Showing papers by "Stefan Vogt published in 2001"

Computed tomography of cryogenic cells

Abstract: COMPUTED TOMOGRAPHY OF CRYOGENIC CELLS G. SCHNEIDER, and E. ANDERSON Center for X-ray Optics, Lawrence Berkeley National Laboratory, One Cyclotron Road MS 2-400, Berkeley, CA 94720, USA S. VOGT, C. KNOCHEL, and D. WEISS Institut fur Rontgenphysik, Universitat Gottingen, Geiststrase 11 D-37073 Gottingen, Germany M. LEGROS, and C. LARABELL Life Sciences, Lawrence Berkeley National Laboratory, One Cyclotron Road MS 6-2100, Berkeley, CA 94720, USA Received (to be inserted Revised by publisher) Soft X-ray microcopy has resolved 30 nm structures in biological cells. To protect the cells from radiation damage caused by X-rays, imaging of the samples has to be performed at cryogenic temperatures, which makes it possible to take multiple images of a single cell. Due to the small numerical aperture of zone plates, X-ray objectives have a depth of focus on the order of several microns. By treating the X-ray microscopic images as projections of the sample absorption, computed tomography (CT) can be performed. Since cryogenic biological samples are resistant to radiation damage, it is possible to reconstruct frozen-hydrated cells imaged with a full-field X-ray microscope. This approach is used to obtain three-dimensional information about the location of specific proteins in cells. To localize proteins in cells, immunolabelling with strongly X-ray absorbing nanoparticles was performed. With the new tomography setup developed for the X-ray microscope XM-1 installed at the ALS, we have performed tomography of immunolabelled frozen-hydrated cells to detect protein distributions inside of cells. As a first example, the distribution of the nuclear protein, male specific lethal 1 (MSL-1) in the Drosophila melanogaster cell was studied. 1. Introduction The structure of proteins can be studied by X-ray crystallography with atomic resolution, but their location in cells remains unknown. With immunolabelling it is possible to localize these proteins in cells. Up to now light microscopy has mainly been used to study their distribution in cells by tagging the investigated protein with fluorophore-conjugated antibodies. While light microscopes allows routine investigation of whole, unsectioned cells, the obtainable resolution is diffraction limited to about 200 nm. In addition, this technique reveals mainly the distribution of the fluorophore-conjugated antibodies whereas most unlabelled cell structure is not clearly visualized. Electron microscopy can reveal cell structures at much higher resolution level, but is limited by the thickness of the sample, i.e. only less than 1 µm thick objects can be imaged. Therefore, no conventional imaging technique exists which can visualize the three- dimensional distribution of proteins inside whole hydrated cells, e.g. in the cell nucleus, with higher than light microscopical resolution. Due to the shorter wavelengths of X-rays than visible light, X-ray microscopy provides higher resolving power than light microscopes. By utilizing the natural absorption contrast between protein and water at photon energies of about 0.5 keV, smallest cell structures of about 30 nm in size embedded in vitreous ice can be detected in X-ray microscope images 1-3 . The aim of this work is to apply computed tomography, which has already been demonstrated using artificial samples 4 , mineralized sheats of bacteria 5 and frozen-hydrated algae 6 , in order to localize specific proteins and organelles in unsectioned, frozen-hydrated cells. 2. Lateral Resolution and Depth of Focus The computed-tomography experiments presented in this work are all based on tilt series of images acquired using the amplitude contrast mode of the TXM. In the amplitude contrast mode, the microscope forms enlarged images of the intrinsic photoelectric absorption contrast of the object. However, the obtained image contrast is influenced both by the condenser illuminating the object and by the imaging X-ray objective. The e-beam written condenser zone plate used for these experiments has an outermost zone width of dr N = 54 nm 7 . At 2.4 nm wavelength, the numerical aperture is given by NA cond = λ / (2 dr N ) = 0.0222.

Tomographic imaging of biological specimens with the cryo transmission X-ray microscope

Abstract: Using the photoelectric absorption contrast between water and protein at 2:4 nm wavelength, cryo X-ray microscopy has visualized protein structures down to 30 nm size in unstained, unsectioned biological specimens. Due to the large depth of focus of the Fresnel zone plate objectives, computed tomography based on a tilt series of X-ray microscopic images can be used to reconstruct the three-dimensional specimen structure. This method has been applied to the green alga Chlamydomonas reinhardtii, and to cell nuclei of male Drosophila melanogaster fruit fly cells. # 2001 Elsevier Science B.V. All rights reserved. PACS: 07.85.Tt; 87.59.Fm; 07.20.Mc

Visualizing specific nuclear proteins in eukaryotic cells using soft X-ray microscopy

Abstract: Specific nuclear proteins in immunogold labeled Drosophila melanogaster cells were visualized by applying soft X-ray microscopy. In addition, first experiments were performed to localize two different labeled nuclear proteins in the same X-ray micrograph by using immunofluorescence microscopy for distinguishing the proteins. # 2001 Elsevier Science B.V. All rights reserved. PACS: 07.85.Y