Targeting of multiple cells in the same block (Dhc KD cells in the follicular epithelium of Drosophila ovaries expressing CD8-mCherry). (A) Workflow. (B) Tiled Z-stack scan of the entire resin block in XY view, with the two targets highlighted in green and red. (C) XZ view of the same block with the two fluorescent targets segmented in the same colors. The block surface before trimming is indicated by the arrowhead. The red target is 28 µm deeper than the green one. Therefore, the two cells have to be exposed sequentially for FIB-SEM acquisition. (D) Z targeting of the first group of CD8-mCherry–positive cells (segmented in green). The image shows the position of the fluorescent cell (segmented) and the block surface (arrowhead) after trimming. (E) Confocal imaging of the block after trimming and laser branding around the first cells of interest (XY view). (F) FIB-SEM acquisition of the same volume. The asterisks in E and F indicate the same cell viewed from orthogonal orientations. (G and H) Tiled Z-stack scan of the same resin block after removing the area imaged by FIB-SEM. Note that the lower left corner of the block, previously containing the green target, is missing. The XZ view in H shows that the second target (red) is now 28 µm deep from the block surface (arrowhead). (I) Z targeting of the second group of cells (segmented in red). The image shows the position of the fluorescent cell (segmented) and the block surface (arrowhead) after the second trimming. (J) Confocal imaging of the block after trimming and laser branding around the second cells of interest (XY view). (K) FIB-SEM acquisition of the same volume. The asterisks in J and K indicate the same cell.