High Resolution 3D X-ray Diffraction Microscope

描述(由申请人提供)：我们建议发展X射线衍射显微镜，目标是对冷冻的水化细胞进行10纳米分辨率的3D成像。这一目标是建立在我们最近在劳伦斯伯克利国家实验室的高级光源(ALS)进行的实验中成功获得30纳米分辨率的冷冻干燥酵母酿酒酵母的2D图像的基础上的。X射线衍射显微镜是最近发展起来的一种方法，它测量非晶态样品的相干衍射图，然后通过迭代算法对其进行定相，从而产生实空间图像。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，它避免了辐射剂量、光学效率和最大收集角度的增加限制，否则由X射线光学系统施加。在NIH的支持下，我们建造了一台新的装置来获取冷冻水化样品的倾斜序列数据，并将其安装在ALS的光束线上。利用该装置，我们采集了一系列冻干酵母的二维衍射图，并获得了30 nm分辨率的重建图像。我们还开始收集冷冻水合酵母的衍射数据，并验证了冷冻水合细胞的辐射耐受性与我们10 nm分辨率的目标是一致的。为了实现对冷冻水化标本进行高分辨率3D成像的目标，我们计划在仪器附近提供样品制备功能，并改进我们的仪器，通过整合扫描成像功能来允许更快速地评估不同的制备方法。我们还计划改进我们的数据采集程序，开发一种新的波束停止，使我们能够更好地捕获低空间频率的数据。我们将改进我们的数据重建方法和软件，以应对3D成像带来的挑战。我们将开展标记研究和相关显微镜研究，以解读通过这种新模式传递的酵母图像。我们将改进我们目前工作的光束线，以提供在更高的光子能量下工作的能力，以便工作在更厚的样品上。我们还将采取步骤，在高级光源建立一个新的设施(包括一个定制设计的波荡器和光束线)，使更广泛的用户社区能够常规使用这项技术。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加深我们对生物结构和功能的理解至关重要。软X射线在成像未切片的水合细胞方面具有基本优势，而衍射显微镜可以最大限度地获取给定辐射暴露下的结构信息。

DESCRIPTION (provided by applicant): 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 beam line 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 beam line 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 beam line) 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.