CellProfiler Pipeline: http://www.cellprofiler.org Version:1 SVNRevision:10415 LoadImages:[module_num:1|svn_version:\'10372\'|variable_revision_number:7|show_window:False|notes:\x5B\x5D] File type to be loaded:individual images File selection method:Text-Exact match Number of images in each group?:3 Type the text that the excluded images have in common:Do not use Analyze all subfolders within the selected folder?:No Input image file location:Default Input Folder\x7CNone Check image sets for missing or duplicate files?:Yes Group images by metadata?:No Exclude certain files?:No Specify metadata fields to group by: Image count:1 Text that these images have in common (case-sensitive):tif Position of this image in each group:1 Extract metadata from where?:None Regular expression that finds metadata in the file name:^(?P.*)_(?P\x5BA-P\x5D\x5B0-9\x5D{2})_s(?P\x5B0-9\x5D) Type the regular expression that finds metadata in the subfolder path:.*\x5B\\\\/\x5D(?P.*)\x5B\\\\/\x5D(?P.*)$ Channel count:1 Group the movie frames?:No Grouping method:Interleaved Number of channels per group:3 Name this loaded image:OrigDAPI Channel number:1 IdentifyPrimaryObjects:[module_num:2|svn_version:\'10372\'|variable_revision_number:7|show_window:True|notes:\x5B\'Using the background method ignores the non-specific cytoplasmic staining in favor of the nuclei, but some adjustment of the threshold correction factor was still needed.\'\x5D] Select the input image:OrigDAPI Name the primary objects to be identified:AllNuclei Typical diameter of objects, in pixel units (Min,Max):20,60 Discard objects outside the diameter range?:Yes Try to merge too small objects with nearby larger objects?:No Discard objects touching the border of the image?:Yes Select the thresholding method:Otsu Global Threshold correction factor:2.5 Lower and upper bounds on threshold:0.000000,1.000000 Approximate fraction of image covered by objects?:0.01 Method to distinguish clumped objects:Shape Method to draw dividing lines between clumped objects:Intensity Size of smoothing filter:10 Suppress local maxima that are closer than this minimum allowed distance:7 Speed up by using lower-resolution image to find local maxima?:Yes Name the outline image:PrimaryOutlines Fill holes in identified objects?:Yes Automatically calculate size of smoothing filter?:Yes Automatically calculate minimum allowed distance between local maxima?:Yes Manual threshold:0.0 Select binary image:None Retain outlines of the identified objects?:No Automatically calculate the threshold using the Otsu method?:Yes Enter Laplacian of Gaussian threshold:0.5 Two-class or three-class thresholding?:Three classes Minimize the weighted variance or the entropy?:Weighted variance Assign pixels in the middle intensity class to the foreground or the background?:Background Automatically calculate the size of objects for the Laplacian of Gaussian filter?:Yes Enter LoG filter diameter:5 Handling of objects if excessive number of objects identified:Continue Maximum number of objects:500 Select the measurement to threshold with:None MeasureObjectSizeShape:[module_num:3|svn_version:\'1\'|variable_revision_number:1|show_window:False|notes:\x5B\x5D] Select objects to measure:AllNuclei Calculate the Zernike features?:No MeasureObjectIntensity:[module_num:4|svn_version:\'10300\'|variable_revision_number:3|show_window:False|notes:\x5B\x5D] Hidden:1 Select an image to measure:OrigDAPI Select objects to measure:AllNuclei DisplayDataOnImage:[module_num:5|svn_version:\'10414\'|variable_revision_number:2|show_window:True|notes:\x5B\'Display the major axis length (ie, surrogate for the diameter for round objects) to see if the nuclei/micronuclei are distinguishable.\'\x5D] Display object or image measurements?:Object Select the input objects:AllNuclei Measurement to display:AreaShape_MajorAxisLength Select the image on which to display the measurements:OrigDAPI Text color:red Name the output image that has the measurements displayed:DisplayImage Font size (points):10 Number of decimals:2 Image elements to save:Image DisplayDataOnImage:[module_num:6|svn_version:\'10414\'|variable_revision_number:2|show_window:True|notes:\x5B\'Same for intensity measures.\'\x5D] Display object or image measurements?:Object Select the input objects:AllNuclei Measurement to display:Intensity_MeanIntensity_OrigDAPI Select the image on which to display the measurements:OrigDAPI Text color:red Name the output image that has the measurements displayed:DisplayImage Font size (points):10 Number of decimals:2 Image elements to save:Image ClassifyObjects:[module_num:7|svn_version:\'10300\'|variable_revision_number:2|show_window:True|notes:\x5B\'You can classify objects based on any of these measurements to see what the measurement distribution and identificaiton based on measurement looks like. While micronuclei may be smaller, it seems that the intensity might be the best metric.