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manual_spots.md

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Manual spot calling

  • To begin, copy three timepoints (for triplicate measurements) from the movie into example_folder/7_manonetwo/.
    • If the VidCod3 commands were used, then multi_T00_MIP.tiff, multi_T04_MIP.tiff, and multi_T09_MIP.tiff, can be transferred.
    • Prepare a z-MIP of each of the timepoints; stack0.tif, stack4.tif, and stack9.tif.
  • Open the *_viewpost.png figure and the first stack timepoint, find the location of each cropped cell and determine by eye whether one or two spots are apparent, recording the results in a spreadsheet.
  • Once the three timepoints have been scored, run the spot obs rep script to combine the manual scores with the cell observations.
  • The spotobsrep_comb script can be used to combine the manual scores from multiple experiments.
  • The obsspotreps_conf_comb script can be used to combine the pooled manual scores with the confirmed distances, for a comparison of manual vs computational spot calling.
    • The confdist_comb script can be used to pool the distances from the manually confirmed computational spot calling.