High Resolution 3D X-ray Diffraction Microscope

我们建议发展X射线衍射显微镜，以实现10 nm分辨率的3D成像， 冷冻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。 一种冷冻干燥的酵母，酿酒酵母，在先进光源进行的实验中， (ALS)劳伦斯伯克利国家实验室 X射线衍射显微术是近年来发展起来的一种方法， 测量非结晶样品，然后通过迭代算法定相以产生实空间图像。 它提供了一种方法，以实现最大可能的空间分辨率在x射线成像，通过避免 辐射剂量增加光学效率和最大收集角的限制， X射线光学系统。在NIH的支持下，我们建立了一个新的装置来获取冷冻的倾斜系列数据， 水化标本，并已安装在ALS的光束线。用这个仪器，我们收集了 一系列冻干酵母的二维衍射图，并获得了30 nm处的重建图像 分辨率我们还开始收集冷冻水合酵母的衍射数据，并已证实， 冷冻水合细胞的辐射耐受性与我们的10nm分辨率的目标相容。 为了实现我们对冷冻水合标本进行高分辨率3D成像的目标，我们计划提供 样品制备能力附近的设备，并改善我们的设备，以允许更快速 通过结合扫描图像能力来评估不同的制剂。我们还计划改善 我们的数据采集程序，通过开发一个新的beamstop，使我们能够更好地捕捉数据在低 空间频率我们将改进我们的数据重建方法和软件来处理 3D成像带来的挑战。我们将进行标记研究和相关的显微镜检查，以便 解释这种新模式提供的酵母图像。我们将改进目前运行的光束线 以提供在更高光子能量下工作的能力，从而对更厚的样品进行工作。我们将 还采取步骤建立一个新的设施（包括一个定制设计的波荡器， 光束线）的先进光源，这将使该技术经常提供给更广泛的用户 社区该项目在ALS战略计划中具有最高优先级。 研究细胞超微结构的改进方法的发展对于增加我们的研究至关重要。 了解生物结构和功能。软x射线具有成像的基本优势 未切片的水合细胞和衍射显微镜可以最大限度地提高结构信息， 得到的辐射剂量。

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.