#Quantification of cytokinesis in a one cell C. elegans embryo# version 1.0

References to the methods used in this application can be found at Positive Feedback Between Contractile Ring Myosin and Ring-Directed Cortical Flow Drives Cytokinesis, Renat Khaliullin, Rebecca Green, Linda Shi, Michael Berns, J. Sebastian Gomez-Cavazos, Arshad Desai, Karen Oegema ##Description## This application consists of two programs.

1. FindRing.py:

   • finds a contractile ring in a z-stack time series. This generates a csv file containing positions and the size of the ring at each time point.

2. divPlaneClass.py (requires the csv file generated by FindRing.py):

   • to validate ring position and size this program generates reconstructed division planes and draws contractile rings detected by FindRing;
   • quantifies the fluorescence intensities along the contractile ring perimeter within arcs of a specified angular range;
   • generates a kymograph of the division plane from the central z plane;
   • calculates cortical fluorescence from the central plane;

##Setup##

• Install python with all dependencies: opencv, scipy, numpy, pillow, matplotlib, pyqt4, lmfit
• FindRing.py parameters (change at the bottom of the file):
  • series = '01' prefix in the file name, i.e. seriesRing.tif (input images)
  • timePointStart = 1 slide number to start ring detection. Make sure that intensity in the equatorial band is higher than at the surrounding cortex
  • flip = False Check that anterior side is at the top, otherwise change to True
  • folder = '' folder name with the images
  • filename = folder+series+'Ring.tif' file name of the images with embryo z stacks
  • nSlices = 30 number of z slices in a stack
  • zPixelScale = 4. number of pixels in the camera per 1um (ie 1um = 250nm x 4 pixels)
  • tol = 8 number of pixels allowed for ring position misalignment
  • chromosomeMarker = False if chromosome marker is present, the algorithm will try to detect and remove them before processing
  • lateStage=False if the ring is half way closed or more at the first slide, change to True
  • kernelSize = 5 the size of the gaussian filter before processing (has to be odd)
  • embryoCenterDrift = 'linear' defines how the center of the embryo in z is calculated;
    • 'independent': calculate center for each timepoint independently (use when the depth is manually adjusted through out imaging);
    • 'linear': the center depth is calculated from a linear fit to detected individual centers (use when a steady drift is observed, like imaging on agarose pads);
    • 'median': calculates median position for the center (use only when there is no drift of the embryo center)
• divPlaneClass parameters (change at the bottom of the file): - fileName = '01Ring.csv' full path to the input file - dts = 30 time between time points - nZ = 30 number of Z planes - dZ = 4. pixels between z - pixelSize = 0.25 pixel size in microns - da = 20. averaging angle in degrees (angle step size) - drS = 0.1 averaging distance inside the ring - drL = 0.3 averaging distance outside the ring

##CONTACT## Renat Khaliullin

renatkh at gmail.com