Volume correlation procedure.

One entire microwell was imaged by light microscopy (DIC and confocal fluorescence microscopy), while one cell at an edge of the same well was imaged by FIB/SEM tomography. a) Overview SEM image of the well of interest after epoxy embedding, and SEM image of the region of interest at the corner of the well after preparing the region for FIB/SEM tomography. The images collected during the tomography experiment are in the x-z plane (yellow square), while the images collected during LM imaging are in the x-y plane, parallel to the well surface (yellow line). Ion images are collected in the x-y plane before each milling step, where the cross serves as a reference marker enabling each slice to be milled with nanometer precision. b) Illustration of how the shape of the microwells can be used to place the volume imaged during FIB/SEM tomography correctly within the larger volume imaged during LM imaging. The ion images are first aligned with the edges of the well as imaged by LM. The perpendicular FIB/SEM tomography images can then be aligned with the ion images, with the first and the last image overlapping with the edge observed in the first and last ion image. c) After alignment the two volumes are correlated, and an overlay between fluorescence and EM images can be done in any plane or volume. In the 2D overlay, an xy projection from the FIB/SEM stack is overlaid on the corresponding fluorescence image, with nuclei displayed in blue and bacteria in red. Scale bars: a: 20μm, c: 5μm.