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Fig. 2. FLOID analysis of migrating keratocytes. TIRF images (a and b) show the location in which dye has spread from the dorsal to ventral cell surface; either at the back of the cell only (single peak, a and c) or both at the front and back (double peak, b and d). Undulations in the ventral cell surface have been shown previously using interference reflection microscopy (Anderson and Cross, 2000 ), and lead to variations in dye excitation according to the distance of the cell surface from the coverslip. White bars (a and b) show the position used for fluorescence intensity scans in c and d, respectively. Arrowhead (b) indicates a region of reduced protrusion at the leading edge, where dye has spread from the dorsal to ventral cell membrane. Comparison of sequentially acquired epi-fluorescent (f) and TIRF (g) images from the same cell, and the overlay (h). Note that TIRF proceeds epi-fluorescent illumination by 750 milliseconds. The white bar (h) shows the position used for fluorescence intensity scans in i. (i) The epi-fluorescence intensity scan (green line) indicates that dye has spread from the back of the cell toward the front, and begun to accumulate at the leading edge (arrowhead). The TIRF intensity scan (red line) also shows that dye has spread from the back of the cell toward the front, but not yet reached the leading edge. Note that accumulated dye in the dorsal membrane has not spread around the leading edge to the ventral surface. Inset shows profile for the whole cell. 86% of cells analyzed (n=138) were found to show the single-peak intensity profile (e). Bars, 10 µm.
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