CellProfiler Pipeline: http://www.cellprofiler.org Version:1 SVNRevision:10997 LoadImages:[module_num:1|svn_version:\'10951\'|variable_revision_number:11|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?:All Input image file location:Default Input Folder\x7C. Check image sets for missing or duplicate files?:No Group images by metadata?:No Exclude certain files?:No Specify metadata fields to group by: Select subfolders to analyze: Image count:2 Text that these images have in common (case-sensitive):originalIHC.jpg Position of this image in each group:blue Extract metadata from where?:None Regular expression that finds metadata in the file name:None Type the regular expression that finds metadata in the subfolder path:None 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:OrigIHC 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):DAB.jpg 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:maskDAB Name this loaded object:Nuclei Retain outlines of loaded objects?:No Name the outline image:LoadedImageOutlines Channel number:1 Rescale intensities?:No ColorToGray:[module_num:2|svn_version:\'10318\'|variable_revision_number:2|show_window:False|notes:\x5B\x5D] Select the input image:OrigIHC Conversion method:Combine Image type\x3A:RGB Name the output image:OrigGray Relative weight of the red channel:1 Relative weight of the green channel:1 Relative weight of the blue channel:1 Convert red to gray?:Yes Name the output image:OrigRed Convert green to gray?:Yes Name the output image:OrigGreen Convert blue to gray?:Yes Name the output image:OrigBlue Channel count:1 Channel number\x3A:Red\x3A 1 Relative weight of the channel:1 Image name\x3A:Channel1 UnmixColors:[module_num:3|svn_version:\'10829\'|variable_revision_number:2|show_window:False|notes:\x5B\x5D] Stain count:2 Color image:OrigIHC Image name:dabIM Stain:Custom Red absorbance:0.2681475 Green absorbance:0.57031 Blue absorbance:0.7764271 Image name:hemIM Stain:Custom Red absorbance:0.574499003 Green absorbance:0.68100744 Blue absorbance:0.45344827 Resize:[module_num:4|svn_version:\'10627\'|variable_revision_number:4|show_window:False|notes:\x5B\x5D] Select the input image:hemIM Name the output image:ResizedH Select resizing method:Resize by a fraction or multiple of the original size Resizing factor:0.50 Width of the final image, in pixels:100 Height of the final image, in pixels:100 Interpolation method:Nearest Neighbor How do you want to specify the dimensions?:Manual Select the image with the desired dimensions:None Additional image count:1 Select the additional image?:dabIM Name the output image:DAB_resize Resize:[module_num:5|svn_version:\'10627\'|variable_revision_number:4|show_window:False|notes:\x5B\x5D] Select the input image:OrigGray Name the output image:ResizedGray Select resizing method:Resize by a fraction or multiple of the original size Resizing factor:0.50 Width of the final image, in pixels:100 Height of the final image, in pixels:100 Interpolation method:Nearest Neighbor How do you want to specify the dimensions?:Manual Select the image with the desired dimensions:None Additional image count:1 Select the additional image?:maskDAB Name the output image:maskDAB_resize ApplyThreshold:[module_num:6|svn_version:\'6746\'|variable_revision_number:5|show_window:False|notes:\x5B\x5D] Select the input image:maskDAB_resize Name the output image:maskDAB_thresh Select the output image type:Binary (black and white) Set pixels below or above the threshold to zero?:Below threshold Subtract the threshold value from the remaining pixel intensities?:No Number of pixels by which to expand the thresholding around those excluded bright pixels:0.0 Select the