Figure S7.
Image analysis pipeline for the determination of lamella size. Fluorescently labeled hemocytes were cultured ex vivo and registered for 30 min at 1-min intervals. (A) Representative image of a hemocyte expressing LifeAct-Ruby and sqh-GFP from a 30-min video (left), in which the signals of all frames were averaged (middle). On the average signal image, the center of the cell was defined (right). The white dashed line represents the adjusted circumference around the center of the cell. (B) The Radial Reslice plugin was run, obtaining the radial signal of both channels (360° of each), represented by the white round arrow. (C) All 360 linear signals were averaged, and a plot profile was obtained for each channel. (D) Obtained data for each plot profile were normalized: maximum signal intensity (Imax) was set to 1, and minimum (Imin) to 0. Each positional coordinate (x) has a defined intensity, where x0 corresponds to the center of the cell. The positional coordinate in the radial axis (xr) was set to 100% of the radius, whereby the signal intensity of the channel corresponding to actin cytoskeleton equaled 0.02 (Intensity radius, Ir), which is slightly above the noise. (E) Normalized values were plotted to demonstrate graphical distribution of signals, and positional identity was converted into percentage of the radius, where xr=100%. A.U., arbitrary units. The dashed line marks the 50% of the maximal signal intensity. These values were used to define the position of the boundaries of each signal. The difference between both boundaries was used to define lamella size (black bracket). (F) To quantitatively compare signal distributions among conditions, the difference (percentage with respect to the radius) of both signals at I = 0.5 (in E) was used as lamella extension beyond Sqh territory. Cg, collagen driver; Act, actin.

Image analysis pipeline for the determination of lamella size. Fluorescently labeled hemocytes were cultured ex vivo and registered for 30 min at 1-min intervals. (A) Representative image of a hemocyte expressing LifeAct-Ruby and sqh-GFP from a 30-min video (left), in which the signals of all frames were averaged (middle). On the average signal image, the center of the cell was defined (right). The white dashed line represents the adjusted circumference around the center of the cell. (B) The Radial Reslice plugin was run, obtaining the radial signal of both channels (360° of each), represented by the white round arrow. (C) All 360 linear signals were averaged, and a plot profile was obtained for each channel. (D) Obtained data for each plot profile were normalized: maximum signal intensity (Imax) was set to 1, and minimum (Imin) to 0. Each positional coordinate (x) has a defined intensity, where x0 corresponds to the center of the cell. The positional coordinate in the radial axis (xr) was set to 100% of the radius, whereby the signal intensity of the channel corresponding to actin cytoskeleton equaled 0.02 (Intensity radius, Ir), which is slightly above the noise. (E) Normalized values were plotted to demonstrate graphical distribution of signals, and positional identity was converted into percentage of the radius, where xr=100%. A.U., arbitrary units. The dashed line marks the 50% of the maximal signal intensity. These values were used to define the position of the boundaries of each signal. The difference between both boundaries was used to define lamella size (black bracket). (F) To quantitatively compare signal distributions among conditions, the difference (percentage with respect to the radius) of both signals at I = 0.5 (in E) was used as lamella extension beyond Sqh territory. Cg, collagen driver; Act, actin.

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