Targeting of tracheal terminal cell in Drosophila larva. (A and B) Tiled confocal Z-scan covering the entire volume of the block containing tissue. The gray-scale image shows a volume rendering of the thresholded autofluorescence signal (green channel) of the block (clearly visible is the autofluorescence of the epidermal cuticle) in XY (A) or XZ (B) views. The segmented cell of interest (in red, the DsRed fluorescence of the cell; in green, autofluorescence of the ECM of the tracheal tube) is highlighted in the boxed area to visualize its location in the block. (C and D) In gray scale, XZ view of the volume rendering of the fluorescence of the block, with the cell of interest segmented. The arrowheads indicate 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 marking the ROI to be acquired by FIB-SEM (segmented in magenta). (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 (G). In F, the green box delimits the area that will be milled during the FIB-SEM experiment, and the green arrow shows the milling direction.