thresholding method:Otsu Global Manual threshold:0.0 Lower and upper bounds on threshold:0.000000,1.000000 Threshold correction factor:1 Approximate fraction of image covered by objects?:0.01 Select the input objects:None 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 the measurement to threshold with:None CorrectIlluminationCalculate:[module_num:7|svn_version:\'10458\'|variable_revision_number:2|show_window:False|notes:\x5B\x5D] Select the input image:ResizedH Name the output image:IllumCorrectedH Select how the illumination function is calculated:Regular Dilate objects in the final averaged image?:No Dilation radius:1 Block size:60 Rescale the illumination function?:Yes Calculate function for each image individually, or based on all images?:Each Smoothing method:Median Filter Method to calculate smoothing filter size:Manually Approximate object size:10 Smoothing filter size:3 Retain the averaged image for use later in the pipeline (for example, in SaveImages)?:Yes Name the averaged image:IllumBlueAvg Retain the dilated image for use later in the pipeline (for example, in SaveImages)?:No Name the dilated image:IllumDilatedH 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 Morph:[module_num:8|svn_version:\'10300\'|variable_revision_number:2|show_window:False|notes:\x5B\x5D] Select the input image:IllumBlueAvg Name the output image:invertH Select the operation to perform:invert Number of times to repeat operation:Once Repetition number:2 Scale:3 EnhanceOrSuppressFeatures:[module_num:9|svn_version:\'10591\'|variable_revision_number:2|show_window:False|notes:\x5B\x5D] Select the input image:invertH Name the output image:FilteredH Select the operation:Enhance Feature size:20 Feature type:Neurites Range of hole sizes:10,20 Morph:[module_num:10|svn_version:\'10300\'|variable_revision_number:2|show_window:False|notes:\x5B\x5D] Select the input image:FilteredH Name the output image:tosegmentH Select the operation to perform:close Number of times to repeat operation:Once Repetition number:2 Scale:3 Select the operation to perform:invert Number of times to repeat operation:Once Repetition number:2 Scale:3 MaskImage:[module_num:11|svn_version:\'10428\'|variable_revision_number:3|show_window:False|notes:\x5B\x5D] Select the input image:tosegmentH Name the output image:MaskNuc Use objects or an image as a mask?:Image Select object for mask:ShrunkenNuclei Select image for mask:maskDAB_thresh Invert the mask?:No IdentifyPrimaryObjects:[module_num:12|svn_version:\'10826\'|variable_revision_number:8|show_window:False|notes:\x5B\x5D] Select the input image:MaskNuc Name the primary objects to be identified:Nuclei Typical diameter of objects, in pixel units (Min,Max):5,40 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:RobustBackground Global Threshold correction factor:1 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:Intensity Size of smoothing filter:1 Suppress local maxima that are closer than this minimum allowed distance:1 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?:No Automatically calculate minimum allowed distance between local maxima?:No Manual threshold:0.0 Select binary image:MorphH 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 MeasureObjectSizeShape:[module_num:13|svn_version:\'1\'|variable_revision_number:1|show_window:False|notes:\x5B\x5D] Select objects to measure:Nuclei Calculate the Zernike features?:No FilterObjects:[module_num:14|svn_version:\'10300\'|variable_revision_number:5|show_window:False|notes:\x5B\x5D] Name the output objects:FilteredNuc Select the object to filter:Nuclei Filter using classifier rules or measurements?:Measurements Select the filtering method:Limits