The permeability barrier is a distinct envelope that forms between the embryo surface and trilaminar eggshell. (A) Embryos placed in Oregon green (OG) phalloidin (left; n = 14), fluorescein (middle; n = 7), or 3,000-D fluorescein dextran (right; n = 10) were imaged by differential interference contrast (DIC; top row) and fluorescence (middle row; green in merge) microscopy. Magnified views of the embryo anterior are shown on the right (yellow dashed lines mark the embryo plasma membrane; yellow arrows point to the edge of the permeability barrier). (B) DIC (top) and fluorescence (middle; red in merge) images of a one cell–stage mitotic embryo expressing mCherry::CPG-2 (n = 9). The embryo anterior is magnified on the right. The contrast of the top DIC image has been adjusted to visualize the edge of the permeability barrier (indicated by the black arrows in the merge). (C, left) A fluorescence image of CPG-2 was acquired immediately before cryoimmobilization and processing for transmission electron microscopy (n = 3). (middle) Alignment of the resulting correlative transmission electron micrograph (TEM) image allows visualization of the edge of the permeability barrier (red arrowheads). (right) A pseudocolored micrograph illustrates the location of the chitin and CPG eggshell layers along with the edge of the permeability barrier. The periembryonic and perivitelline spaces and the first polar body are also labeled. (D) Transmission electron micrographs of a control embryo showing the location of the polar bodies extruded during anaphase of meiosis I (embedded in the CPG layer; see also C) and during anaphase of meiosis II (in the periembryonic space). (E) Merged DIC and fluorescence images of the anterior of an embryo at the two-cell stage expressing mCherry::CPG-2. Green lines mark the location of the permeability barrier. Time indicates minutes and seconds past the first frame. (F) DIC and confocal fluorescence images of control (n = 12) and partial kca-1(RNAi) (n = 12) embryos expressing mCherry::CPG-2. (middle row) Embryos were also exposed to FITC dye to monitor the integrity of the permeability barrier (green lines). The embryo surface is also marked (dashed yellow lines). (G) Confocal fluorescence images of control (left) and partial kca-1(RNAi) (right) embryos expressing mCherry::CPG-2. The mCherry::CPG-2 in the perivitelline (left) or periembryonic (right) spaces was photobleached (red circle inside dashed white box, magnified on the left) at 0 s, and the embryo was continuously imaged every 1 or 2 s (left and right, respectively) to monitor recovery (quantified in graphs below). Bars: (A–C [left], E, and G) 10 µm; (C [middle and right], D, and F) 1 µm.