FIG. 4.

ROI identification by correlation of features in cryo-LM and SEM images. (A) cryo-LM image of OCF5 worm, rotated to align with (B, C), below. The vulva is easily identified (A, arrowhead, also Fig.3B, top right). (B) Snapshot of freeze-substituted and resin-embedded worm.(C) Transmitted light microscopic image of the final semi-thin section of the worm stained with toluidine blue before SEM and FIB-SEM imaging. (D) SEM image of top surface of remaining resin block, revealing exposed worm and feature corresponding to vulva (arrow).(E) Higher magnification of worm, boxed area and thick arrow indicate area of protective Pt pad and FIB-SEM acquisition. (F) Polished “cliff face” of protected ROI. The appearance of a void (arrowhead) indicates a soon-to-be-revealed embryo. (G) FIB-SEM image section through targeted metaphase (*) and telophase nuclei (#). The plane of the images (F) and (G) is orthogonal to (A)–(E). Scale bars: (A, D): 100 μm, (E): 50 μm, (F, G): 10 μm.