Saved Pipeline, in file AnalysisPIPE.txt, Saved on 26-Jun-2008 Pixel Size: 1 Pipeline: LoadImages LoadSingleImage CorrectIllumination_Apply CorrectIllumination_Apply CorrectIllumination_Apply IdentifyPrimAutomatic IdentifySecondary IdentifyTertiarySubregion MeasureImageIntensity MeasureImageIntensity MeasureImageIntensity MeasureCorrelation MeasureImageQuality MeasureObjectAreaShape MeasureObjectIntensity MeasureObjectIntensity MeasureObjectIntensity MeasureTexture MeasureTexture MeasureTexture MeasureTexture MeasureTexture MeasureTexture MeasureObjectNeighbors MeasureObjectNeighbors ExportToDatabase CreateBatchFiles Module #1: LoadImages revision - 2 How do you want to load these files? Text-Regular expressions Type the text that one type of image has in common (for TEXT options), or their position in each group (for ORDER option): d0.DIB What do you want to call these images within CellProfiler? OrigDNA Type the text that one type of image has in common (for TEXT options), or their position in each group (for ORDER option): d1.DIB What do you want to call these images within CellProfiler? OrigpH3 Type the text that one type of image has in common (for TEXT options), or their position in each group (for ORDER option): d2.DIB What do you want to call these images within CellProfiler? OrigActin Type the text that one type of image has in common (for TEXT options), or their position in each group (for ORDER option): / What do you want to call these images within CellProfiler? / If using ORDER, how many images are there in each group (i.e. each field of view)? 3 What type of files are you loading? individual images Analyze all subfolders within the selected folder? Yes Enter the path name to the folder where the images to be loaded are located. Type period (.) for default image folder. . Note - If the movies contain more than just one image type (e.g., brightfield, fluorescent, field-of-view), add the GroupMovieFrames module. . Module #2: LoadSingleImage revision - 4 This module loads one image for *all* cycles that will be processed. Typically, however, a different module (LoadImages) is used to load new sets of images during each cycle of processing. n/a Enter the path name to the folder where the images to be loaded are located. Type period (.) for the default image folder, or type ampersand (&) for the default output folder. . What image file do you want to load? Include the extension, like .tif AS_09125_050117050001_P24f05d0ILLUM.mat What do you want to call that image? IllumDNA What image file do you want to load? Include the extension, like .tif AS_09125_050117050001_P24f05d1ILLUM.mat What do you want to call that image? IllumpH3 What image file do you want to load? Include the extension, like .tif AS_09125_050117050001_P24f05d2ILLUM.mat What do you want to call that image? IllumActin What image file do you want to load? Include the extension, like .tif NO FILE LOADED What do you want to call that image? Do not load Module #3: CorrectIllumination_Apply revision - 3 What did you call the image to be corrected? OrigDNA What do you want to call the corrected image? DNA What did you call the illumination correction function image to be used to carry out the correction (produced by another module or loaded as a .mat format image using Load Single Image)? IllumDNA How do you want to apply the illumination correction function? Divide If you chose division, Choose rescaling method. No rescaling Module #4: CorrectIllumination_Apply revision - 3 What did you call the image to be corrected? OrigpH3 What do you want to call the corrected image? pH3 What did you call the illumination correction function image to be used to carry out the correction (produced by another module or loaded as a .mat format image using Load Single Image)? IllumpH3 How do you want to apply the illumination correction function? Divide If you chose division, Choose rescaling method. No rescaling Module #5: CorrectIllumination_Apply revision - 3 What did you call the image to be corrected? OrigActin What do you want to call the corrected image? Actin What did you call the illumination correction function image to be used to carry out the correction (produced by another module or loaded as a .mat format image using Load Single Image)? IllumActin How do you want to apply the illumination correction function? Divide If you chose division, Choose rescaling method. No rescaling Module #6: IdentifyPrimAutomatic revision - 12 What did you call the images you want to process? DNA What do you want to call the objects identified by this module? Nuclei Typical diameter of objects, in pixel units (Min,Max): 3,30 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 an automatic thresholding method or enter an absolute threshold in the range [0,1]. To choose a binary image, select "Other" and type its name. Choosing 'All' will use the Otsu Global method to calculate a single threshold for the entire image group. The other methods calculate a threshold for each image individually. "Set interactively" will allow you to manually adjust the threshold during the first cycle to determine what will work well. Otsu Adaptive Threshold correction factor 1 Lower and upper bounds on threshold, in the range [0,1] .125,1 For MoG thresholding, what is the approximate fraction of image covered by objects? 0.01 Method to distinguish clumped objects (see help for details): Intensity Method to draw dividing lines between clumped objects (see help for details): Intensity Size of smoothing filter, in pixel units (if you are distinguishing between clumped objects). Enter 0 for low resolution images with small objects (~< 5 pixel diameter) to prevent any image smoothing. 