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:tif,tiff,flex movies File selection method:Text-Regular expressions Number of images in each group?:2 Type the text that the excluded images have in common:Do not use Analyze all subfolders within the selected folder?:Yes Input image file location:Default Input Folder\x7C. Check image sets for missing or duplicate files?: 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):.flex Position of this image in each group:1 Extract metadata from where?:Both Regular expression that finds metadata in the file name:^(?P.*).flex Type the regular expression that finds metadata in the subfolder path:(?P.*) Channel count:2 Group the movie frames?:Yes Grouping method:Interleaved Number of channels per group:2 Name this loaded image:GFP Channel number:1 Name this loaded image:RFP Channel number:2 CorrectIlluminationCalculate:[module_num:2|svn_version:\'10300\'|variable_revision_number:2|show_window:False|notes:\x5B\x5D] Select the input image:RFP Name the output image:IllumRFP Select how the illumination function is calculated:Background Dilate objects in the final averaged image?:No Dilation radius:1 Block size:60 Rescale the illumination function?:No Calculate function for each image individually, or based on all images?:All\x3A First cycle Smoothing method:No smoothing Method to calculate smoothing filter size:Automatic Approximate object size:10 Smoothing filter size:10 Retain the averaged image for use later in the pipeline (for example, in SaveImages)?:No Name the averaged image:IllumBlueAvg Retain the dilated image for use later in the pipeline (for example, in SaveImages)?:No Name the dilated image:IllumBlueDilated Automatically calculate spline parameters?:Yes Background mode:auto Number of spline points:5 Background threshold:2 Image resampling factor:2 Maximum number of iterations:40 Residual value for convergence:0.001 CorrectIlluminationCalculate:[module_num:3|svn_version:\'10300\'|variable_revision_number:2|show_window:False|notes:\x5B\x5D] Select the input image:GFP Name the output image:IllumGFP Select how the illumination function is calculated:Background Dilate objects in the final averaged image?:No Dilation radius:1 Block size:60 Rescale the illumination function?:No Calculate function for each image individually, or based on all images?:Each Smoothing method:No smoothing Method to calculate smoothing filter size:Automatic Approximate object size:10 Smoothing filter size:10 Retain the averaged image for use later in the pipeline (for example, in SaveImages)?:No Name the averaged image:IllumBlueAvg Retain the dilated image for use later in the pipeline (for example, in SaveImages)?:No Name the dilated image:IllumBlueDilated Automatically calculate spline parameters?:Yes Background mode:auto Number of spline points:5 Background threshold:2 Image resampling factor:2 Maximum number of iterations:40 Residual value for convergence:0.001 CorrectIlluminationApply:[module_num:4|svn_version:\'10300\'|variable_revision_number:3|show_window:False|notes:\x5B\x5D] Select the input image:RFP Name the output image:CorrRFP Select the illumination function:IllumRFP Select how the illumination function is applied:Subtract Select the input image:GFP Name the output image:CorrGFP Select the illumination function:IllumGFP Select how the illumination function is applied:Subtract MeasureImageQuality:[module_num:5|svn_version:\'10368\'|variable_revision_number:3|show_window:False|notes:\x5B\x5D] Select an image to measure:RFP Check for blur?:Yes Window size for blur measurements:20 Check for saturation?:Yes Calculate threshold?:Yes Select a thresholding method:Otsu Global Typical fraction of the image covered by objects:0.1 Calculate quartiles and sum of radial power spectrum?:Yes 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 Select an image to measure:GFP Check for blur?:Yes Window size for blur measurements:20 Check for saturation?:Yes Calculate threshold?:Yes Select a thresholding method:Otsu Global Typical fraction of the image covered by objects:0.1 Calculate quartiles and sum of radial power spectrum?:Yes 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 RescaleIntensity:[module_num:6|svn_version:\'Unknown\'|variable_revision_number:2|show_window:False|notes:\x5B\x5D] Select the input image:CorrGFP Name the output image:RescaledGFP 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:7|svn_version:\'Unknown\'|variable_revision_number:2|show_window:False|notes:\x5B\x5D] Select the input image:CorrRFP Name the output image:RescaledRFP 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:8|svn_version:\'10372\'|variable_revision_number:7|show_window:True|notes:\x5B\'\'\x5D] Select the input image:RescaledRFP Name the primary objects to be identified:Nuclei Typical diameter of objects, in pixel units (Min,Max):7,40 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:1.3 Lower and upper bounds on threshold:0.006,1.0 Approximate fraction of image covered by objects?:0.01 Method to distinguish clumped objects:Intensity Method to draw dividing lines between clumped objects:Intensity Size of smoothing filter:20 Suppress local maxima that are closer than this minimum allowed distance:20 Speed up by using lower-resolution image to find local maxima?:No Name the outline image:NucleiOutlines Fill holes in identified objects?:Yes Automatically calculate size of smoothing filter?:No Automatically calculate minimum allowed distance between local maxima?:No 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 IdentifyPrimaryObjects:[module_num:9|svn_version:\'10372\'|variable_revision_number:7|show_window:True|notes:\x5B\x5D] Select the input image:RescaledRFP Name the primary objects to be identified:Cell Typical diameter of objects, in pixel units (Min,Max):40,95 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:1.3 Lower and upper bounds on threshold:0.006,1.0 Approximate fraction of