Supplementary Methods

Photobleaching for CLEM experiments In order to effectively remove all FM1-43 derived photoconversion products in the bleach region of CLEM experiments, we scanned our rectangular region(s) of interest ten times at maximal laser power. In samples fixed immediately after photobleaching > 99% of vesicles in the bleach region of serial sections were PC− (Fig. 3a). We established that the bleach protocol was not detrimental to bouton health by demonstrating their ability to take up and then release FM1-43 following photobleaching (Supplementary Fig. 2).