Select the objects that contain the filtered objects:Nuclei 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:AreaShape_Area Filter using a minimum measurement value?:Yes Minimum value:1 Filter using a maximum measurement value?:Yes Maximum value:300 ExpandOrShrinkObjects:[module_num:15|svn_version:\'10830\'|variable_revision_number:1|show_window:False|notes:\x5B\x5D] Select the input objects:FilteredNuc Name the output objects:ShrunkenNuclei Select the operation:Add partial dividing lines between objects 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 ConvertObjectsToImage:[module_num:16|svn_version:\'10807\'|variable_revision_number:1|show_window:False|notes:\x5B\x5D] Select the input objects:ShrunkenNuclei Name the output image:NucleiImage Select the color type:Color Select the colormap:Default MeasureObjectSizeShape:[module_num:17|svn_version:\'1\'|variable_revision_number:1|show_window:False|notes:\x5B\x5D] Select objects to measure:ShrunkenNuclei Calculate the Zernike features?:Yes MeasureGranularity:[module_num:18|svn_version:\'10300\'|variable_revision_number:3|show_window:False|notes:\x5B\x5D] Image count:1 Object count:1 Select an image to measure:ResizedH Subsampling factor for granularity measurements:0.25 Subsampling factor for background reduction:0.25 Radius of structuring element:5 Range of the granular spectrum:10 Select objects to measure:ShrunkenNuclei MeasureTexture:[module_num:19|svn_version:\'1\'|variable_revision_number:2|show_window:False|notes:\x5B\x5D] Hidden:1 Hidden:1 Hidden:1 Select an image to measure:ResizedH Select objects to measure:ShrunkenNuclei Texture scale to measure:3 Measure Gabor features?:Yes Number of angles to compute for Gabor:4 MeasureObjectRadialDistribution:[module_num:20|svn_version:\'10798\'|variable_revision_number:1|show_window:False|notes:\x5B\x5D] Hidden:1 Hidden:1 Hidden:1 Select an image to measure:ResizedH Select objects to measure:ShrunkenNuclei Object to use as center?:These objects Select objects to use as centers:None Number of bins:4 MaskImage:[module_num:21|svn_version:\'10428\'|variable_revision_number:3|show_window:False|notes:\x5B\x5D] Select the input image:ResizedGray Name the output image:MaskNuc Use objects or an image as a mask?:Objects Select object for mask:ShrunkenNuclei Select image for mask:None Invert the mask?:No Resize:[module_num:22|svn_version:\'10627\'|variable_revision_number:4|show_window:False|notes:\x5B\x5D] Select the input image:NucleiImage Name the output image:ResizedOriH Select resizing method:Resize by a fraction or multiple of the original size Resizing factor:2 Width of the final image, in pixels:100 Height of the final image, in pixels:100 Interpolation method:Nearest Neighbor How do you want to specify the dimensions?:Manual Select the image with the desired dimensions:None Additional image count:0 SaveImages:[module_num:23|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:ResizedOriH Select the objects to save:Nuclei Select the module display window to save:None Select method for constructing file names:From image filename Select image name for file prefix:OrigIHC Enter single file name:OrigBlue Do you want to add a suffix to the image file name?:Yes Text to append to the image name:_NUC_MASK Select file format to use:jpg Output file location:Default Output Folder\x7CNone Image bit depth:8 Overwrite existing files without warning?:Yes Select how often to save:Every cycle Rescale the images? :No Save as grayscale or color image?:Grayscale Select colormap:Default Store file and path information to the saved image?:No Create subfolders in the output folder?:No SaveImages:[module_num:24|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:OrigIHC