FIB-SEM

Prior to FIB-SEM imaging, blocks were mounted on SEM stubs (EMS cat# 75220) with conductive carbon adhesive tape (EMS cat#77816). Additional straps of conductive tape were applied to the blocks, to connect the stub and the block surface. An 8-10nm layer of Ir was deposited on the surface of the block using compact coating unit (CCU) - 010 (Safematic GmbH, Switzerland). Samples were then mounted onto a Helios NanoLab DualBeam 600 microscope (Thermofisher Scientific, USA). After finding ROIs in the SEM, 0.5-1μm of Pt was deposited on top of the ROI using either an ion beam (30kV,0.46-0.92nA), or with an additional electron beam deposition step (2kV, 5.5nA) prior to ion beam deposition. To expose the cross-section of the ROI, a frontal trench was milled with the ion beam (30kV, 21nA); trench dimensions varied with respect to ROI dimensions. Samples were subsequently imaged using a Crossbeam 550 system (Carl Zeiss GmbH, Germany) at a 54° tilt angle and a 5 mm working distance. Prior to serial surface imaging, cross-sections were polished with an ion beam (30kV, 1.5-7nA) and then milled in serial section mode (30kV, 0.7-1.5nA), at different dose factors (5-8) depending on the specific specimen. SEM micrographs were acquired under 2kV, 350pA. A combination of detectors, in-lens secondary electrons (inLens)/ energy-selective backscattered electrons (EsB) or EsB/secondary electrons type 2 (SE2), were used for image acquisition. Detection was determined per acquisition with respect to the image quality obtained in every specific stack.

Image processing: collected data was filtered using unsharp masking or local contrast enhancement (CLAHE) followed by smoothing (if needed) in Fiji. All other processing steps were performed using Amira version 2019.3 (Thermofisher Scientific, USA). Alignment was done with Align Slices module, allowing translation only, and refined manually to correct for misaligned slices. Segmentation of the vesicles was made semi-automatically using a brush tool with a threshold mask, followed by interpolation. The threshold was adjusted to include the volume inside the vesicles. The fusion pore area was segmented from the negative curvatures formed at the connection with the plasma membrane. Segmentation was performed every 4^th slice of the stack unless new features appeared (especially in crumpled vesicles), then a smaller gap was used to include the new feature in the interpolation. The gaps were then interpolated, and the interpolation was corrected manually in cases of mis-interpolated features. A surface was generated from the segmented data using the Generate Surface module, followed by volume and surface area calculations using the Surface Area Volume module.