CellProfiler Pipeline: http://www.cellprofiler.org Version:3 DateRevision:20160418141927 GitHash:9969f42 ModuleCount:16 HasImagePlaneDetails:False Images:[module_num:1|svn_version:\'Unknown\'|variable_revision_number:2|show_window:False|notes:\x5B\'To begin creating your project, use the Images module to compile a list of files and/or folders that you want to analyze. You can also specify a set of rules to include only the desired files in your selected folders.\'\x5D|batch_state:array(\x5B\x5D, dtype=uint8)|enabled:True|wants_pause:False] : Filter images?:Images only Select the rule criteria:and (extension does isimage) (directory doesnot containregexp "\x5B\\\\\\\\\\\\\\\\/\x5D\\\\\\\\.") Metadata:[module_num:2|svn_version:\'Unknown\'|variable_revision_number:4|show_window:False|notes:\x5B\'The Metadata module optionally allows you to extract information describing your images (i.e, metadata) which will be stored along with your measurements. This information can be contained in the file name and/or location, or in an external file.\'\x5D|batch_state:array(\x5B\x5D, dtype=uint8)|enabled:True|wants_pause:False] Extract metadata?:No Metadata data type:Text Metadata types:{} Extraction method count:1 Metadata extraction method:Extract from file/folder names Metadata source:File name Regular expression:^(?P.*)_(?P\x5BA-P\x5D\x5B0-9\x5D{2})_s(?P\x5B0-9\x5D)_w(?P\x5B0-9\x5D) Regular expression:(?P\x5B0-9\x5D{4}_\x5B0-9\x5D{2}_\x5B0-9\x5D{2})$ Extract metadata from:All images Select the filtering criteria:and (file does contain "") Metadata file location: Match file and image metadata:\x5B\x5D Use case insensitive matching?:No NamesAndTypes:[module_num:3|svn_version:\'Unknown\'|variable_revision_number:6|show_window:False|notes:\x5B\'The NamesAndTypes module allows you to assign a meaningful name to each image by which other modules will refer to it.\'\x5D|batch_state:array(\x5B\x5D, dtype=uint8)|enabled:True|wants_pause:False] Assign a name to:All images Select the image type:Color image Name to assign these images:ColorImage Match metadata:\x5B\x5D Image set matching method:Order Set intensity range from:Image metadata Assignments count:1 Single images count:0 Maximum intensity:255.0 Select the rule criteria:and (file does contain "") Name to assign these images:DNA Name to assign these objects:Cell Select the image type:Grayscale image Set intensity range from:Image metadata Retain outlines of loaded objects?:No Name the outline image:LoadedOutlines Maximum intensity:255.0 Groups:[module_num:4|svn_version:\'Unknown\'|variable_revision_number:2|show_window:False|notes:\x5B\'The Groups module optionally allows you to split your list of images into image subsets (groups) which will be processed independently of each other. Examples of groupings include screening batches, microtiter plates, time-lapse movies, etc.\'\x5D|batch_state:array(\x5B\x5D, dtype=uint8)|enabled:True|wants_pause:False] Do you want to group your images?:No grouping metadata count:1 Metadata category:None ColorToGray:[module_num:5|svn_version:\'Unknown\'|variable_revision_number:3|show_window:False|notes:\x5B\x5D|batch_state:array(\x5B\x5D, dtype=uint8)|enabled:True|wants_pause:False] Select the input image:ColorImage Conversion method:Split Image type:Channels Name the output image:OrigGray Relative weight of the red channel:1.0 Relative weight of the green channel:1.0 Relative weight of the blue channel:1.0 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 Convert hue to gray?:Yes Name the output image:OrigHue Convert saturation to gray?:Yes Name the output image:OrigSaturation Convert value to gray?:Yes Name the output image:OrigValue Channel count:3 Channel number:Red\x3A 1 Relative weight of the channel:1.0 Image name:TCF7 Channel number:Green\x3A 2 Relative weight of the channel:1.0 Image name:CD8 Channel number:Blue\x3A 3 Relative weight of the channel:1.0 Image name:DAPI EnhanceOrSuppressFeatures:[module_num:6|svn_version:\'Unknown\'|variable_revision_number:5|show_window:False|notes:\x5B\x5D|batch_state:array(\x5B\x5D, dtype=uint8)|enabled:True|wants_pause:False] Select the input image:DAPI Name the output image:FilteredDAPI Select the operation:Enhance Feature size:20 Feature type:Speckles Range of hole sizes:1,10 Smoothing scale:2.0 Shear angle:0.0 Decay:0.95 Enhancement method:Tubeness Speed and accuracy:Fast / hexagonal IdentifyPrimaryObjects:[module_num:7|svn_version:\'Unknown\'|variable_revision_number:10|show_window:False|notes:\x5B\x5D|batch_state:array(\x5B\x5D, dtype=uint8)|enabled:True|wants_pause:False] Select the input image:FilteredDAPI Name the primary objects to be identified:Nuclei Typical