Figure 3.

The approach taken to systematically evaluate the different algorithms of analyzing a focus. (A) Illustration of our approach of increasing the amount of signal in a focus, while simultaneously maintaining a constant total fluorescence value in the cell. For each increment we simulate a diffraction limited focus with more fluorescence content (red line), while also subtracting that amount of fluorescence from the cellular signal (blue line). We schematically illustrate the effect on the focus and cytoplasmic signal. (B) A sample temporal montage of a complete simulation from no signal in a focus (far left side of A) until the signal consists only of a focus (far right side of A). (C) Three sample simulations of different focus fluorescence content together with their corresponding line profile plots. Scale bars: 1 μm. Note that the cellular background is measured and not simulated. To see this figure in color, go online.