Procedure for intravital CLEM.

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Procedure for intravital CLEM. Procedure for intravital CLEM. (A) Intravital microscopy of bone marrow within murine calvarium. (B) Single frame from time-lapse imaging of mTmG-Tomato–expressing mouse showing a megakaryocyte positive for glycoprotein 1b (green) entering a sinusoid. (C) Extended focus-stitched tile scan of the calvarium showing the marrow (red) and vasculature (white), which will be used to locate the area observed during intravital imaging (box) and the location of the protrusion (*). (D) Calvarium is removed and immersion-fixed. (E) Calvarium is decalcified, stained, and embedded in resin. (F) Resin-embedded calvarium showing the same area viewed for live imaging (box) and the location of the protrusion (*). (G) The desired area trimmed down for sectioning. (H) Thick section showing sinusoids (SL), bone marrow (BM), and bone (B), which when viewed in combination with z-stacks acquired during live imaging allows determination of the z-height within the tissue and hence is when serial 300-nm sections should be taken. (I) Serial 300-nm sections taken just before the region of interest is reached. (J) Low-magnification TEM image confirming the location viewed with intravital imaging has been found. (K) Selected TEM images from the serial sections at the protrusion location. (L) Alignment of the images acquired from the serial sections. (M) Segmentation of structures of interest within the aligned stack of images. (N) 3D rendering to generate a model of the structures. (O, P) Models generated from the segmented image stack. (Q) Tomography can be performed on sections corresponding to regions of interest within the model. (R) This allows high-resolution models to be generated revealing cytoskeletal structure. (S) Model generated from large-volume tomography showing MT (red) arrangement. Scale bars represent 20 μm in (B, K, O, P); 500 μm in (C, F); 100 μm in (H); 50 μm in (J); and 2 μm in (S). Edward Brown et al. LSA 2018;1:e201800061 © 2018 Brown et al.