diameter of objects, in pixel units (Min,Max):15,50 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 Method to distinguish clumped objects:Shape 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:7.0 Speed up by using lower-resolution image to find local maxima?:Yes Name the outline image:PrimaryOutlines Fill holes in identified objects?:After both thresholding and declumping Automatically calculate size of smoothing filter for declumping?:Yes Automatically calculate minimum allowed distance between local maxima?:Yes Retain outlines of the identified objects?:No Automatically calculate the threshold using the Otsu method?:Yes Enter Laplacian of Gaussian threshold:0.5 Automatically calculate the size of objects for the Laplacian of Gaussian filter?:Yes Enter LoG filter diameter:5.0 Handling of objects if excessive number of objects identified:Continue Maximum number of objects:500 Threshold setting version:2 Threshold strategy:Adaptive Thresholding method:Otsu Select the smoothing method for thresholding:Automatic Threshold smoothing scale:1.0 Threshold correction factor:1.0 Lower and upper bounds on threshold:0.0,1.0 Approximate fraction of image covered by objects?:0.01 Manual threshold:0.0 Select the measurement to threshold with:None Select binary image:None Masking 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 Method to calculate adaptive window size:Image size Size of adaptive window:10 Use default parameters?:Default Lower outlier fraction:0.05 Upper outlier fraction:0.05 Averaging method:Mean Variance method:Standard deviation # of deviations:2.0 RescaleIntensity:[module_num:8|svn_version:\'Unknown\'|variable_revision_number:2|show_window:False|notes:\x5B\x5D|batch_state:array(\x5B\x5D, dtype=uint8)|enabled:True|wants_pause:False] Select the input image:CD8 Name the output image:RescaledCD8 Rescaling method:Stretch each image to use the full intensity range Method to calculate the minimum intensity:Custom Method to calculate the maximum intensity:Custom Lower intensity limit for the input image:0.0 Upper intensity limit for the input image:1.0 Intensity range for the input image:0.0,1.0 Intensity range for the output image:0.0,1.0 Method to rescale pixels below the lower limit:Mask pixels Custom value for pixels below lower limit:0.0 Method to rescale pixels above the upper limit:Mask pixels Custom value for pixels above upper limit:0.0 Select image to match in maximum intensity:None Divisor value:1.0 Divisor measurement:None MeasureObjectIntensity:[module_num:9|svn_version:\'Unknown\'|variable_revision_number:3|show_window:False|notes:\x5B\x5D|batch_state:array(\x5B\x5D, dtype=uint8)|enabled:True|wants_pause:False] Hidden:1 Select an image to measure:RescaledCD8 Select objects to measure:Nuclei FilterObjects:[module_num:10|svn_version:\'Unknown\'|variable_revision_number:7|show_window:False|notes:\x5B\x5D|batch_state:array(\x5B\x5D, dtype=uint8)|enabled:True|wants_pause:False] Name the output objects:CD8PosNuclei Select the object to filter:Nuclei Select the filtering mode: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:Elsewhere...\x7C Rules file name:rules.txt Class number:1 Measurement count:1 Additional object count:0 Assign overlapping child to:Both parents Select the measurement to filter by:Intensity_MeanIntensityEdge_RescaledCD8 Filter using a minimum measurement value?:Yes Minimum value:0.1 Filter using a maximum measurement value?:No Maximum value:1.0 IdentifyPrimaryObjects:[module_num:11|svn_version:\'Unknown\'|variable_revision_number:10|show_window:False|notes:\x5B\x5D|batch_state:array(\x5B\x5D, dtype=uint8)|enabled:True|wants_pause:False] Select the input image:TCF7 Name the primary objects to be identified:TCF7Nuclei Typical diameter of objects, in pixel units (Min,Max):10,40 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 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:7.0 Speed up by using lower-resolution image to find local maxima?:Yes Name the outline image:PrimaryOutlines Fill holes in identified objects?:After both thresholding and declumping Automatically calculate size of smoothing filter for declumping?:Yes Automatically calculate minimum allowed distance between local maxima?:Yes Retain outlines of the identified objects?:No Automatically calculate the threshold using the Otsu method?:Yes Enter Laplacian of Gaussian threshold:0.5 Automatically calculate the size of objects for the Laplacian of Gaussian filter?:Yes Enter LoG filter diameter:5.0 Handling of objects if excessive number of objects identified:Continue Maximum number of objects:500 Threshold setting version:2 Threshold strategy:Automatic Thresholding