Full Volume Correlation of the ROI.

<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

2PEM Imaging of D2.0R Cells Xenotransplanted in Mouse Ear Skin.

<p>A. Two weeks after injection with LifeAct-Ypet expressing D2.0R cells, a tumor is formed in the mouse ear skin (top left panel, outlined in green). The green fluorescent signal of the D2.0R cells can be observed in the tumor region (top right panel). A histological cross-section through the tumor region was obtained from the second ear injected at the same time with the same cell type. The Trichrome Masson staining colors the collagen in blue (bottom panel). B. Maximum projection of a z-stack obtained from an area in the periphery of the invasive tumor (top left panel A, white square). The D2.0R cells are shown in green, and the tumor is outlined in yellow. A background signal from the Evans Blue (red) can be observed in the vicinity of the blood vessels, potentially caused by the ‘leaky’ nature of the tumor vessels, its uptake by residing immune cells and the repeated injections. Scale bar: 50 µm. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114448#pone.0114448.s006" target="_blank">Movie S4</a>. C. Higher magnification z-projection of the cell of interest boxed in B. Scale bar: 10 µm. D. Time-lapse imaging of a D2.0R tumor cell in vivo. The different panels depict, in black against a white background, the fluorescent signal of the actin in the cell at the indicated time-points. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114448#pone.0114448.s006" target="_blank">Movie S4</a>. E. Color-coded map of the structural changes of the cell of interest (boxed in B and C) over time. Scale bar: 10 µm.</p

Correlating Intravital Multi-Photon Microscopy to 3D Electron Microscopy of Invading Tumor Cells Using Anatomical Reference Points

<div><p>Correlative microscopy combines the advantages of both light and electron microscopy to enable imaging of rare and transient events at high resolution. Performing correlative microscopy in complex and bulky samples such as an entire living organism is a time-consuming and error-prone task. Here, we investigate correlative methods that rely on the use of artificial and endogenous structural features of the sample as reference points for correlating intravital fluorescence microscopy and electron microscopy. To investigate tumor cell behavior <i>in vivo</i> with ultrastructural accuracy, a reliable approach is needed to retrieve single tumor cells imaged deep within the tissue. For this purpose, fluorescently labeled tumor cells were subcutaneously injected into a mouse ear and imaged using two-photon-excitation microscopy. Using near-infrared branding, the position of the imaged area within the sample was labeled at the skin level, allowing for its precise recollection. Following sample preparation for electron microscopy, concerted usage of the artificial branding and anatomical landmarks enables targeting and approaching the cells of interest while serial sectioning through the specimen. We describe here three procedures showing how three-dimensional (3D) mapping of structural features in the tissue can be exploited to accurately correlate between the two imaging modalities, without having to rely on the use of artificially introduced markers of the region of interest. The methods employed here facilitate the link between intravital and nanoscale imaging of invasive tumor cells, enabling correlating function to structure in the study of tumor invasion and metastasis.</p></div

Correlation based on Sequential LM and EM imaging to Target a Single Tumor Cell.

<p>A. 3D view of a 2PEM z-stack of mouse ear skin tissue, 7 days post-injection with GFP-expressing tumor cells (green). Vessels are stained with Evans Blue (red). The arrowhead indicates the part of the blood vessel that is also visible in the thick section shown in panel D. The image is obtained using the 3D Viewer plugin in Fiji. Scale bar: 50 µm. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114448#pone.0114448.s004" target="_blank">Movie S2</a>. B. 3D model of the imaged volume. The cell of interest, boxed in panel A and B, is segmented in green, the other cells are segmented in grey. The vessels are shown in red. Scale bar: 50 µm. C. Cartoon of the sectioning procedure. The resin embedded sample is sectioned from the direction indicated with the arrow. To approach the ROI, 180 thick sections (500 nm) were obtained from the sample ('Approach and Correlation'). Next, 10 consecutive series of 10 thin (60 nm) and two thick (500 nm) sections were obtained from the ROI. Thick sections were used for correlation and the thin sections were imaged with EM. D. The cell of interest (box) was identified in a thick toluidine blue stained section of the ROI. The arrowhead points to a cross-section of the vessel depicted in A with a white arrowhead. Scale bar: 50 µm. E: Low magnification EM image of the cell boxed in panel D. The cell appears highly polarized and contains protrusive structures, showed at higher magnification in panel F. Scale bar in E: 5 µm, in F: 2 µm. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114448#pone.0114448.s004" target="_blank">Movie S2</a>.</p

Outline of the Correlative Workflow.

