We performed two-photon imaging in the mouse visual cortex as described previously27 (link),30 (link) by recording calcium responses to visual stimuli consisting of drifting gratings in each of 16 directions. We then acquired an in vivo fluorescent anatomical volume after injecting the tail vein with SR101 (100 mM) to label vasculature. The animal was perfused transcardially (2% paraformaldehyde/2.5% glutaraldehyde) and the brain was processed for serial-section TEM. Serial thin (<50 nm) sections were cut, picked up on pioloform-coated slot grids, and then post-stained with uranyl acetate and lead citrate. 1,215 serial sections were imaged at 120 kV on a JEOL 1200 EX with a custom scintillator atop optical-quality leaded vacuum glass at the end of a custom-built vacuum chamber extension. Custom software controlled automated x–y stage motion and image acquisition with a 2x2 array of CCD cameras (Imperx IPX-11M5) and Zeiss lenses. Images suitable for circuit reconstruction were acquired at a net rate of 5–8 MPix/s. Camera images were aligned in 2–D by registering adjacent camera images and dewarping, followed by histogram equalization and stitching. Then adjacent sections were registered and 3-D deformations were equalized in aligning the EM volume. Axonal and dendritic arbours of the functionally characterized neurons were manually reconstructed using TrakEM2 and objects were classified using classical criteria33 . Neurons or dendritic fragments receiving synapses from multiple functionally characterized cells were included in analysis of convergence. For each synapse participating in a convergence, a second individual (blind to the original reconstruction) traced the pre- and the post-synaptic processes, starting from the synapse. Segmentation that diverged between the two tracers was excluded from further analysis. Cumulative synaptic proximity (CSP) of pairs of axons was calculated by centring a 3-D Gaussian density function at each synaptic bouton and taking the sum of their dot products over all pairs of synapses.