Quantifying Synaptic Ultrastructure Through Electron Microscopy

Electron microscopy was performed as previously described (Watanabe et al., 2013 (link)). Freezing was performed on a Leica EMpact2 (Leica, Wetzlar, Germany). To stimulate neurotransmission animals were exposed to blue (488 nm) LED light for 20ms and frozen 50ms later. Thirty-three nm serial sections were taken and imaged using a Hitachi H-7100 transmission electron microscope equipped with a Gatan Orius digital camera (Gatan, Pleasanton, CA). Micrographs were analyzed in ImageJ using a program for morphological analysis of synapses (Watanabe et al., 2020 (link)). Scripts available at: https://github.com/shigekiwatanabe/SynapsEM (copy archived at Watanabe, 2022 ).