\'\x5D] Should each classification decision be based on a single measurement or on the combination of a pair of measurements?:Single measurement Hidden:2 Select the object to be classified:AllNuclei Select the measurement to classify by:AreaShape_MajorAxisLength Select bin spacing:Custom-defined bins Number of bins:2 Lower threshold:0 Use a bin for objects below the threshold?:Yes Upper threshold:1 Use a bin for objects above the threshold?:Yes Enter the custom thresholds separating the values between bins:35 Give each bin a name?:No Enter the bin names separated by commas:None Retain an image of the objects classified by their measurements, for use later in the pipeline (for example, in SaveImages)?:No Name the output image:ClassifiedNuclei Select the object to be classified:AllNuclei Select the measurement to classify by:Intensity_MeanIntensity_OrigDAPI Select bin spacing:Custom-defined bins Number of bins:3 Lower threshold:0 Use a bin for objects below the threshold?:Yes Upper threshold:1 Use a bin for objects above the threshold?:Yes Enter the custom thresholds separating the values between bins:0.5 Give each bin a name?:No Enter the bin names separated by commas:None Retain an image of the objects classified by their measurements, for use later in the pipeline (for example, in SaveImages)?:No Name the output image:ClassifiedNuclei Enter the object name:AllNuclei Select the first measurement:AreaShape_Area Method to select the cutoff:Custom Enter the cutoff value:0.5 Select the second measurement:None Method to select the cutoff:Mean Enter the cutoff value:0.5 Use custom names for the bins?:No Enter the low-low bin name:low_low Enter the low-high bin name:low_high Enter the high-low bin name:high_low Enter the high-high bin name:high_high Retain an image of the objects classified by their measurements, for use later in the pipeline (for example, in SaveImages)?:No Enter the image name:None FilterObjects:[module_num:8|svn_version:\'10300\'|variable_revision_number:5|show_window:True|notes:\x5B"You can filter the objects based on any of the prior measurements. Here, a threshold of 0.5 is used to define the micronuclei. For the 10415 version of CP, ClassifyObjects doesn\'t report the counts, so FilterObjects is being used as a workaround (see http\x3A//cellprofiler.org/forum/viewtopic.php?f=14&t=1143)."\x5D] Name the output objects:MicroNuclei Select the object to filter:AllNuclei Filter using classifier rules or measurements?:Measurements Select the filtering method:Limits Select the objects that contain the filtered objects:None Retain outlines of the identified objects?:No Name the outline image:FilteredObjects Rules file location:Default Input Folder\x7CNone Rules file name:rules.txt Measurement count:1 Additional object count:0 Select the measurement to filter by:Intensity_MeanIntensity_OrigDAPI Filter using a minimum measurement value?:Yes Minimum value:0.5 Filter using a maximum measurement value?:No Maximum value:1 FilterObjects:[module_num:9|svn_version:\'10300\'|variable_revision_number:5|show_window:True|notes:\x5B\'A similar procedure for the regular nuclei.\'\x5D] Name the output objects:MicroNuclei Select the object to filter:AllNuclei Filter using classifier rules or measurements?:Measurements Select the filtering method:Limits Select the objects that contain the filtered objects:None Retain outlines of the identified objects?:No Name the outline image:FilteredObjects Rules file location:Default Input Folder\x7CNone Rules file name:rules.txt Measurement count:1 Additional object count:0 Select the measurement to filter by:Intensity_MeanIntensity_OrigDAPI Filter using a minimum measurement value?:No Minimum value:0.5 Filter using a maximum measurement value?:Yes Maximum value:0.5 ExportToSpreadsheet:[module_num:10|svn_version:\'10251\'|variable_revision_number:7|show_window:True|notes:\x5B\x5D] Select or enter the column delimiter:Comma (",") Prepend the output file name to the data file names?:Yes Add image metadata columns to your object data file?:No Limit output to a size that is allowed in Excel?:No Select the columns of measurements to export?:No Calculate the per-image mean values for object measurements?:No Calculate the per-image median values for object measurements?:No Calculate the per-image standard deviation values for object measurements?:No Output file location:Default Output Folder\x7CNone Create a GenePattern GCT file?:No Select source of sample row name:Metadata Select the image to use as the identifier:None Select the metadata to use as the identifier:None Export all measurements?:Yes Press button to select measurements to export: Data to export:Do not use Combine these object measurements with those of the previous object?:No File name:DATA.csv Use the object name for the file name?:Yes