Determining The Optimal Values For The Flowj Plugin

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3. StackReg each 50 frame movie (Plugins > Registration > StackReg > Transformation: Rigid Body). This is done to correct for stage drift. 4. Stretch images by 2 on time axis (Plugins > Transform > TransformJ > Scale > x-factor for scaling: 1, y-factor for scaling: 1 z-factor for scaling: 2 > Interpolation scheme: quintic B-spline). Based on a separate analysis, this also improves the tracking the rapidly movement mitochondria. 5. Determine the optimal values for the FlowJ plugin. Crop out a small section of a movie that has the motion of interest and is representative for the data set. Directly measure the motion of interest using a kymograph or other trusted approach to establish “ground truth” flow. As an additional control, it may be helpful to construct a parallel movie where the motion of interest is absent. Set Σs = 2 and Σt =2, systematically vary τ and Σw and produce x y flow maps using the “Vary Parameters and Save” function in FlowJ. First examine how the output varies as a function of Σw. Pick the smallest value that generates a clear flow field for the moving object, then for that value of Σw examine the output as a function of τ. Find a value that fully suppresses flow associated with image noise, but does not suppress the flow fields associated with mitochondria. A straightforward way to do this is to examine the flow fields in a region of an image that both contains and lacks mitochondria. Take these values and conduct spot tests on different movies in the

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