Settings
- Auto Threshold: Select to use automatic grey-level thresholding of images for cell segmentation. A separate threshold will be calculated for each frame.
- Grey Level Threshold: Specify a constant threshold to apply to all frames if Auto Threshold is not selected.
- Spatial Resolution: Specify the spatial resolution of the input data in microns per pixel.
- Frames per Minute: Specify the temporal resolution of the input data in frames per minute.
- Minimum Trajectory Length: The minimum number of frames for which a cell must be tracked in order for it to be output in the results.
- Minimum Object Size: The minimum size an object must exceed to be considered a cell.
- Generate Visualisations: Select to generate movies showing cell curvature and velocity, along with all bleb detections superimposed on the original input.
- Generate Morphology Data: Select to output a time-varying, morphological analysis of the whole cell.
- Generate Signal Distribution: For each cell, output a map illustrating the intensity of fluorescence in the sig channel relative to distance to the cell centre over time. This may be used, for example, to demonstrate that a protein is more concentrated at the cell periphery, or that the localization of a protein changes over time. WARNING: this can be time consuming!
- Thresholding Method: The algorithm used to automatically calculate a grey-level threshold for segmentation.
- Smoothing Filter Radius: Set the width of the Gaussian filter used to de-noise individual cytosolic frames prior to segmentation.
- Erosion Iterations: Specify the number of erosion operations applied to a segmented cell mask in a given frame before it is used as the seed for segmentation in the next frame. Try increasing this value if cell segmentation is proving problematic, but bear in mind that this will increase execution time.
- Spatial Filter Radius: Set the width of the smoothing filter applied to velocity maps in the spatial dimension. If adjacent blebs are being detected as a single bleb, try reducing this value.
- Temporal Filter Radius: Set the width of the smoothing filter applied to velocity maps in the temporal dimension.
- Cortex Depth: The width of the region over which fluorescence values in the sig channel are quantified (the region bounded by the green lines in the image of the GUI above).
- Visualisation Line Thickness: Determines the width of lines in all output visualisations.
- Minimum/Maximum Display Velocity: The upper/lower limits applied to velocity values in colour velocity maps.
- Analyse Individual Protrusions: Select to enable analysis of individual protrusions.
- Detect Blebs or Filopodia: Self explanatory!
- Max/Min Filopodia Size: Anticipated size range for filopodia to be detected.
- Curvature Window: The minimum spacing, in pixels traced along the cell boundary, between adjacent bleb anchor points.
- Min Curvature Threshold: The maximum allowable angle that may be subtended at a point on the cell boundary for it to be considered a bleb anchor point.
- Cut-Off Time: The maximum length of time, in seconds, that any one protrusion will be tracked for.
- Display Plots: Graph and show results of bleb analysis.
- Use Signal Threshold: Select to apply a threshold value to signal analysis. Any pixel values below this threshold will be considered noise and excluded from the analysis.
- Signal Threshold Factor: Set the signal threshold factor. The signal threshold is calculated as (mean signal value in signal map) + (signal threshold factor) x (standard deviation of values in signal map)
- Signal Map Threshold: Set the minimum fraction of pixels within a cortical signal region that must be above the signal threshold for the bleb to be included in the analysis.