeast, and have obtained reconstructed images at 30 nm resolution. We have also begun to collect diffraction data from frozen hydrated yeast, and have verified that the radiation tolerance of frozen hydrated cells is compatible with our goal of 10 nm resolution. To reach our goal of high resolution 3D imaging of frozen hydrated specimens, we plan on providing specimen preparation capabilities near the apparatus, and to improve our apparatus to allow for more rapid evaluation of different preparations by incorporating a scanned image capability. We also plan on improving our data acquisition procedures by developing a new beamstop to allow us to better capture data at low spatial frequencies. We will improve our data reconstruction approaches and software to deal with the challenges posed by 3D imaging. We will carry out labeling studies and correlative microscopy so as to interpret images of yeast delivered by this new modality. We will improve the beamline we presently operate at to provide the capability to work at higher photon energies so as to work with thicker specimens. We will also take steps toward the establishment of a new facility (including a custom designed undulator and beamline) at the Advanced Light Source that will make the technique routinely available to the wider user community. This project has top priority in the ALS Strategic Plan. Development of improved methods for studying cellular ultrastructure is crucial for increasing our understanding of biological structure and function. Soft x rays have fundamental advantages for imaging unsectioned, hydrated cells, and diffraction microscopy can maximize the structural information that can be obtained for a given radiation exposure.