Soft X-Ray Nanotomography of Cryo Specimens

摘要9986819 Chris J. Jacobsen SUNY Stony BrookSoft低温样本的X射线纳米断层扫描 高分辨率成像在理解细胞分子机制方面取得了巨大进展。 可见光显微镜可用于观察活细胞中的分子运输，分辨率为细胞直径的二十分之一至百分之一。 电子显微镜可用于观察组织切片中抗体标记的标记物的位置，其尺寸尺度对应于几种蛋白质大分子。 X射线晶体学可用于以接近原子分辨率研究单个蛋白质或病毒。 在这些强大的方法中，软X射线显微镜为整个湿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可以达到可见光显微镜的3-10倍，并提供了观察有机分子不同键合状态的可能性。 为了充分利用这种潜力，需要冷冻水合细胞的断层成像方法。 在美国国家科学基金会的支持下，开发出了一种能够对冷冻水合生物标本进行成像的透射X射线显微镜。 该显微镜的第一个结果产生了成纤维细胞的100 x100 x250 nm图像。 该图像是通过将样品旋转通过波带片透镜的焦深而获得的。 这一奖项将有可能扩大这种方法的解决方案。 为了实现这种更高的分辨率，有必要避开高分辨率、高数值孔径光学器件的焦深的固有限制。 这将通过采用衍射层析成像的成熟方法来完成，以在每个层析成像倾斜处记录全息图，并通过反向传播获得三维重建。 在实验上，该奖项将资助在机动测角系统中开发高倾斜低温转移试样保持器。 所获得的全息图将被放大到CCD相机使用菲涅耳波带片。 通过使用这种方法，它将有可能获得5至10微米厚的冷冻水化标本的层析数据集，在现有的波带片的分辨率极限：目前为30 nm，未来为10-20 nm。 这种仪器的开发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间分辨率信息。 这些图像将为细胞功能提供新的见解。 该奖项还将支持物理学生在生物成像方面的培训。

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.