CPP pipeline for cell segmentation and feature extraction used in De Groot et al. Large-scale image-based profiling of single-cell phenotypes in arrayed CRISPR-Cas9 gene perturbation screens Pixel Size: 1 Pipeline: LoadImages IlluminationCorrectionPelkmans IlluminationCorrectionPelkmans IlluminationCorrectionPelkmans IlluminationCorrectionPelkmans IdentifyPrimAutomatic DiscardObjectsBySize IdentifySecondaryIterative ExpandOrShrink ExpandOrShrink IdentifyTertiarySubregion IdentifyTertiarySubregion SaveSegmentedObjects SaveSegmentedObjects SaveSegmentedObjects SaveSegmentedObjects MeasureObjectNeighborsPelkmans MeasureObjectAreaShape MeasureObjectAreaShape SubtractBackgroundPelkmans SubtractBackgroundPelkmans SubtractBackgroundPelkmans SubtractBackgroundPelkmans MeasureObjectIntensity MeasureObjectIntensity MeasureObjectIntensity MeasureObjectIntensity MeasureObjectIntensity MeasureObjectIntensity MeasureObjectIntensity MeasureObjectIntensity MeasureTexture MeasureTexture MeasureTexture MeasureTexture MeasureTexture MeasureTexture MeasureTexture MeasureTexture MeasureTexture MeasureTexture MeasureTexture MeasureTexture CreateBatchFiles Module #1: LoadImages revision - 1 How do you want to load these files? Text-Exact match Type the text that one type of image has in common (for TEXT options), or their position in each group (for ORDER option): C01. What do you want to call these images within CellProfiler? OrigBlue Type the text that one type of image has in common (for TEXT options), or their position in each group (for ORDER option): C02. What do you want to call these images within CellProfiler? OrigGreen Type the text that one type of image has in common (for TEXT options), or their position in each group (for ORDER option): C03. What do you want to call these images within CellProfiler? OrigRed Type the text that one type of image has in common (for TEXT options), or their position in each group (for ORDER option): C04. What do you want to call these images within CellProfiler? OrigFarRed 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? / 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? / 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? / 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? / 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? / 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? / 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? / 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? / 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? / 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? / 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? / 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? / 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? / 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? / 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? / 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 Are you loading image or movie files? Image If you are loading a movie, what is the extension? avi Analyze all subfolders within the selected folder? No Enter the path name to the folder where the images to be loaded are located. Type period (.) for default image folder. . Module #2: IlluminationCorrectionPelkmans revision - 4 What did you call the images to be used to calculate the illumination functions (input)? OrigBlue How do you want to call the corrected images (output)? CorrBlue Do you also want to smooth mean/std statistics values before using them for correction? No If yes, enter the size of the Gaussian filter used for smoothing. 50 If yes, enter the factor 'x' for computing the sigma of the Gaussian filter, where sigma = x*size. 0.5 Enter the relative path name to the folder where the illumination correction files are located (starting with "./../"). Type "Pre" (Pre) to load files from previous multiplexing cycle. Type period (.) for default directory. . Module #3: IlluminationCorrectionPelkmans revision - 4 What did you call the images to be used to calculate the illumination functions (input)? OrigGreen How do you want to call the corrected images (output)? CorrGreen Do you also want to smooth mean/std statistics values before using them for correction? No If yes, enter the size of the Gaussian filter used for smoothing. 50 If yes, enter the factor 'x' for computing the sigma of the Gaussian filter, where sigma = x*size. 0.5 Enter the relative path name to the folder where the illumination correction files are located (starting with "./../"). Type "Pre" (Pre) to load files from previous multiplexing cycle. Type period (.) for default directory. . Module #4: IlluminationCorrectionPelkmans revision - 4 What did you call the images to be used to calculate the illumination functions (input)? OrigRed How do you want to call the corrected images (output)? CorrRed Do you also want to smooth mean/std statistics values before using them for correction? No If yes, enter the size of the Gaussian filter used for smoothing. 50 If yes, enter the factor 'x' for computing the sigma of the Gaussian filter, where sigma = x*size. 0.5 Enter the relative path name to the folder where the illumination correction files are located (starting with "./../"). Type "Pre" (Pre) to load files from previous multiplexing cycle. Type period (.) for default directory. . Module #5: IlluminationCorrectionPelkmans revision - 4 What did you call the images to be used to calculate the illumination functions (input)? OrigFarRed How do you want to call the corrected images (output)? CorrFarRed Do you also want to smooth mean/std statistics values before using them for correction? No If yes, enter the size of the Gaussian filter used for smoothing. 