SBF SEM data was collected using a

SBF SEM data was collected using a 3View2XP (Gatan Inc.)attached to a Sigma VP SEM (Zeiss). Inverted backscatteredelectron images were acquired through the entire extent ofthe region of interest. For each 50nm slice, a low-resolutionoverview image (pixel size of 50nm using a 1.5μs dwell time)and several high-resolution images of the different regionsof interest (indicated magnification 5000x, pixel size of 6-7nm using a 1.5μs dwell time) were acquired. The overviewimage was used to relocate the region of interest defined bythe confocal images of the sections. The SEM was operatedin variable pressure mode at 5Pa. The 30μm aperture wasused, at an accelerating voltage of 2kV. Typically, between300 and 1000 slices were necessary for an entire region ofinterest.As data was collected in variable pressure mode, only minoradjustments in image alignment were needed, particularlywhere the field of view was altered in order to track the cellof interest. All the images were converted to tiff in DigitalMicrograph (Gatan Inc.), and tiff stacks were automaticallyaligned using TrakEM2, a FIJI framework plug-in (Cardonaet al. 2012). Manual segmentations were done in TrakEM2.For Fig. 1D, labels were exported as tiff for visualization in3D in ClearVolume, a FIJI framework plug-in (46). For Sup.Fig. 4B, they were exported as Amira labels for visualizationin 3D in Amira Software (Thermo Fisher Scientific). Movie2 was generated in FIJI, Movie 4 in Amira, both were compressedin Quick Time Pro with the H.264 encoder.