High Resolution 3D X-ray Diffraction Microscope

描述（由申请人提供）：我们建议开发X射线衍射显微镜，以实现冷冻水合细胞10 nm分辨率3D成像的目标。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先进光源（ALS）实验中成功获得30 nm分辨率的冻干酵母（酿酒酵母）2D图像的基础上。X射线衍射显微术是近年来发展起来的一种方法，它首先测量非晶体样品的相干衍射图样，然后通过迭代算法进行相位调整，从而产生实空间图像。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，避免辐射剂量增加的限制，光学效率和最大收集角，否则由X射线光学系统。在美国国立卫生研究院的支持下，我们已经建立了一个新的设备来获取冷冻水合标本的倾斜系列数据，并将其安装在ALS的光束线上。利用该装置，我们收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。为了达到我们的冷冻水合标本的高分辨率3D成像的目标，我们计划在设备附近提供标本制备能力，并改进我们的设备，以允许通过结合扫描图像能力更快速地评估不同的制剂。我们还计划通过开发一种新的光束停止器来改进我们的数据采集程序，以使我们能够更好地在低空间频率下捕获数据。我们将改进数据重建方法和软件，以应对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，以解释这种新方式提供的酵母图像。我们将改进我们目前操作的光束线，以提供在更高光子能量下工作的能力，从而能够处理更厚的样品。我们还将采取措施，在高级光源处建立一个新的设施（包括一个定制设计的波荡器和光束线），使该技术定期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对生物结构和功能的理解至关重要。软x射线具有成像未切片，水合细胞的基本优势，衍射显微镜可以最大限度地提高结构信息，可以获得一个给定的辐射曝光。

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.