image covered by objects?:0.01 Method to distinguish clumped objects:Intensity 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:40 Speed up by using lower-resolution image to find local maxima?:No Name the outline image:CellOutlines Fill holes in identified objects?:Yes Automatically calculate size of smoothing filter?:No Automatically calculate minimum allowed distance between local maxima?:No 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?: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 RelateObjects:[module_num:10|svn_version:\'10300\'|variable_revision_number:2|show_window:True|notes:\x5B\x5D] Select the input child objects:Nuclei Select the input parent objects:Cell Calculate distances?:None Calculate per-parent means for all child measurements?:No Calculate distances to other parents?:No Parent name:None ExpandOrShrinkObjects:[module_num:11|svn_version:\'10300\'|variable_revision_number:1|show_window:False|notes:\x5B\x5D] Select the input objects:Nuclei Name the output objects:NucleiExpanded Select the operation:Expand objects by a specified number of pixels Number of pixels by which to expand or shrink:15 Fill holes in objects so that all objects shrink to a single point?:No Retain the outlines of the identified objects for use later in the pipeline (for example, in SaveImages)?:Yes Name the outline image:NucleiExpandedOutlines Crop:[module_num:12|svn_version:\'10300\'|variable_revision_number:2|show_window:False|notes:\x5B\x5D] Select the input image:GFP Name the output image:GFP_CropByNucleus 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:NucleiExpanded IdentifyPrimaryObjects:[module_num:13|svn_version:\'10372\'|variable_revision_number:7|show_window:False|notes:\x5B\x5D] Select the input image:GFP_CropByNucleus Name the primary objects to be identified:Nucleoli Typical diameter of objects, in pixel units (Min,Max):5,30 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:1.3 Lower and upper bounds on threshold:0.006,1.0 Approximate fraction of image covered by objects?:0.01 Method to distinguish clumped objects:Intensity 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:5 Speed up by using lower-resolution image to find local maxima?:No Name the outline image:NucleoliOutlines Fill holes in identified objects?:Yes Automatically calculate size of smoothing filter?:No Automatically calculate minimum allowed distance between local maxima?:No Manual threshold:0.0 Select binary image:Otsu Global Retain outlines of the identified objects?:Yes Automatically calculate the threshold using the Otsu method?:Yes Enter Laplacian of Gaussian threshold:.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 RelateObjects:[module_num:14|svn_version:\'10300\'|variable_revision_number:2|show_window:True|notes:\x5B\x5D] Select the input child objects:Nuclei Select the input parent objects:Nucleoli Calculate distances?:None Calculate per-parent means for all child measurements?:No Calculate distances to other parents?:No Parent name:None MeasureObjectSizeShape:[module_num:15|svn_version:\'1\'|variable_revision_number:1|show_window:False|notes:\x5B\x5D] Select objects to measure:Nuclei Select objects to measure:Cell Select objects to measure:Nucleoli Calculate the Zernike features?:Yes MeasureObjectIntensity:[module_num:16|svn_version:\'10300\'|variable_revision_number:3|show_window:False|notes:\x5B\x5D] Hidden:2 Select an image to measure:GFP Select an image to measure:RFP Select objects to measure:Nuclei Select objects to measure:Cell Select objects to measure:Nucleoli OverlayOutlines:[module_num:17|svn_version:\'10300\'|variable_revision_number:2|show_window:True|notes:\x5B\x5D] Display outlines on a blank image?:No Select image on which to display outlines:RescaledRFP Name the output image:Cell&NucleiOutlinesOnRescaledRFP Select outline display mode:Color Select method to determine brightness of outlines:Max of image Width of outlines:1 Select outlines to display:NucleiOutlines Select outline color:Green Select outlines to display:CellOutlines Select outline color:White OverlayOutlines:[module_num:18|svn_version:\'10300\'|variable_revision_number:2|show_window:True|notes:\x5B\x5D] Display outlines on a blank image?:No Select image on which to display outlines:RescaledGFP Name the output image:NucleoliOutlinesOnRescaledGFP Select outline display mode:Color Select method to determine brightness of outlines:Max of image Width of outlines:1 Select outlines to display:NucleoliOutlines Select outline color:Red SaveImages:[module_num:19|svn_version:\'10411\'|variable_revision_number:6|show_window:False|notes:\x5B\x5D] Select the type of image to save:Image Select the image to save:Cell&NucleiOutlinesOnRescaledRFP Select the module display window to save:None Select method for constructing file names:Sequential numbers Select image name for file prefix:None Enter file prefix:\\g_CellsNuclei_ Do you want to add a suffix to the image file name?:No Text to append to the image name: Select file format to use:tiff 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 Select colormap:gray Update file names within CellProfiler?:Yes Create subfolders in the output folder?:No SaveImages:[module_num:20|svn_version:\'10411\'|variable_revision_number:6|show_window:False|notes:\x5B\x5D] Select the type of image to save:Image Select the image to save:NucleoliOutlinesOnRescaledGFP Select the module display window to save:None Select method for constructing file names:Sequential numbers Select image name for file prefix:RFP Enter file prefix:\\g_Nucleoli_ Do you want to add a suffix to the image file name?:Yes Text to append to the image name:_GreenFiltNucRings Select file format to use:tiff 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 Select colormap:gray Update file names within CellProfiler?:Yes Create subfolders in the output folder?:No ExportToSpreadsheet:[module_num:21|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