<p>A. Cartoon of the sectioning approach. Following 2PEM, NIRB and processing as described before, a few thick sections were produced from the sample to verify the orientation of the block and the presence of the NIRB markings (‘Approach’). The sectioning direction is indicated with an arrow. As soon as the first NIRB marks were spotted in the thick sections, we proceeded with obtaining thin 240 nm sections for electron tomography (‘Correlation+EM imaging ROI’). These sections were not only employed for EM imaging of the ROI, but also provided the structural features that were used for correlation between 2PEM and EM. B. Graphic representation of the landmark-based correlation procedure. The prisms with the blue and green ribs represent similar 3D volumes that differ in size, shape and orientation, representing the samples pre- and post-EM-processing. In order to predict the position of the ROI (ball, visible in volume 1) in volume 2, similar points (landmarks) in both datasets are identified (‘Correlation’). These landmarks are overlaid in 3D (‘Registration’) enabling warping of volume 1 into volume 2 and thus also projecting the position of the ROI in volume 2 (‘Docking’). C. Collagen fibers can be observed in both the 2PEM z-stack (top row panels, 2PEM) and the EM images of the serial sections (middle row, TEM). In the TEM images, the collagen is visible as electron dense fibers running in between the cellular material. Each column shows a 2PEM and an EM image obtained at a similar position in the sample, pre- and post EM processing. The numbers in the top left corner of the panels indicate their position in z to the image shown in the first column. The 2PEM dataset is stretched in z relative to the TEM dataset, causing the larger z-steps between the 2PEM images in the different rows. The bottom row shows the overlay of the 2PEM image (blue) and the TEM image (grey levels). Scale bars: 100 µm. D-F. The landmark-registration procedure is performed in Amira, using 3D models of collagen fibers as a reference. The selected landmarks (colored ‘targets’) indicate corresponding points in both datasets. D. 3D model of the collagen fibers (blue) and the cell of interest (green), obtained from a high magnification 2PEM z-stack of the cell of interest. E: 3D model of the collagen fibers (grey), obtained from a z-stack of EM images. F. The 3D visualization software Amira was used to overlay the landmarks and perform non-linear transformations to the 2PEM model to warp it into the EM model. G-I: Landmark-registration between the 2PEM and EM datasets enables retrieval and electron tomography of the cell of interest (green). G. Following the registration of the 2PEM in the EM dataset, the cell of interest could be docked within the z-stack of EM images. Scale bar: 10 µm. H. The docked cell of interest is outlined in green on a TEM section of the ROI. The nucleus of the cell is visible. Scale bar: 3 µm. K. Virtual slice through the tomogram obtained in the area that is boxed in I. The collagen fibers in the top left corner are clearly recognizable owing to their striated structure and high electron density. Scale bar: 500 nm. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114448#pone.0114448.s006" target="_blank">Movie S4</a>.</p