method:Otsu Select the smoothing method for thresholding:Automatic Threshold smoothing scale:1.0 Threshold correction factor:1.0 Lower and upper bounds on threshold:0.0,1.0 Approximate fraction of image covered by objects?:0.01 Manual threshold:0.0 Select the measurement to threshold with:None Select binary image:None Masking 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 Method to calculate adaptive window size:Image size Size of adaptive window:10 Use default parameters?:Default Lower outlier fraction:0.05 Upper outlier fraction:0.05 Averaging method:Mean Variance method:Standard deviation # of deviations:2.0 MaskObjects:[module_num:12|svn_version:\'Unknown\'|variable_revision_number:2|show_window:False|notes:\x5B\x5D|batch_state:array(\x5B\x5D, dtype=uint8)|enabled:True|wants_pause:False] Select objects to be masked:CD8PosNuclei Name the masked objects:TCF7PosCD8PosNuclei Mask using a region defined by other objects or by binary image?:Objects Select the masking object:TCF7Nuclei Select the masking image:None Handling of objects that are partially masked:Remove depending on overlap Fraction of object that must overlap:0.3 Numbering of resulting objects:Renumber Retain outlines of the resulting objects?:No Name the outline image:MaskedOutlines Invert the mask?:No CalculateMath:[module_num:13|svn_version:\'Unknown\'|variable_revision_number:2|show_window:False|notes:\x5B\x5D|batch_state:array(\x5B\x5D, dtype=uint8)|enabled:True|wants_pause:False] Name the output measurement:Count_CD8Pos_TCFNeg Operation:Subtract Select the minuend measurement type:Image Select the minuend objects:None Select the minuend measurement:Count_CD8PosNuclei Multiply the above operand by:1.0 Raise the power of above operand by:1.0 Select the subtrahend measurement type:Image Select the subtrahend objects:None Select the subtrahend measurement:Count_TCF7PosCD8PosNuclei Multiply the above operand by:1.0 Raise the power of above operand by:1.0 Take log10 of result?:No Multiply the result by:1.0 Raise the power of result by:1.0 Add to the result:0.0 Constrain the result to a lower bound?:No Enter the lower bound:0.0 Constrain the result to an upper bound?:No Enter the upper bound:1.0 OverlayOutlines:[module_num:14|svn_version:\'Unknown\'|variable_revision_number:3|show_window:False|notes:\x5B\x5D|batch_state:array(\x5B\x5D, dtype=uint8)|enabled:True|wants_pause:False] Display outlines on a blank image?:No Select image on which to display outlines:ColorImage Name the output image:OrigOverlay Outline display mode:Color Select method to determine brightness of outlines:Max of image Width of outlines:1 Select outlines to display:None Select outline color:#F8FF2D Load outlines from an image or objects?:Objects Select objects to display:CD8PosNuclei Select outlines to display:None Select outline color:#F1F7F7 Load outlines from an image or objects?:Objects Select objects to display:TCF7PosCD8PosNuclei SaveImages:[module_num:15|svn_version:\'Unknown\'|variable_revision_number:11|show_window:False|notes:\x5B\x5D|batch_state:array(\x5B\x5D, dtype=uint8)|enabled:True|wants_pause:True] 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:ColorImage Enter single file name:OrigBlue Number of digits:4 Append a suffix to the image file name?:Yes Text to append to the image name:_Overlay Saved file format:tif Output file location:Default Output Folder\x7C Image bit depth:8-bit integer Overwrite existing files without warning?:No When to save:Every cycle Rescale the images? :Yes Save as grayscale or color image?:Grayscale Select colormap:gray Record the file and path information to the saved image?:No Create subfolders in the output folder?:No Base image folder:Elsewhere...\x7C Saved movie format:avi ExportToSpreadsheet:[module_num:16|svn_version:\'Unknown\'|variable_revision_number:11|show_window:False|notes:\x5B\x5D|batch_state:array(\x5B\x5D, dtype=uint8)|enabled:True|wants_pause:False] Select the column delimiter:Comma (",") Add image metadata columns to your object data file?:No Limit output to a size that is allowed in Excel?:No Select the measurements to export:Yes 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\x7C 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 measurement types?:No :Image\x7CCount_TCF7Nuclei,Image\x7CCount_Nuclei,Image\x7CCount_CD8PosNuclei,Image\x7CCount_TCF7PosCD8PosNuclei,Image\x7CMath_Count_CD8Pos_TCFNeg Representation of Nan/Inf:NaN Add a prefix to file names?:No Filename prefix:MyExpt_ Overwrite existing files without warning?:No Data to export:Image Combine these object measurements with those of the previous object?:No File name:DATA.csv Use the object name for the file name?:Yes