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Fig. 1. The computer-assisted DIAS 4.0 software program provides 3D reconstructions of living cells that include color-coded representations of the general cell body, nucleus, pseudopodia and filopodia. (A) The in-focus edges of cell compartments and filopodia are outlined in each of 60 optical sections beginning at the substratum and ending 20 µm above the substratum. Cells are imaged by differential interference contrast (DIC) microscopy. The set of 60 optical sections is collected in a 2-second period and the procedure repeated at intervals of 4 seconds. The outline of the in-focus cell body containing particulate cytoplasm is color-coded green, outlines of the in-focus pseudopodial protrusions containing non-particulate cytoplasm are color-coded blue, the outline of the nucleus is color-coded fuchsia, filopodial segments emanating from the general cell body and pseudopodia are color-coded red and filopodia emanating from the uropod, referred to here as tail fibers, are color-coded yellow. Only the first 12 outlined optical sections, beginning at the substratum (0 µm) are presented here, of a cell turning towards an aggregation stream in response to a gradient of chemoattractant late in the Dictyostelium aggregation process. (B) Reconstruction of the outlined cell viewed at 10°, 30° and 55° from three rotational vantage points. The arrows in each rotation illustrated at the top of the panels reflect the posterior-anterior axis deduced from the prior history of cellular translocation. The capacity to view the cell from different angles provides a more complete picture of filopod location and interaction with the substratum. The cell body is a transparent mesh and color-coded green, the nucleus is color-coded fuchsia, the pseudopodial regions are color-coded gray, the filopodia are color-coded red and the tail fibers emanating from the uropod are color-coded yellow. Bar, 5 µm.
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