Soft X-Ray Nanotomography of Cryo Specimens

冷冻标本的软 X 射线纳米断层扫描

• 批准号: 9986819
• 负责人: Chris Jacobsen
• 金额: $ 66.2万
• 依托单位: SUNY at Stony Brook
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-03-15 至 2003-02-28
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9986819&HistoricalAwards=false
• 关键词: Soft; Ray; Nanotomography; Cryo; Specimens

ABSTRACT9986819Chris J. JacobsenSUNY Stony BrookSoft X-ray Nanotomography of Cryo Specimens High resolution imaging has led to dramatic advances in understanding the molecular machinery of cells. Visible light microscopy can be used to observe molecular traffic in living cells at a resolution of a twentieth to a hundredth of a cell diameter. Electron microscopy can be used to observe the location of antibody-tagged labels in tissue sections at a size scale corresponding to several protein macromolecules. X-ray crystallography can be used to study individual proteins or viruses at close to atomic resolution. Amongst these powerful approaches, soft x-ray microscopy provides new opportunities for imaging whole, wet cells. Samples suitable for soft x-ray microscopy can be 10-50 times thicker than electron microscopy samples. The soft x-ray microscope can obtain a resolution of 3-10 times that of visible light microscopy, and offers the possibility of observing the different bonding states of organic molecules. To fully exploit this potential, methods for tomographic imaging of frozen hydrated cells are required. With support from the National Science Foundation, a transmission x-ray microscope that is able to image frozen hydrated biological specimens has been developed. The first results from this microscope have yielded a 100x100x250 nm image of a fibroblast cell. This image was obtained by rotating the specimen through the depth of focus of a zone plate lens. This award will make it possible to extend the resolution of this approach. To achieve this higher resolution, it is necessary to circumvent the intrinsic limit of the depth of focus of high resolution, high numerical aperture optics. This will be done by adopting well-developed methods of diffraction tomography to record a hologram at each tomographic tilt and obtain a three dimensional reconstruction by backpropagation. Experimentally, this award will fund the development of a high tilt cryogenic transfer specimen holder in a motorized goniometer system. The holograms obtained will be magnified onto a CCD camera using a Fresnel zone plate. By using this approach, it will be possible to obtaining tomographic datasets of 5 to 10 micrometer thick frozen hydrated specimens at the resolution limit of available zone plates: 30 nm at present, and 10-20 nm in the future. Development of this instrumentation will provide biologists with intermediate resolution information on the three dimensional organization of structures in a cell that are difficult to fluorescently label. These images will provide new insights into cell function. This award will also support the training of physics students in biological imaging.

克里斯·J·雅各布森纽约州立大学石材布鲁克索夫特冷冻标本的X射线纳米层析成像高分辨率成像在理解细胞的分子机制方面取得了巨大的进步。可见光显微镜可以用来观察活细胞中的分子交通，分辨率为细胞直径的二十分之一到百分之一。电子显微镜可以用来观察抗体标记的标记在组织切片中的位置，其大小与几个蛋白质大分子相对应。X射线结晶学可以用来研究单个蛋白质或病毒，其分辨率接近原子分辨率。在这些强大的方法中，软X射线显微镜为完整、潮湿的细胞成像提供了新的机会。适用于软X射线显微镜的样品可以比电子显微镜样品厚10-50倍。软X射线显微镜的分辨率是可见光显微镜的3-10倍，为观察有机分子的不同成键状态提供了可能。为了充分开发这一潜力，需要对冷冻的水化细胞进行断层成像。在国家科学基金会的支持下，一种能够对冷冻水合生物标本成像的透射式X射线显微镜已经开发出来。这台显微镜的第一批结果已经产生了成纤维细胞的100x100x250 nm的图像。这幅图像是通过波带片透镜的焦深旋转样品获得的。这项裁决将使延长这一办法的决议成为可能。为了达到更高的分辨率，有必要绕过高分辨率、高数值孔径光学系统的焦深限制。这将通过采用成熟的衍射层析成像方法来实现，在每次层析倾斜时记录全息图，并通过反向传播获得三维重建。在实验方面，该奖项将资助开发电动测角仪系统中的高倾斜低温转移样品架。获得的全息图将被放大到使用菲涅耳波带片的CCD相机上。利用这种方法，将有可能获得5-10微米厚的冷冻水化样品的层析数据集，其分辨率极限为：目前为30 nm，未来为10-20 nm。这种仪器的发展将为生物学家提供关于细胞中难以荧光标记的结构的三维组织的中等分辨率信息。这些图像将提供对细胞功能的新见解。该奖项还将支持对物理学学生进行生物成像方面的培训。