CellProfiler Pipeline: http://www.cellprofiler.org Version:1 SVNRevision:11710 LoadImages:[module_num:1|svn_version:\'11587\'|variable_revision_number:11|show_window:False|notes:\x5B\'This CellProfiler Pipeline expects input TIFF files with fluorescent staining of DNA, the Golgi, and one that allows approximate identification of cell boundaries. \', \'The Regular Expression in this module may need to be edited depending on the naming scheme of your files.\', \'Pipeline identifies nuclei, cells, and Golgi, groups Golgi for each cell into a single object, and calculates the area of the bounding ellipse. It also outputs images with outlines of the identified Golgi and nuclei to allow checking.\', \'Seth Field 03-31-11\'\x5D] File type to be loaded:individual images File selection method:Text-Exact match Number of images in each group?:1 Type the text that the excluded images have in common:Do not use Analyze all subfolders within the selected folder?:None Input image file location:Default Input Folder\x7CNone Check image sets for missing or duplicate files?:No Group images by metadata?:No Exclude certain files?:No Specify metadata fields to group by:Drug Select subfolders to analyze: Image count:4 Text that these images have in common (case-sensitive):RFP Position of this image in each group:1 Extract metadata from where?:File name Regular expression that finds metadata in the file name:(?P.*)_(?P.*)_(?P\x5B0-9\x5D{1,2}).*\x5Btif\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:2 Load the input as images or objects?:Images Name this loaded image:Golgi Name this loaded object:Nuclei Retain outlines of loaded objects?:No Name the outline image:NucleiOutlines Channel number:1 Rescale intensities?:Yes Text that these images have in common (case-sensitive):DAP Position of this image in each group:2 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 Load the input as images or objects?:Images Name this loaded image:DNA Name this loaded object:Nuclei Retain outlines of loaded objects?:No Name the outline image:LoadedImageOutlines Channel number:1 Rescale intensities?:Yes Text that these images have in common (case-sensitive):Cy5 Position of this image in each group:3 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 Load the input as images or objects?:Images Name this loaded image:G3 Name this loaded object:Nuclei Retain outlines of loaded objects?:No Name the outline image:LoadedImageOutlines Channel number:1 Rescale intensities?:Yes Text that these images have in common (case-sensitive):GFP Position of this image in each group:4 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 Load the input as images or objects?:Images Name this loaded image:GFP Name this loaded object:Nuclei Retain outlines of loaded objects?:No Name the outline image:LoadedImageOutlines Channel number:1 Rescale intensities?:Yes RescaleIntensity:[module_num:2|svn_version:\'6746\'|variable_revision_number:2|show_window:False|notes:\x5B\x5D] Select the input image:Golgi Name the output image:RescaledGolgi Select rescaling method:Stretch each image to use the full intensity range How do you want to calculate the minimum intensity?:Custom How do you want to calculate the maximum intensity?:Custom Enter the lower limit for the intensity range for the input image:0 Enter the upper limit for the intensity range for the input image:1 Enter the intensity range for the input image:0.000000,1.000000 Enter the desired intensity range for the final, rescaled image:0.000000,1.000000 Select method for rescaling pixels below the lower limit:Mask pixels Enter custom value for pixels below lower limit:0 Select method for rescaling pixels above the upper limit:Mask pixels Enter custom value for pixels below upper limit:0 Select image to match in maximum intensity:None Enter the divisor:1 Select the measurement to use as a divisor:None RescaleIntensity:[module_num:3|svn_version:\'6746\'|variable_revision_number:2|show_window:False|notes:\x5B\x5D] Select the input image:DNA Name the output image:RescaledDNA Select rescaling method:Stretch each image to use the full intensity range How do you want to calculate the minimum intensity?:Custom How do you want to calculate the maximum intensity?:Custom Enter the lower limit for the intensity range for the input image:0 Enter the upper limit for the intensity range for the input image:1 Enter the intensity range for the input image:0.000000,1.000000 Enter the desired intensity range for the final, rescaled image:0.000000,1.000000 Select method for rescaling pixels below the lower limit:Mask pixels Enter custom value for pixels below lower limit:0 Select method for rescaling pixels above the upper limit:Mask pixels Enter custom value for pixels below upper limit:0 Select image to match in maximum intensity:None Enter the divisor:1 Select the measurement to use as a divisor:None