Targeting of H2B-mCherry –expressing mammary gland organoids grown in Matrigel. (A and B) Tiled Z-stack confocal acquisition of the resin block. The gray-scale image shows a 3D rendering of the mCherry signal. The autofluorescence allows the identification of the block edges and surface. XY and XZ projection views of the volume are shown in A and B, respectively. The dashed box indicates the organoid of interest. (C) XZ views of the organoid in A and its distance to the block surface (arrowheads) during iterative imaging/trimming cycles to approach the ROI in Z. (D) Confocal bright field (BF) image of the block surface after laser branding. (E) SEM image of the block surface showing the laser mark. Note that the biological sample is not yet exposed at the surface, making the branding the only reference to target FIB-SEM acquisition. (F) FIB-SEM acquisition of the full organoid, achieved at 15 nm × 15 nm × 20 nm voxel size. Orthogonal slices through the volume are shown. (G) Segmentation of the organoid (in red) and of a representative single cell (green). At this stage of the organoid development, the cells acquire a complex organization. The cell highlighted has contact to the Matrigel on two sides and forms the lumen with a lateral portion of its protrusion. (H) Overlay of the nuclei segmented from the EM volume (white) and from the fluorescence stack (red) shows precise alignment of the datasets, allowing single-cell identification. (I–N) Targeting of a mitotic telophase event within an organoid. I and J show the targeting of an organoid (dashed box). After exposing the organoid, a mitotic event could be identified (arrowheads in K and L), and these cells were then targeted for FIB-SEM acquisition at high resolution. (M and N) Overlay between the fluorescence dataset and a slice of the FIB-SEM volume.