3D ultrastructural organization of whole Chlamydomonas reinhardtii cells studied by nanoscale soft x-ray tomography

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

Toward detecting and identifying macromolecules in a cellular context: Template matching applied to electron tomograms

Electron tomography is the only technique available that allows us to visualize the three-dimensional structure of unfixed and unstained cells currently with a resolution of 6–8 nm, but with the prospect to reach 2–4 nm. This raises the possibility of detecting and identifying specific macromolecular complexes within their cellular context by virtue of their structural signature. Templates derived from the high-resolution structure of the molecule under scrutiny are used to search the reconstructed volume. Here we outline and test a computationally feasible two-step procedure: In a first step, mean-curvature motion is used for segmentation, yielding subvolumes that contain with a high probability macromolecules in the expected size range. Subsequently, the particles contained in the subvolumes are identified by cross-correlation, using a set of three-dimensional templates. With simulated and real tomographic data we demonstrate that such an approach is feasible and we explore the detection limits. Even structurally similar particles, such as the thermosome, GroEL, and the 20S proteasome can be identified with high fidelity. This opens up exciting prospects for mapping the territorial distribution of macromolecules and for analyzing molecular interactions in situ

Ultrastructural details in wild type and cw15⁺ cell tomograms.

<p>(A) Sections of flagella in the tomogram of the wild type cell. The arrow indicates a microtubule doublet. Bar, 1 µm. Inset: enlarged view showing flagellar membrane and microtubule doublets; bar, 200 nm. (B) Basal apparatus with distal connecting fiber (arrow) connecting the two basal bodies in the wild type cell. Pm, plasma membrane; Cw, cell wall. Bar, 1 µm. (C) Nuclear envelope membranes in the tomogram of the wild type cell. Bar, 1 µm. Inset: enlarged view showing inner and outer nuclear envelope membrane; bar, 200 nm. (D) Nuclear envelope membranes in the tomogram of the cw15⁺ cell. Bar, 1 µm. Inset: enlarged view showing inner and outer nuclear envelope membrane; bar, 200 nm. (E) Putative ER membrane (arrow) emanating from the nuclear envelope of the cw15⁺ cell. Bar, 500 nm. (F) Contrast rich cisternae structure (black arrow) which could be part of the Golgi apparatus in the cw15⁺ cell. Bar, 500 nm. Inset, possible vesicle budding site (white arrow). Bar, 200 nm. (G) Ultrastructural details of the chloroplast of the cw15⁺ cell: thylakoids and granae (arrow) and pyrenoid (Py). Bar, 1 µm. (H) Thylakoids and plastid envelope membranes of the chloroplast of the cw15⁺ cell. Bar, 500 nm.</p

Tomographic reconstruction using IMOD, Bsoft and Alignator reconstruction software applied to the <i>Chlamydomonas</i> cw15⁺ dataset shown in Fig. 1B.

<p>(A) Representative tomographic sections using the fiducial based Etomo package included in IMOD. Bar, 2 µm. (B) Representative tomographic sections using the back-projection based alignment procedure Bsoft. (C) Representative tomographic sections using the fiducial less alignment approach Alignator. (D) Z-section generated with Alignator. A number of organelles can be identified: Chloroplast (Cp) with the starch containing pyrenoid (Py) and thylakoids (Th); nucleus (Nc) with nucleolus (Ncl); vacuoles (Va); mitochondria (Mt); lipid bodies (Li). Bar, 2 µm.</p

Soft x-ray dataset of <i>Chlamydomonas reinhardtii</i> wild type and cw15⁺ strain used for tomographic reconstruction.

<p>(A) Wild type strain tilt-series taken from −60° to +60° and 0° image after acquisition. Low tilt angles were exposed for 2 s, higher tilt (40° onwards) for 8 s. All angles provide good structural resolution and were all used for the reconstruction of the tomograms. Bar, 2 µm. (B) cw15⁺ strain tilt-series −60° to +60° and 0° image after acquisition were taken as above. Bar, 2 µm.</p

Segmentation of cellular structures in <i>Chlamydomonas</i> cells.

<p>(A) Segmentation of a wild type cell tomogram. Top, stereo pairs in front view and after 180° rotation. Bottom, from left to right: chloroplast with pyrenoid; mitochondria; flagella; nucleus with nucleolus; vacuoles; lipid bodies. Bars, 2 µm. The tomogram and segmented volumes of the same cell are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053293#pone.0053293.s006" target="_blank">Videos S4</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053293#pone.0053293.s007" target="_blank">S5</a> (B) Segmentation of a cw15⁺ cell tomogram. Segmented volumes are presented as in A. The tomogram and segmented volumes of the same cell are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053293#pone.0053293.s005" target="_blank">Videos S3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053293#pone.0053293.s008" target="_blank">S6</a>.</p

Demonstration: VR-HYPERSPACE - the innovative use of virtual reality to increase comfort by changing the perception of self and space

Our vision is that regardless of future variations in the interior of airplane cabins, we can utilize ever-advancing state-of-the-art virtual and mixed reality technologies with the latest research in neuroscience and psychology to achieve high levels of comfort for passengers. Current surveys on passenger's experience during air travel reveal that they are least satisfied with the amount and effectiveness of their personal space, and their ability to work, sleep or rest. Moreover, considering current trends it is likely that the amount of available space is likely to decrease and therefore the passenger's physical comfort during a flight is likely to worsen significantly. Therefore, the main challenge is to enable the passengers to maintain a high level of comfort and satisfaction while being placed in a restricted physical space