Targeting of a clone of Drosophila ovary FCs expressing Dhc RNAi and CD8-mCherry. (A and B) Tiled Z-stack confocal acquisition of the resin block. The gray-scale image shows a rendering of the red signal. A few mCherry-expressing cell clones are visible (e.g., arrowhead) as well as the autofluorescence background of the Ficoll-containing freezing medium. The target of the FIB-SEM acquisition is highlighted by the dashed box, and segmentation is shown to facilitate its visualization. A and B show XY and XZ views of the block, respectively. (C and D) In gray scale, XZ view of the volume rendering of the fluorescence of the block, with the cells of interest segmented in two different colors. The arrowheads show the position of the block surface before (C) and after (D) trimming. (E) XY view of the same volume that shows the two-photon branding of the block surface limiting the ROI to be acquired by FIB-SEM (magenta segmentation). (F and G) Images of the block during FIB-SEM run setup: 50-pA FIB image acquired with secondary electron detector (F) and 1.5-keV 700-pA SEM image acquired with ESB detector. A crack at the interface between the basal membrane of the ovary and the empty resin is visible here. This happened frequently during the branding of Drosophila oocyte samples but did not affect the structure of the FCs. (H–M) Overlays of the fluorescence (H and K) and segmented FIB-SEM (I and L) datasets show the precision of the registration in different orientations.