Extended Data Fig. 2: Workflow of in vivo imaging followed by correlated ex vivo electron microscopy.

(a) Workflow of correlated light and electron microscopy (CLEM) to identify the in vivo- imaged dendrites for EM. From top left moving clockwise: in vivo images of dendrites imaged during an experiment are acquired a final time for reference of the most updated structural information. Vascular maps are then generated by 2p in vivo imaging of TRITC-Dextran (injected retro-orbitally on final day of in vivo imaging), capturing an ~200𝜇m z-stack in the area surrounding the dendrite of interest for detailed structural landmarks (panel 2 and white square in panel 3) and a low-zoom (usually 1 zoom/ ~1000𝜇m², panel 3) to provide the larger structural context with respect to the vasculature. Arrows indicate the vasculature used for aligning the subsequent images. After the animal is perfused with fixative, thin sections are cut, and bright-field imaging is used to find the vasculature and approximate the location of the dendrite (white square in panel 4). Confocal images are then acquired using the slice containing the target vasculature so as to ensure the location of the target dendrite (white square in panel 5). The slice of interest is then processed for EM, thereby permanently rendering the tissue photo-inaccessible. The tissue is then exposed to iterative steps of X-Ray tomography and trimming (guided by software-based non-rigid alignment) so as to isolate the dendrite within a small volume amenable to EM. Shown is an alignment of the confocal stack with the x-ray tomogram. Colored arrowheads point to the dendrites of interest identified in subsequent images. Serial-section scanning-blockface EM is then performed on the final tissue block, and the confocal images are aligned to the output using the accumulated fiducial structures over previous imaging modalities. The final result (panel 8) is an aligned stack of confocal fluorescence and EM data, allowing a fluorescence overlay onto the EM stack. Using this alignment, the target dendrite and constituent spines are then reconstructed using the in vivo 2p image as reference for the domains of interest. These experiments were repeated independently in n = 4 mice with comparable results (shown below). (b) After reconstruction of the target dendrite(s) and spines (left), the synaptically connected axonal partner of each spine was traced to the full extent of the imaging volume or to the furthest point of confident identification (right). In this volume, 59 axons were successfully reconstructed to various extents. (c) Results of all remaining CLEM experiments from the current study. Left, in vivo 2-photon average projections, aligned EM data, and the corresponding reconstruction of the target dendrites are shown. Reconstruction images are angled so as to accentuate the correspondence between 2p and EM. Middle, zoomed-in visualizations of the reconstructions of target dendrites prior to reconstruction of synaptically connected axons. The number of axons reconstructed in each volume were: 137 (top), 74 (middle), and 60 (bottom), for a total of 330 axons (including the 59 axons from (b)). Note that the axonal partners of spines near the ends of dendrites were often not reconstructed due to their proximity to the edge of the sample volume.