Evaluation of Trabecular Microstructure of Cancellous Bone Using Quarter-Detector Computed Tomography

Quarter-detector computed tomography (QDCT) is an ultra-high-spatial-resolution imaging technique. This study aimed to verify the validity of trabecular structure evaluation using a QDCT scanner in the diagnosis of osteoporosis. We used a cancellous bone specimen image of the second lumbar vertebrae of an adult male with moderate osteoporosis. To obtain QDCT images, we created a three-dimensional model from micro-CT images of the specimen. Statistical analysis was performed on the relationship between micro-CT and QDCT imaging modalities. The differences between micro-CT and QDCT were assessed based on their significance with respect to the calculated mean measurements using the Mann–Whitney test. Single regression analysis was performed using linear regression, with micro-CT and QDCT as the explanatory and objective variables, respectively, to determine the relationship of the measured values between the two modalities. By applying the necessary correction to the micro-CT measured values, it is possible to perform an analysis equivalent to micro-CT, which offers higher spatial resolution than QDCT. We found evidence that if QDCT can be used, trabecular structure evaluation may contribute to image diagnosis to evaluate practical bone fragility

Three-Dimensional Ultrastructural Study of Oil and Astaxanthin Accumulation during Encystment in the Green Alga <em>Haematococcus pluvialis</em>

<div><p><em>Haematococcus pluvialis</em> is a freshwater species of green algae and is well known for its accumulation of the strong antioxidant astaxanthin, which is used in aquaculture, various pharmaceuticals, and cosmetics. High levels of astaxanthin are present in cysts, which rapidly accumulate when the environmental conditions become unfavorable for normal cell growth. It is not understood, however, how accumulation of high levels of astaxanthin, which is soluble in oil, becomes possible during encystment. Here, we performed ultrastructural 3D reconstruction based on over 350 serial sections per cell to visualize the dynamics of astaxanthin accumulation and subcellular changes during the encystment of <em>H. pluvialis</em>. This study showcases the marked changes in subcellular elements, such as chloroplast degeneration, in the transition from green coccoid cells to red cyst cells during encystment. In green coccoid cells, chloroplasts accounted for 41.7% of the total cell volume, whereas the relative volume of astaxanthin was very low (0.2%). In contrast, oil droplets containing astaxanthin predominated in cyst cells (52.2%), in which the total chloroplast volume was markedly decreased (9.7%). Volumetric observations also demonstrated that the relative volumes of the cell wall, starch grains, pyrenoids, mitochondria, the Golgi apparatus, and the nucleus in a cyst cell are smaller than those in green coccid cells. Our data indicated that chloroplasts are degraded, resulting in a net-like morphology, but do not completely disappear, even at the red cyst stage.</p> </div

3D TEM images of subcellular components.

<p><b>A</b>, <b>C</b>, <b>E</b>, <b>G</b>, <b>I</b>, and <b>K</b> represent a green coccoid cell; <b>B</b>, <b>D</b>, <b>F</b>, <b>H</b>, <b>J</b>, and <b>L</b> represent a cyst cell. <b>A</b> and <b>B</b>. 3D reconstruction of chloroplasts with pyrenoids, mitochondria, and/or starch grains. <b>C</b> and <b>D</b>. 3D reconstruction of astaxanthin distribution. <b>E</b> and <b>F</b>. 3D reconstruction of starch grains with the nucleus. <b>G</b> and <b>H</b>. 3D reconstruction of Golgi bodies with the nucleus. <b>I</b> and <b>J</b>. 3D reconstruction of mitochondria (with the nucleus in J). <b>K</b> and <b>L</b>. 3D reconstruction of pyrenoids and starch capsules. All subcellular components are denoted by different colors as indicated in the color legends. Scale bar in all images: 5 µm.</p

interference contrast (DIC) image and subcellular localization of lipids, astaxanthin, and chlorophyll in an astaxanthin-rich <i>H. pluvialis</i> cell.

<p>Nile Red (NR) signal, astaxanthin (AXT), and chlorophyll (CHL) autofluorescence are yellow, red, and green, respectively. Two overlaid images (NR+AXT and NR+AXT+CHL) are shown. Note that the Nile Red signals are colocalized with astaxanthin, shown in orange. Scale bar: 10 µm.</p

Transmission electron micrographs of green coccoid cells in <i>H. pluvialis</i>.

<p><b>A</b>. General ultrastructure. The cell wall is surrounded by extracellular matrix (arrowheads). Arrows indicate astaxanthin granules. <b>B</b>. Chloroplast and pyrenoid. <b>C</b>. High-magnification view of astaxanthin granules (arrows). <b>D, E</b>. One-layer thylakoids with a regular arrangement. C, chloroplast; CW, cell wall; N, nucleus; P, pyrenoid. Scale bars in A and B–E: 5 µm and 1 µm, respectively.</p

3D TEM images of whole cells.

<p><b>A</b>. Cut-away image of a green coccoid cell. <b>B</b>. Cut-away image of a cyst cell. All subcellular components are denoted by different colors as indicated in the color legends. Scale bar: 5 µm. (See also Movies S1 and S2 for supporting information.)</p

Transmission electron micrographs of <i>H. pluvialis</i> cyst cells.

<p><b>A</b>. General ultrastructure of cyst cells, showing small granules that contain astaxanthin. <b>B</b>. General ultrastructure of a cyst cell, showing astaxanthin accumulation in oil droplets. <b>C</b>. General ultrastructure of a cyst cell, showing large oil droplets. Chloroplasts localize in the interspace between oil droplets (arrows). <b>D</b>. Some oil droplets are fused. <b>E</b>. High-magnification view of chloroplasts. C, chloroplast; N, nucleus; OD, oil droplet. Scale bars in A–D and E: 5 µm and 0.5 µm, respectively.</p

Transmission electron micrographs of intermediate <i>H. pluvialis</i> cells.

<p><b>A</b>. General ultrastructure. <b>B</b>. High-magnification view of astaxanthin oil droplets. <b>C</b>. Partial degradation of thylakoids (arrow). <b>D</b>. High-magnification view of thylakoid degradation (arrows). C, chloroplast; CW, cell wall; N, nucleus; OD; oil droplet; P, pyrenoid; SC, starch capsule; SG, starch grain. Scale bars in A and B–D: 5 µm and 1 µm, respectively.</p

Life cycle of <i>H. pluvialis</i>.

<p><b>A</b>. Fluorescence microscopy images, showing the 1- to 32-cell stages, and the flagellated stage. DIC: differential interference contrast image; SYBR: SYBR Green I-stained cells (green); CHL: chlorophyll autofluorescence (red); and Overlay: overlaid images of SYBR and CHL. <b>B</b>. Illustration of life cycle of <i>H. pluvialis</i>. Refresh: when old cultures are transplanted into fresh medium, coccoid cells undergo cell division to form flagellated cells within the mother cell wall. Germination: Flagellated cells settle and become coccoid cells. Continuous and/or strong light accelerate the accumulation of astaxanthin during encystment (red arrows).</p

Relative volumes of <i>H. pluvialis</i> subcellular components.

<p>Subcellular components are indicated by colors in pie charts. <b>A</b>. Relative volumes of subcellular components in a green coccoid cell. <b>B</b>. Relative volumes of subcellular components in a cyst cell.</p