4 Suppress local maxima within this distance, (a positive integer, in pixel units) (if you are distinguishing between clumped objects) 4 Speed up by using lower-resolution image to find local maxima? (if you are distinguishing between clumped objects) Yes Enter the following information, separated by commas, if you would like to use the Laplacian of Gaussian method for identifying objects instead of using the above settings: Size of neighborhood(height,width),Sigma,Minimum Area,Size for Wiener Filter(height,width),Threshold / What do you want to call the outlines of the identified objects (optional)? NucOutlines Do you want to fill holes in identified objects? Yes Do you want to run in test mode where each method for distinguishing clumped objects is compared? No Module #7: IdentifySecondary revision - 3 What did you call the primary objects you want to create secondary objects around? Nuclei What do you want to call the objects identified by this module? Cells Select the method to identify the secondary objects (Distance - B uses background; Distance - N does not): Propagation What did you call the images to be used to find the edges of the secondary objects? For DISTANCE - N, this will not affect object identification, only the final display. Actin Select an automatic thresholding method or enter an absolute threshold in the range [0,1]. To choose a binary image, select "Other" and type its name. Choosing 'All' will use the Otsu Global method to calculate a single threshold for the entire image group. The other methods calculate a threshold for each image individually. Set interactively will allow you to manually adjust the threshold during the first cycle to determine what will work well. Otsu Adaptive Threshold correction factor .7 Lower and upper bounds on threshold, in the range [0,1] 0.05,1 For MoG thresholding, what is the approximate fraction of image covered by objects? 10 For DISTANCE, enter the number of pixels by which to expand the primary objects [Positive integer] 10 For PROPAGATION, enter the regularization factor (0 to infinity). Larger=distance,0=intensity 0.05 What do you want to call the outlines of the identified objects (optional)? CellOutlines Do you want to run in test mode where each method for identifying secondary objects is compared? No Module #8: IdentifyTertiarySubregion revision - 1 What did you call the larger identified objects? Cells What did you call the smaller identified objects? Nuclei What do you want to call the new subregions? Cytoplasm What do you want to call the outlines of the identified objects (optional)? Do not save Module #9: MeasureImageIntensity revision - 2 What did you call the images you want to process? DNA Ignore pixels below this intensity level (Range = 0-1) 0 Ignore pixels above this intensity level (Range = 0-1) 1 Exclude pixels within this many pixels of an excluded bright object 0 Module #10: MeasureImageIntensity revision - 2 What did you call the images you want to process? pH3 Ignore pixels below this intensity level (Range = 0-1) 0 Ignore pixels above this intensity level (Range = 0-1) 1 Exclude pixels within this many pixels of an excluded bright object 0 Module #11: MeasureImageIntensity revision - 2 What did you call the images you want to process? Actin Ignore pixels below this intensity level (Range = 0-1) 0 Ignore pixels above this intensity level (Range = 0-1) 1 Exclude pixels within this many pixels of an excluded bright object 0 Module #12: MeasureCorrelation revision - 3 Choose at least two image types to measure correlations between: DNA (All pairwise correlations will be measured) pH3 Actin Do not use Choose objects within which to measure the correlations (Choosing Image will measure correlations across the entire images) Image Nuclei Cells Cytoplasm Do not use Do not use Module #13: MeasureImageQuality revision - 1 Do you want to check the following selected images for image quality (called blur earlier)? Yes If you chose to check images for image quality above, enter the window size of LocalFocusScore measurement (A suggested value is 2 times ObjectSize)? 20 Which grayscale image would you like to use to check for saturation? OrigDNA Which grayscale image would you like to use to calculate a suggested threshold? OrigDNA Which automatic thresholding method would you like to use to calculate a suggested threshold? Otsu Global Which grayscale image would you like to use to check for saturation? OrigpH3 Which grayscale image would you like to use to calculate a suggested threshold? OrigpH3 Which automatic thresholding method would you like to use to calculate a suggested threshold? Otsu Global Which grayscale image would you like to use to check for saturation? OrigActin Which grayscale image would you like to use to calculate a suggested threshold? OrigActin Which automatic thresholding method would you like to use to calculate a suggested threshold? Otsu Global Which grayscale image would you like to use to check for saturation? Do not use Which grayscale image would you like to use to calculate a suggested threshold? Do not use Which automatic thresholding method would you like to use to calculate a suggested threshold? Otsu Global Module #14: MeasureObjectAreaShape revision - 3 What did you call the objects that you want to measure? Nuclei Cells Cytoplasm Do not use Do not use Do not use Do not use Would you like to calculate the Zernike features for each object (with lots of objects, this can be very slow)? Yes Module #15: MeasureObjectIntensity revision - 2 What did you call the greyscale images you want to measure? DNA What did you call the objects that you want to measure? Nuclei Cells Cytoplasm Do not use Do not use Do not use Module #16: MeasureObjectIntensity revision - 2 What did you call the greyscale images you want to measure? pH3 What did you call the objects that you want to measure? Nuclei Cells Cytoplasm Do not use Do not use Do not use Module #17: MeasureObjectIntensity revision - 2 What did you call the greyscale images you want to measure? Actin What did you call the objects that you want to measure? Nuclei Cells Cytoplasm Do not use Do not use Do not use Module #18: MeasureTexture revision - 2 What did you call the greyscale images you want to measure? DNA What did you call the objects that you want to measure? Image Nuclei Cells Cytoplasm Do not use Do not use What is the scale of texture? 