Soft X-Ray Nanotomography of Cryo Specimens

Suny At Stony Brook, Stony Brook NY

Investigators

Abstract

ABSTRACT 9986819 Chris J. Jacobsen SUNY Stony Brook Soft 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.