Soft X-Ray Nanotomography of Cryo Specimens

高分辨率成像技术在理解细胞的分子机制方面取得了巨大的进步。可见光显微镜可用于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。电子显微镜可以观察到抗体标记的标签在组织切片中的位置，其大小与几种蛋白质大分子相对应。x射线晶体学可用于研究接近原子分辨率的单个蛋白质或病毒。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射线显微镜可以获得可见光显微镜3-10倍的分辨率，并提供了观察有机分子不同成键状态的可能性。为了充分利用这一潜力，需要冷冻水合细胞的层析成像方法。在美国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x射线显微镜已经开发出来。该显微镜的第一个结果产生了成纤维细胞100 × 100 × 250 nm的图像。该图像是通过通过带板透镜的聚焦深度旋转试样获得的。这一奖项将有可能扩大这种方法的解决方案。为了达到更高的分辨率，必须克服高分辨率、高数值孔径光学系统固有的聚焦深度限制。这将通过采用成熟的衍射层析成像方法在每次层析倾斜处记录全息图并通过反向传播获得三维重建来实现。在实验方面，该奖项将资助在电动测角仪系统中开发高倾斜低温转移试样夹。获得的全息图将通过菲涅耳带片放大到CCD相机上。通过这种方法，将有可能获得5至10微米厚的冷冻水合标本的层析数据集，其分辨率限制在可用的带板：目前为30 nm，未来为10-20 nm。该仪器的开发将为生物学家提供难以荧光标记的细胞中结构的三维组织的中等分辨率信息。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专业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.