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      3D Ultrastructural Organization of Whole Chlamydomonas reinhardtii Cells Studied by Nanoscale Soft X-Ray Tomography

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          Abstract

          The complex architecture of their structural elements and compartments is a hallmark of eukaryotic cells. The creation of high resolution models of whole cells has been limited by the relatively low resolution of conventional light microscopes and the requirement for ultrathin sections in transmission electron microscopy. We used soft x-ray tomography to study the 3D ultrastructural organization of whole cells of the unicellular green alga Chlamydomonas reinhardtii at unprecedented spatial resolution. Intact frozen hydrated cells were imaged using the natural x-ray absorption contrast of the sample without any staining. We applied different fiducial-based and fiducial-less alignment procedures for the 3D reconstructions. The reconstructed 3D volumes of the cells show features down to 30 nm in size. The whole cell tomograms reveal ultrastructural details such as nuclear envelope membranes, thylakoids, basal apparatus, and flagellar microtubule doublets. In addition, the x-ray tomograms provide quantitative data from the cell architecture. Therefore, nanoscale soft x-ray tomography is a new valuable tool for numerous qualitative and quantitative applications in plant cell biology.

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          Most cited references 38

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          Far-field optical nanoscopy.

           Stefan Hell (2007)
          In 1873, Ernst Abbe discovered what was to become a well-known paradigm: the inability of a lens-based optical microscope to discern details that are closer together than half of the wavelength of light. However, for its most popular imaging mode, fluorescence microscopy, the diffraction barrier is crumbling. Here, I discuss the physical concepts that have pushed fluorescence microscopy to the nanoscale, once the prerogative of electron and scanning probe microscopes. Initial applications indicate that emergent far-field optical nanoscopy will have a strong impact in the life sciences and in other areas benefiting from nanoscale visualization.
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            Soft X-ray microscopes and their biological applications.

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              Three-dimensional cellular ultrastructure resolved by X-ray microscopy.

              We developed an X-ray microscope using partially coherent object illumination instead of previously used quasi-incoherent illumination. The design permitted the incorporation of a cryogenic tilt stage, enabling tomography of frozen-hydrated, intact adherent cells. We obtained three-dimensional reconstructions of mouse adenocarcinoma cells at ∼36-nm (Rayleigh) and ∼70-nm (Fourier ring correlation) resolution, which allowed us to visualize the double nuclear membrane, nuclear pores, nuclear membrane channels, mitochondrial cristae and lysosomal inclusions.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                1932-6203
                2012
                31 December 2012
                : 7
                : 12
                Affiliations
                [1 ]Institut für Zellbiologie, Universität Bayreuth, Bayreuth, Germany
                [2 ]Helmholtz-Zentrum für Materialien und Energie GmbH, Institute for Soft Matter and Functional Materials, Berlin, Germany
                [3 ]Frankfurt Institute for Molecular Life Sciences and Institute of Biophysics, Goethe University Frankfurt, Frankfurt am Main, Germany
                University of Hyderabad, India
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Conceived and designed the experiments: EH PG. Performed the experiments: EH PG MK. Analyzed the data: EH PG BT MK ASF BW. Contributed reagents/materials/analysis tools: PG SW BT GS MK ASF. Wrote the paper: EH GS BW.

                Article
                PONE-D-12-19435
                10.1371/journal.pone.0053293
                3534036
                23300909

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                Page count
                Pages: 9
                Funding
                The authors thank the Helmholtz center for beam time allocation. This publication was funded by the German Research Foundation (DFG) and the University of Bayreuth in the funding program Open Access Publishing. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. No additional founding was required to publish this study.
                Categories
                Research Article
                Biology
                Biochemistry
                Cytochemistry
                Organelles
                Model Organisms
                Plant and Algal Models
                Chlamydomonas Reinhardtii
                Molecular Cell Biology
                Cellular Structures
                Subcellular Organelles
                Plant Cell Biology
                Plant Science
                Plants
                Algae
                Plant Cell Biology

                Uncategorized

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