1 Module #19: MeasureTexture revision - 2 What did you call the greyscale images you want to measure? DNA What did you call the objects that you want to measure? Image Nuclei Cells Cytoplasm Do not use Do not use What is the scale of texture? 3 Module #20: MeasureTexture revision - 2 What did you call the greyscale images you want to measure? pH3 What did you call the objects that you want to measure? Image Nuclei Cells Cytoplasm Do not use Do not use What is the scale of texture? 1 Module #21: MeasureTexture revision - 2 What did you call the greyscale images you want to measure? pH3 What did you call the objects that you want to measure? Image Nuclei Cells Cytoplasm Do not use Do not use What is the scale of texture? 3 Module #22: MeasureTexture revision - 2 What did you call the greyscale images you want to measure? Actin What did you call the objects that you want to measure? Image Nuclei Cells Cytoplasm Do not use Do not use What is the scale of texture? 1 Module #23: MeasureTexture revision - 2 What did you call the greyscale images you want to measure? Actin What did you call the objects that you want to measure? Image Nuclei Cells Cytoplasm Do not use Do not use What is the scale of texture? 3 Module #24: MeasureObjectNeighbors revision - 5 What did you call the objects whose neighbors you want to measure? Nuclei Objects are considered neighbors if they are within this distance, in pixels. If you want your objects to be touching before you count neighbors (for instance, in an image of tissue), use the ExpandOrShrink module to expand your objects: 20 What do you want to call the objects colored by number of neighbors, which are compatible for converting to a color image using the Convert To Image and Save Images modules? Do not save What do you want to call the image of the objects with grayscale values corresponding to the number of neighbors, which is compatible for saving in .mat format using the Save Images module for further analysis in Matlab? Do not save This text is left for consistency with prior versions. Any saved settings shown to the right are not used. Yes Module #25: MeasureObjectNeighbors revision - 5 What did you call the objects whose neighbors you want to measure? Cells Objects are considered neighbors if they are within this distance, in pixels. If you want your objects to be touching before you count neighbors (for instance, in an image of tissue), use the ExpandOrShrink module to expand your objects: 20 What do you want to call the objects colored by number of neighbors, which are compatible for converting to a color image using the Convert To Image and Save Images modules? Do not save What do you want to call the image of the objects with grayscale values corresponding to the number of neighbors, which is compatible for saving in .mat format using the Save Images module for further analysis in Matlab? Do not save This text is left for consistency with prior versions. Any saved settings shown to the right are not used. Yes Module #26: ExportToDatabase revision - 4 What type of database do you want to use? MySQL For MySQL only, what is the name of the database to use? 2007_08_23_NIRHT What prefix should be used to name the tables in the database (should be unique per experiment, or leave "/" to have generic Per_Image and Per_Object tables)? / What prefix should be used to name the SQL files? SQL_ Enter the directory where the SQL files are to be saved. Type period (.) to use the default output folder. . Module #27: CreateBatchFiles revision - 7 Is your cluster using the MATLAB version or the compiled version of CPCluster? Compiled How many cycles should be in each batch? 99999 What prefix should be used to name the batch files? BatchPIPE_ If you chose the MATLAB option, what is the path to the CellProfiler folder on the cluster machines? Leave a period (.) to use the parent of the default module folder. . What is the path to the image folder on the cluster machines? Leave a period (.) to use the default image folder. . What is the path to the folder where batch output should be written on the cluster machines? Leave a period (.) to use the default output folder. . What is the path to the folder where you want to save the batch files? Leave a period (.) to use the default output directory. . What is the path to the folder where the batch data file will be saved on the cluster machines? Leave a period (.) to use the default output folder. . If pathnames are specified differently between the local and cluster machines, enter that part of the pathname from the local machine's perspective, omitting trailing slashes. Otherwise, leave a period (.) /Volumes/imaging_analysis If pathnames are specified differently between the local and cluster machines, enter that part of the pathname from the cluster machines' perspective, omitting trailing slashes. Otherwise, leave a period (.) /imaging/analysis Note: This module must be the last one in the analysis pipeline.