Visualization of the organelle aggregate seen in EPCD and LPCD stage cells. (A) DIC image depicting aggregates of mitochondria and chloroplasts in early-PCD (EPCD) stage cells. (B) Higher magnification DIC image from EPCD stage cells. Aggregate marked by black arrow (C) TEM image of EPCD stage cells depicting an aggregate of electron dense material inside the vacuole of a lace plant cell; note the membranous body in the vacuole. (D) Higher magnification TEM image of an EPCD stage cell, displaying aggregate of electron dense material in the vacuole, marked with a black arrow. Also note the organelle material was bound by a single membrane in the vacuole, marked with a red arrow. (E) Low magnification confocal micrograph displaying EPCD stage cells stained with FM1-43, sequestered to the phospholipid bilayer. Note the aggregates of membrane-bound organelles within each cell. (F) High magnification confocal micrograph depicting LPCD stage cells stained with FM1-43. Note the presence of the organelle aggregate and some vesiculation in the cells. (G) Confocal micrograph of EPCD stage cells stained with CMXRos, which stains mitochondria with intact ΔΨm (mitochondrial membrane potential). Note the aggregates are stained red, indicating the presence of mitochondria. (H) High magnification confocal micrograph of EPCD cells stained with CMXRos. Note that the aggregates are stained red, indicating the presence of viable mitochondria. (I) 3D volume view confocal image of LPCD stage cells co-stained with FM1-43 and propidium iodide (PI). Fifty successive z-stack images were compiled into a single 3D image using the deconvolution demonstration from Nikon Instruments. Note the presence of the aggregate within the vacuole of the cell, denoted with white arrow. Also note surrounding vesicles. (J) Secondary view of panel I, also depicting the aggregate, stained with FM1-43 within the vacuole of LPCD lace plant cells, denoted by white arrow. Scale bars: A = 100 μm, B = 10 μm, C = 50 μm, D = 25 μm E = 150 μm, F = 25 μm, G = 50 μm, H-J = 25 μm.