Select the objects to save:Nuclei Select the module display window to save:None Select method for constructing file names:From image filename Select image name for file prefix:OrigIHC Enter single file name:OrigBlue Do you want to add a suffix to the image file name?:Yes Text to append to the image name:_original_ihc Select file format to use:jpg Output file location:Default Output Folder\x7CNone Image bit depth:8 Overwrite existing files without warning?:Yes Select how often to save:Every cycle Rescale the images? :No Save as grayscale or color image?:Grayscale Select colormap:Default Store file and path information to the saved image?:No Create subfolders in the output folder?:No ExportToSpreadsheet:[module_num:25|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?:Yes Calculate the per-image mean values for object measurements?:Yes Calculate the per-image median values for object measurements?:No Calculate the per-image standard deviation values for object measurements?:Yes 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:Image\x7CCount_Nuclei,Image\x7CCount_ShrunkenNuclei,Image\x7CCount_FilteredNuc,Image\x7CGroup_Index,Image\x7CGroup_Number,Image\x7CExecutionTime_21MaskImage,Image\x7CExecutionTime_17MeasureObjectSizeShape,Image\x7CExecutionTime_13MeasureObjectSizeShape,Image\x7CExecutionTime_11MaskImage,Image\x7CExecutionTime_04Resize,Image\x7CExecutionTime_14FilterObjects,Image\x7CExecutionTime_18MeasureGranularity,Image\x7CExecutionTime_15ExpandOrShrinkObjects,Image\x7CExecutionTime_20MeasureObjectRadialDistribution,Image\x7CExecutionTime_19MeasureTexture,Image\x7CExecutionTime_10Morph,Image\x7CExecutionTime_02ColorToGray,Image\x7CExecutionTime_06ApplyThreshold,Image\x7CExecutionTime_23SaveImages,Image\x7CExecutionTime_03UnmixColors,Image\x7CExecutionTime_16ConvertObjectsToImage,Image\x7CExecutionTime_12IdentifyPrimaryObjects,Image\x7CExecutionTime_09EnhanceOrSuppressFeatures,Image\x7CExecutionTime_24SaveImages,Image\x7CExecutionTime_08Morph,Image\x7CExecutionTime_07CorrectIlluminationCalculate,Image\x7CExecutionTime_05Resize,Image\x7CExecutionTime_01LoadImages,Image\x7CExecutionTime_22Resize,Image\x7CMD5Digest_maskDAB,Image\x7CMD5Digest_OrigIHC,Image\x7CFileName_maskDAB,Image\x7CFileName_OrigIHC,Image\x7CScaling_maskDAB,Image\x7CScaling_OrigIHC,Image\x7CModuleError_21MaskImage,Image\x7CModuleError_17MeasureObjectSizeShape,Image\x7CModuleError_13MeasureObjectSizeShape,Image\x7CModuleError_11MaskImage,Image\x7CModuleError_04Resize,Image\x7CModuleError_14FilterObjects,Image\x7CModuleError_18MeasureGranularity,Image\x7CModuleError_15ExpandOrShrinkObjects,Image\x7CModuleError_20MeasureObjectRadialDistribution,Image\x7CModuleError_19MeasureTexture,Image\x7CModuleError_10Morph,Image\x7CModuleError_02ColorToGray,Image\x7CModuleError_06ApplyThreshold,Image\x7CModuleError_23SaveImages,Image\x7CModuleError_03UnmixColors,Image\x7CModuleError_16ConvertObjectsToImage,Image\x7CModuleError_12IdentifyPrimaryObjects,Image\x7CModuleError_09EnhanceOrSuppressFeatures,Image\x7CModuleError_24SaveImages,Image\x7CModuleError_08Morph,Image\x7CModuleError_07CorrectIlluminationCalculate,Image\x7CModuleError_05Resize,Image\x7CModuleError_01LoadImages,Image\x7CModuleError_22Resize,Image\x7CPathName_maskDAB,Image\x7CPathName_OrigIHC,Image\x7CThreshold_OrigThreshold_maskDAB_thresh,Image\x7CThreshold_OrigThreshold_Nuclei,Image\x7CThreshold_SumOfEntropies_maskDAB_thresh,Image\x7CThreshold_SumOfEntropies_Nuclei,Image\x7CThreshold_WeightedVariance_maskDAB_thresh,Image\x7CThreshold_WeightedVariance_Nuclei,Image\x7CThreshold_FinalThreshold_maskDAB_thresh,Image\x7CThreshold_FinalThreshold_Nuclei,ShrunkenNuclei\x7CGranularity_10_ResizedH,ShrunkenNuclei\x7CGranularity_1_ResizedH,ShrunkenNuclei\x7CGranularity_3_ResizedH,ShrunkenNuclei\x7CGranularity_2_ResizedH,ShrunkenNuclei\x7CGranularity_5_ResizedH,ShrunkenNuclei\x7CGranularity_4_ResizedH,ShrunkenNuclei\x7CGranularity_7_ResizedH,ShrunkenNuclei\x7CGranularity_6_ResizedH,ShrunkenNuclei\x7CGranularity_9_ResizedH,ShrunkenNuclei\x7CGranularity_8_ResizedH,ShrunkenNuclei\x7CNumber_Object_Number,ShrunkenNuclei\x7CTexture_DifferenceEntropy_ResizedH_3,ShrunkenNuclei\x7CTexture_InfoMeas1_ResizedH_3,ShrunkenNuclei\x7CTexture_DifferenceVariance_ResizedH_3,ShrunkenNuclei\x7CTexture_SumVariance_ResizedH_3,ShrunkenNuclei\x7CTexture_Gabor_ResizedH_3,ShrunkenNuclei\x7CTexture_AngularSecondMoment_ResizedH_3,ShrunkenNuclei\x7CTexture_Entropy_ResizedH_3,ShrunkenNuclei\x7CTexture_Correlation_ResizedH_3,ShrunkenNuclei\x7CTexture_SumAverage_ResizedH_3,ShrunkenNuclei\x7CTexture_Variance_ResizedH_3,ShrunkenNuclei\x7CTexture_InverseDifferenceMoment_ResizedH_3,ShrunkenNuclei\x7CTexture_SumEntropy_ResizedH_3,ShrunkenNuclei\x7CTexture_Contrast_ResizedH_3,ShrunkenNuclei\x7CTexture_InfoMeas2_ResizedH_3,ShrunkenNuclei\x7CLocation_Center_X,ShrunkenNuclei\x7CLocation_Center_Y,ShrunkenNuclei\x7CAreaShape_Perimeter,ShrunkenNuclei\x7CAreaShape_Orientation,ShrunkenNuclei\x7CAreaShape_Area,ShrunkenNuclei\x7CAreaShape_Solidity,ShrunkenNuclei\x7CAreaShape_Zernike_1_1,ShrunkenNuclei\x7CAreaShape_Zernike_0_0,ShrunkenNuclei\x7CAreaShape_Zernike_3_1,ShrunkenNuclei\x7CAreaShape_Zernike_3_3,ShrunkenNuclei\x7CAreaShape_Zernike_2_0,ShrunkenNuclei\x7CAreaShape_Zernike_2_2,ShrunkenNuclei\x7CAreaShape_Zernike_5_1,ShrunkenNuclei\x7CAreaShape_Zernike_5_3,ShrunkenNuclei\x7CAreaShape_Zernike_5_5,ShrunkenNuclei\x7CAreaShape_Zernike_4_0,ShrunkenNuclei\x7CAreaShape_Zernike_4_2,ShrunkenNuclei\x7CAreaShape_Zernike_4_4,ShrunkenNuclei\x7CAreaShape_Zernike_7_1,ShrunkenNuclei\x7CAreaShape_Zernike_7_3,ShrunkenNuclei\x7CAreaShape_Zernike_7_5,ShrunkenNuclei\x7CAreaShape_Zernike_7_7,ShrunkenNuclei\x7CAreaShape_Zernike_6_0,ShrunkenNuclei\x7CAreaShape_Zernike_6_2,ShrunkenNuclei\x7CAreaShape_Zernike_6_4,ShrunkenNuclei\x7CAreaShape_Zernike_6_6,ShrunkenNuclei\x7CAreaShape_Zernike_9_1,ShrunkenNuclei\x7CAreaShape_Zernike_9_3,ShrunkenNuclei\x7CAreaShape_Zernike_9_5,ShrunkenNuclei\x7CAreaShape_Zernike_9_7,ShrunkenNuclei\x7CAreaShape_Zernike_9_9,ShrunkenNuclei\x7CAreaShape_Zernike_8_0,ShrunkenNuclei\x7CAreaShape_Zernike_8_2,ShrunkenNuclei\x7CAreaShape_Zernike_8_4,ShrunkenNuclei\x7CAreaShape_Zernike_8_6,ShrunkenNuclei\x7CAreaShape_Zernike_8_8,ShrunkenNuclei\x7CAreaShape_EulerNumber,ShrunkenNuclei\x7CAreaShape_Compactness,ShrunkenNuclei\x7CAreaShape_Extent,ShrunkenNuclei\x7CAreaShape_Eccentricity,ShrunkenNuclei\x7CAreaShape_FormFactor,ShrunkenNuclei\x7CAreaShape_MinorAxisLength,ShrunkenNuclei\x7CAreaShape_MajorAxisLength,ShrunkenNuclei\x7CAreaShape_Center_X,ShrunkenNuclei\x7CAreaShape_Center_Y,ShrunkenNuclei\x7CRadialDistribution_RadialCV_ResizedH_3of4,ShrunkenNuclei\x7CRadialDistribution_RadialCV_ResizedH_4of4,ShrunkenNuclei\x7CRadialDistribution_RadialCV_ResizedH_1of4,ShrunkenNuclei\x7CRadialDistribution_RadialCV_ResizedH_2of4,ShrunkenNuclei\x7CRadialDistribution_FracAtD_ResizedH_3of4,ShrunkenNuclei\x7CRadialDistribution_FracAtD_ResizedH_4of4,ShrunkenNuclei\x7CRadialDistribution_FracAtD_ResizedH_1of4,ShrunkenNuclei\x7CRadialDistribution_FracAtD_ResizedH_2of4,ShrunkenNuclei\x7CRadialDistribution_MeanFrac_ResizedH_3of4,ShrunkenNuclei\x7CRadialDistribution_MeanFrac_ResizedH_4of4,ShrunkenNuclei\x7CRadialDistribution_MeanFrac_ResizedH_1of4,ShrunkenNuclei\x7CRadialDistribution_MeanFrac_ResizedH_2of4 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