Confocal microscopy

Images were acquired using a Zeiss confocal laser scanning microscope (Zeiss LSM 780 and Zeiss LSM 880, Carl Zeiss) equipped with a 63× oil immersion objective lens of numerical aperture 1.4. The refractive index of oil was 1.51. The sampling size of each image acquired on the Zeiss microscope was 90 nm per pixel for the LSM 780 and 44 nm per pixel for the LSM 880, while using a zoom of 3. Sections of 380 nm along the z-axis were imaged with an axial sampling of 30–70 nm. For tile scan imaging to differentiate proximal and distal synapses, a single plane tile scan was taken with the help of a confocal microscope (Zeiss LSM 880) keeping the cell body at the center and sampling at 190 nm to cover a total area of 270 × 270 μm²