<p>A. The flowchart lists the general steps involved in the correlative workflow. B. Left panel: installation of the anesthetized mouse on the LM stage. The ear with the transplanted fluorescent tumor cells (right panel, cartoon) is mounted in a custom-built holder. C. Z-projection of a typical 2PEM dataset obtained from the mouse ear that was injected with GFP-expressing tumor cells (green). SHG signal of the collagen fibers is shown in blue. Evans Blue stains blood vessels and is depicted in red. Scale bar: 100 µm. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114448#pone.0114448.s003" target="_blank">Movie S1</a>. D. Cartoon of the NIRB process representing the imaged volume containing a vessel (red), collagen fibers (blue), and tumor cells (green). Our NIRB-procedure entails drawing a frame at the surface of the skin with a high-powered laser, above and away from the ROI. The imaged volumes presented in this work were in between 60 and 200 µm in depth and ranging from 270 to 440 µm in xy. E. Following NIRB, the same volume is imaged again with 2PEM. The NIRB marks visible in this z-projection are traced in white. Scale bar: 100 µm. Asterisks point to hair follicles. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114448#pone.0114448.s003" target="_blank">Movie S1</a>. F. After chemical fixation of the mouse ear sample, NIRB markings remain temporarily autofluorescent and their location can be mapped. Asterisks pointing to hair follicles (as in E). G. Following EM processing, the embedded sample can be trimmed and sectioned by ultramicrotomy. Serial thick sections (500 nm) are placed on glass slides to be imaged by light microscopy (‘sections on LM slide’). Serial thin sections (60 to 240 nm) are mounted on slot grids (‘sections on TEM grid’) allowing for TEM observation and/or electron tomography.</p

Correlating 2PEM to 3D EM of D2.0R Cells using Collagen Fibers as a Reference.

<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

Immuno-EM of IMPDH localisation.

<p>Representative electron micrographs of CHO cells treated with (A) vehicle or (B and C) 2 µM MPA for 4 h. Cells were fixed, processed and labelled with anti-panIMPDH antibody and gold-labelled anti-mouse secondary antibody, as outlined in methods. Plasma membrane (PM), Nucleus (N) and mitochondria (M) are indicated. Scale bars = 0.5 µm.</p

Investigating a role for the Bateman domain in IMPDH clustering.

<p>(A) Superimposed structure of IMPDH2 (1B3O; yellow) with IMPDH1 (1JCN; red). Ligands have been removed for clarity. N labels the N-terminus. (B) Micrographs of CHO cells transiently expressing HA-IMPDH proteins (as labelled), treated with vehicle (control) or 1 µM MPA for 4 h. Cells were fixed and labelled for HA (HA-IMPDH; green) and nuclei were counterstained with DAPI (blue). Representative of at least four independent experiments. (C) Representative micrographs of CHO cells transiently expressing IMPDH1/IMPDH2 chimera (as labelled). Cells were fixed and stained for HA (HA-IMPDH; green) and nuclei were counterstained with DAPI (blue). Scale bars = 10 µm. (D) Table shows qualitative scoring of chimera subcellular distribution pattern according to the similarity to IMPDH1 (macrostructure formation) or IMPDH2 (diffuse) distribution. Assignments were based on three independent experiments. Shown by the schematics are the IMPDH1 (red) and IMPDH2 (yellow) regions of chimera constructs, with internal numbering referring to IMPDH1 sequence boundary and the striped boxes indicating the CBS dimer.</p

IMPDH activity in the presence of nucleotides.

<p>Activity of purified His-IMPDH proteins following 20 min pre-incubation with 1 mM or 5 mM nucleotides (ATP, AMP, XMP) and normalised to control, no addition, activity. Shown is the mean ± SEM (n = 3, **<i>p</i><0.01, ***<i>p</i><0.001 <i>cf.</i> to the control activity).</p

R224P mutation affects ATP mediated protease protection and affects spontaneous clustering.

<p>(A) Representative Coomassie stained gel of a protease protection assay with His-IMPDH1 proteins. INM stands for IMP, NAD and MPA. (B) Quantitation of remaining full-length protein from protease protection assay presented as percent protection. Shown is the mean ± SEM and the number of experiments (n) is indicated below the graph. (*<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001 <i>cf.</i> the control (digested), # <i>p</i><0.01 <i>cf.</i> IMPDH1. (C) Representative micrographs of CHO cells transiently expressing HA-IMPDH1, HA-IMPDH1 R224P or HA-IMPDH1 D226N. Cells were fixed and stained for HA (HA-IMPDH; green) and nuclei were counterstained with DAPI (blue). Representative of at least four independent experiments. Scale bar = 10 µm. (D) CHO cells transiently expressing HA-IMPDH proteins, as indicated, were treated with either vehicle, 2 µM MPA for 4 h or 2 µM MPA for 4 h and supplemented with 100 µM guanosine for the final 2 h. Cells were fixed and stained with anti-HA antibody. From random fields, 50–80 labelled healthy cells were counted, in a blinded manner, and the classification of the subcellular distribution of the protein classified as diffuse (blue), in spicules (red) or macrostructures (green). Representative of two independent experiments.</p