Full Volume Correlation of the ROI.

Abstract

<p>A. 3D view of a 2PEM z-stack of mouse ear tissue, 7 days post-injection with GFP-expressing tumor cells (green). Vessels are stained with Evans Blue (red). Scale bar: 100 µm. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114448#pone.0114448.s005" target="_blank">Movie S3</a>. B. 3D model of the area that is indicated with a box in A. The cells of interest are segmented in green and numbered (1–6). The vessels are shown in red. The vessel bifurcation close to the skin surface is made transparent to reveal the cells underneath. Scale bar: 50 µm. C. X-Z slices through the 3D 2PEM volume, at the height of the cells of interest (circled). The numbers on the left bottom correspond to the numbers used in panel B to label the cells of interest. These views show the relative position of the cells with respect to the blood vessels (red) and the collagen-rich dermis (blue). D. Cartoon of the sectioning procedure. To approach the ROI and monitor the progression, serial thick sections were produced from the sample (the arrow indicates the sectioning direction) and imaged with LM (‘Approach+Correlation’). Approximating the location of the cells of interest, 240 nm sections for electron tomography were obtained (‘EM imaging ROI’). E. 500 nm toluidine-blue-stained sections that were obtained at different depths within the sample (the relative z-distance from the first section is indicated in top right corner of each panel). The vessels are segmented in red, hair follicles in brown. Scale bar: 100 µm. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114448#pone.0114448.s005" target="_blank">Movie S3</a>. F. 3D model of the hair follicles (brown), vessels (red) and NIRB markings (purple) segmented in the serial thick sections. Scale bar: 50 µm. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114448#pone.0114448.s005" target="_blank">Movie S3</a>. G. Overlay of a 3D model of the 2PEM z-stack (based on A, magenta) and a model of the vessels segmented in the serial thick sections (based on F, yellow). Overlapping areas are highlighted in white. Asterisks indicate vessel-forks that are visible in both datasets. Scale bar: 50 µm. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114448#pone.0114448.s005" target="_blank">Movie S3</a>. H. Correlation between 2PEM, LM and EM enabled retrieving the cells of interest (numbering in bottom right corner, as in B and C) in the sequence of serial thin sections. The electron transparent areas in proximity of the cells indicate a local absence of extracellular matrix components. The cause of this missing material remains to be elucidated. I. A selected area in the 2PEM dataset, showing a cell of interest (number 4 in B, C and H). J. Selection of the TEM images of cell 4, shown in panel I. The cell is modeled in green and the vessel lumen in red. For each image, we indicated the z-distance from the section where the cells was first spotted. Scale bar: 10 µm. K. Cell 4 (green) and the blood vessel lumen (red) are segmented in the serial sections (J). A 3D model of this segmentation reveals the shape of the cell, which closely resembles the 2PEM view of the same cell, shown in I. Scale bar: 10 µm. L-N: Electron tomography of a tumor cell (number 2 in B,C, and H), shown at low magnification in panel L. At the position of the box in L, a tomogram was obtained. Scale bar: 5 µm. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114448#pone.0114448.s005" target="_blank">Movie S3</a>. M. One selected virtual slice of the tomogram shows the complex architecture of the cell membrane and the fiber-like structure of extracellular matrix (ECM). N: The high image contrast of the ECM fibers enables semi-automatic segmentation of the fibers (shown in blue) within the 3D dataset. Scale bars in M and N: 500 nm. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114448#pone.0114448.s005" target="_blank">Movie S3</a>.</p