RescaleIntensity:[module_num:4|svn_version:\'6746\'|variable_revision_number:2|show_window:False|notes:\x5B\x5D] Select the input image:G3 Name the output image:RescaledG3 Select rescaling method:Stretch each image to use the full intensity range How do you want to calculate the minimum intensity?:Custom How do you want to calculate the maximum intensity?:Custom Enter the lower limit for the intensity range for the input image:0 Enter the upper limit for the intensity range for the input image:1 Enter the intensity range for the input image:0.000000,1.000000 Enter the desired intensity range for the final, rescaled image:0.000000,1.000000 Select method for rescaling pixels below the lower limit:Mask pixels Enter custom value for pixels below lower limit:0 Select method for rescaling pixels above the upper limit:Mask pixels Enter custom value for pixels below upper limit:0 Select image to match in maximum intensity:None Enter the divisor:1 Select the measurement to use as a divisor:None IdentifyPrimaryObjects:[module_num:5|svn_version:\'10826\'|variable_revision_number:8|show_window:True|notes:\x5B\'threshold correction 1.3 more stringent (>1)\'\x5D] Select the input image:RescaledDNA Name the primary objects to be identified:Nuclei Typical diameter of objects, in pixel units (Min,Max):40,180 Discard objects outside the diameter range?:Yes Try to merge too small objects with nearby larger objects?:Yes Discard objects touching the border of the image?:Yes Select the thresholding method:Otsu Global Threshold correction factor:1.3 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:Shape 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:NuclearOutlines 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?:Two classes Minimize the weighted variance or the entropy?:Weighted variance Assign pixels in the middle intensity class to the foreground or the background?:Foreground 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 IdentifySecondaryObjects:[module_num:6|svn_version:\'10826\'|variable_revision_number:7|show_window:True|notes:\x5B\x5D] Select the input objects:Nuclei Name the objects to be identified:Cell Select the method to identify the secondary objects:Propagation Select the input image:G3 Select the thresholding method:Otsu Global Threshold correction factor:0.9 Lower and upper bounds on threshold:0.000000,1.000000 Approximate fraction of image covered by objects?:0.01 Number of pixels by which to expand the primary objects:10 Regularization factor:0.08 Name the outline image:CellOutlines Manual threshold:0.0 Select binary image:None Retain outlines of the identified secondary objects?:Yes 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?:Foreground Discard secondary objects that touch the edge of the image?:Yes Discard the associated primary objects?:Yes Name the new primary objects:FilteredNuclei Retain outlines of the new primary objects?:Yes Name the new primary object outlines:FilteredNucleiOutlines Select the measurement to threshold with:None Fill holes in identified objects?:Yes ExpandOrShrinkObjects:[module_num:7|svn_version:\'10830\'|variable_revision_number:1|show_window:False|notes:\x5B\x5D] Select the input objects:Cell Name the output objects:ShrunkenCells1 Select the operation:Shrink objects by a specified number of pixels Number of pixels by which to expand or shrink:1 Fill holes in objects so that all objects shrink to a single point?:Yes Retain the outlines of the identified objects for use later in the pipeline (for example, in SaveImages)?:No Name the outline image:ShrunkenNucleiOutlines Crop:[module_num:8|svn_version:\'10804\'|variable_revision_number:2|show_window:False|notes:\x5B\x5D] Select the input image:RescaledGolgi Name the output image:CropGolgitoCells Select the cropping shape:Objects Select the cropping method:Coordinates Apply which cycle\'s cropping pattern?:Every Left and right rectangle positions:0,end Top and bottom rectangle positions:0,end Coordinates of ellipse center:500,500 Ellipse radius, X direction:400 Ellipse radius, Y direction:200 Use Plate Fix?:No Remove empty rows and columns?:No Select the masking image:None Select the image with a cropping mask:None Select the objects:ShrunkenCells1 IdentifyPrimaryObjects:[module_num:9|svn_version:\'10826\'|variable_revision_number:8|show_window:True|notes:\x5B\x5D] Select the input image:CropGolgitoCells Name the primary objects to be identified:OrigGolgi Typical diameter of objects, in pixel units (Min,Max):2,40 Discard objects outside the diameter range?:No Try to merge too small objects with nearby larger objects?:Yes Discard objects touching the border of the image?:No Select the thresholding method:RobustBackground PerObject Threshold correction factor:1.6 Lower and upper bounds on threshold:0.000000,1.000000 Approximate fraction of image covered by objects?:0.01 Method to distinguish clumped objects:Intensity Method to draw