Targets were selected based on the SynCAM 1 signal. Three-dimensional SIM images were acquired with the OMX V4 Blaze system (GE Healthcare, UK), using the 405 nm, 488 nm, 568 nm, and 647 nm laser lines, a 60× 1.42 N.A. oil objective (Olympus), an immersion oil with a refractive index of 1.518 and standard emission filters at 125 nm z-sectioning. Multicolour registration with an error below 40 nm was done using 100 nm fluorescent beads (TetraSpeck, T7284, Thermo Fisher Scientific). Images were acquired with the DeltaVision OMX acquisition software (GE Healthcare), and images were reconstructed with softWoRx (GE Healthcare). Parameters for the reconstruction can be found in the Supporting information (S1 Files ). The quality of 3D SIM reconstructions was tested with SIMcheck [54 (link)]. The superresolution channels 642, 568, and 488 show a good signal-to-noise ratio and no signs of hexagonal artefacts. Fast Fourier transformed images uncover a high amount of information below the diffraction limit. Due to the limited brightness and stability of the Alexa Fluor 405 dye, the signal-to-noise ratio and resolution in the 405 nm channel were limited. We thus decided to use this channel only for vGlut1 as a reference for the segmentation of mature synapses and not as a structural superresolution readout. An image acquisition parameter log file is included in S1 Files .
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