dividing lines between clumped objects:Propagate 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:OrigGolgiOutline Fill holes in identified objects?:No 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?:Yes 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 ConvertObjectsToImage:[module_num:10|svn_version:\'10807\'|variable_revision_number:1|show_window:False|notes:\x5B\x5D] Select the input objects:OrigGolgi Name the output image:GolgiBinaryImage Select the color type:Binary (black & white) Select the colormap:Default MeasureObjectRadialDistribution:[module_num:11|svn_version:\'10798\'|variable_revision_number:1|show_window:False|notes:\x5B\x5D] Hidden:1 Hidden:1 Hidden:2 Select an image to measure:GolgiBinaryImage Select objects to measure:Cell Object to use as center?:Other objects Select objects to use as centers:FilteredNuclei Number of bins:4 Number of bins:2 MeasureObjectIntensity:[module_num:12|svn_version:\'10816\'|variable_revision_number:3|show_window:False|notes:\x5B\x5D] Hidden:2 Select an image to measure:G3 Select an image to measure:GFP Select objects to measure:FilteredNuclei Select objects to measure:Cell Select objects to measure:OrigGolgi RelateObjects:[module_num:13|svn_version:\'10300\'|variable_revision_number:2|show_window:True|notes:\x5B\x5D] Select the input child objects:OrigGolgi Select the input parent objects:Cell Calculate distances?:Centroid Calculate per-parent means for all child measurements?:Yes Calculate distances to other parents?:Yes Parent name:FilteredNuclei GrayToColor:[module_num:14|svn_version:\'10341\'|variable_revision_number:2|show_window:False|notes:\x5B\x5D] Select a color scheme:RGB Select the input image to be colored red:RescaledGolgi Select the input image to be colored green:RescaledG3 Select the input image to be colored blue:RescaledDNA Name the output image:ColorImage Relative weight for the red image:2 Relative weight for the green image:1 Relative weight for the blue image:1 Select the input image to be colored cyan:Leave this black Select the input image to be colored magenta:Leave this black Select the input image to be colored yellow:Leave this black Select the input image that determines brightness:Leave this black Relative weight for the cyan image:1 Relative weight for the magenta image:1 Relative weight for the yellow image:1 Relative weight for the brightness image:1 Select the input image to add to the stacked image:None OverlayOutlines:[module_num:15|svn_version:\'10672\'|variable_revision_number:2|show_window:True|notes:\x5B\x5D] Display outlines on a blank image?:No Select image on which to display outlines:ColorImage Name the output image:OrigOverlay Select outline display mode:Color Select method to determine brightness of outlines:Max of image Width of outlines:1 Select outlines to display:OrigGolgiOutline Select outline color:Yellow Select outlines to display:FilteredNucleiOutlines Select outline color:Red Select outlines to display:CellOutlines Select outline color:White SaveImages:[module_num:16|svn_version:\'10822\'|variable_revision_number:7|show_window:False|notes:\x5B\x5D] Select the type of image to save:Image Select the image to save:OrigOverlay Select the objects to save:None Select the module display window to save:None Select method for constructing file names:From image filename Select image name for file prefix:Golgi Enter single file name:OrigBlue Do you want to add a suffix to the image file name?:No Text to append to the image name:jpg Select file format to use:jpg Output file location:Default Output Folder\x7CNone Image bit depth:8 Overwrite existing files without warning?:No Select how often to save:Every cycle Rescale the images? :No Save as grayscale or color image?:Grayscale Select colormap:gray Store file and path information to the saved image?:No Create subfolders in the output folder?:No ExportToSpreadsheet:[module_num:17|svn_version:\'10880\'|variable_revision_number:7|show_window:False|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?:Yes 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, using default file names?:Yes Press button to select measurements to export:UnifiedGolgi\x7CParent_Cell,UnifiedGolgi\x7CParent_Golgi,UnifiedGolgi\x7CNumber_Object_Number,UnifiedGolgi\x7CLocation_Center_X,UnifiedGolgi\x7CLocation_Center_Y,UnifiedGolgi\x7CAreaShape_Perimeter,UnifiedGolgi\x7CAreaShape_Orientation,UnifiedGolgi\x7CAreaShape_Area,UnifiedGolgi\x7CAreaShape_Solidity,UnifiedGolgi\x7CAreaShape_EulerNumber,UnifiedGolgi\x7CAreaShape_Compactness,UnifiedGolgi\x7CAreaShape_Extent,UnifiedGolgi\x7CAreaShape_Eccentricity,UnifiedGolgi\x7CAreaShape_FormFactor,UnifiedGolgi\x7CAreaShape_MinorAxisLength,UnifiedGolgi\x7CAreaShape_MajorAxisLength,UnifiedGolgi\x7CAreaShape_Center_X,UnifiedGolgi\x7CAreaShape_Center_Y,UnifiedGolgi\x7CMath_GolgiArea/CellArea 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