50 If yes, enter the factor 'x' for computing the sigma of the Gaussian filter, where sigma = x*size. 0.5 Enter the relative path name to the folder where the illumination correction files are located (starting with "./../"). Type "Pre" (Pre) to load files from previous multiplexing cycle. Type period (.) for default directory. . Module #6: IdentifyPrimAutomatic revision - 12 What did you call the images you want to process? CorrBlue What do you want to call the objects identified by this module? Nuclei Typical diameter of objects, in pixel units (Min,Max): 35,55 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 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 Global Threshold correction factor 1 Lower and upper bounds on threshold, in the range [0,1] 0,1 For MoG thresholding, what is the approximate percentage of image covered by objects? 10 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. Automatic Suppress local maxima within this distance, (a positive integer, in pixel units) (if you are distinguishing between clumped objects) Automatic 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 Weiner Filter(height,width),Threshold / What do you want to call the outlines of the identified objects (optional)? Do not save 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: DiscardObjectsBySize revision - 1 What objects should be discarded? Nuclei What is the minimum allowed object area? 800 Do you want to set a different minimum allowed object area for objects touching the border? No If yes, enter the number of pixels that define the border region. 50 If yes, what is the minimum allowed object area for objects within the border region? 0 Module #8: IdentifySecondaryIterative 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 What did you call the images to be used to find the edges of the secondary objects? CorrFarRed Lower and upper bounds on threshold, in the range [0,1]. Pixels with an intensity below the lower bound will never be part of a secondary object. 0.00195,1 Threshold correction factors. In descending ranking. eg 0.9 0.8 0.7 0.9 0.7 0.6 0.58 0.55 0.50 0.45 0.4 0.35 0.3 0.25 Module #9: ExpandOrShrink revision - 2 What did you call the objects that you want to expand or shrink? Nuclei What do you want to call the expanded or shrunken objects? ExpandedNuclei Were the objects identified using an Identify Primary or Identify Secondary module (note: shrinking results are not perfect with Secondary objects)? Primary Do you want to expand or shrink the objects? Expand Enter the number of pixels by which to expand or shrink the objects, or "Inf" to either shrink to a point or expand until almost touching, or 0 (the number zero) to simply add partial dividing lines between objects that are touching (experimental feature). 5 What do you want to call the outlines of the identified objects (optional)? Do not save Module #10: ExpandOrShrink revision - 2 What did you call the objects that you want to expand or shrink? Nuclei What do you want to call the expanded or shrunken objects? ShrunkenNuclei Were the objects identified using an Identify Primary or Identify Secondary module (note: shrinking results are not perfect with Secondary objects)? Primary Do you want to expand or shrink the objects? Shrink Enter the number of pixels by which to expand or shrink the objects, or "Inf" to either shrink to a point or expand until almost touching, or 0 (the number zero) to simply add partial dividing lines between objects that are touching (experimental feature). 5 What do you want to call the outlines of the identified objects (optional)? Do not save Module #11: IdentifyTertiarySubregion revision - 1 What did you call the larger identified objects? Cells What did you call the smaller identified objects? ExpandedNuclei What do you want to call the new subregions? Cytoplasm Do you want to enforce an unambiguous 1:1:1 relation between objects? Recommended for Nuclei, Cells and Cytoplasm. Yes What do you want to call the outlines of the identified objects (optional)? Do not save Module #12: IdentifyTertiarySubregion revision - 1 What did you call the larger identified objects? ExpandedNuclei What did you call the smaller identified objects? ShrunkenNuclei What do you want to call the new subregions? NuclearPeriphery Do you want to enforce an unambiguous 1:1:1 relation between objects? Recommended for Nuclei, Cells and Cytoplasm. Yes What do you want to call the outlines of the identified objects (optional)? Do not save Module #13: SaveSegmentedObjects revision - 1 For which object? Nuclei Module #14: SaveSegmentedObjects revision - 1 For which object? Cells Module #15: SaveSegmentedObjects revision - 1 For which object? Cytoplasm Module #16: SaveSegmentedObjects revision - 1 For which object? NuclearPeriphery Module #17: MeasureObjectNeighborsPelkmans revision - 4 What do you want to analyse the borders of? Cells How many pixels do you maximally expand away from object? 20 How many pixels do you minimally expand away from object? 2 Module #18: MeasureObjectAreaShape revision - 3 What did you call the objects that you want to measure? Nuclei Cells Do not use 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 #19: MeasureObjectAreaShape revision - 3 What did you call the objects that you want to measure? Cytoplasm NuclearPeriphery Do not use 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)? No Module #20: SubtractBackgroundPelkmans revision - 1 What did you call the image to be corrected? CorrBlue What do you want to call the corrected image? SubsBlue Select an a method for calculating the background value. Single Value For the Iterative Threshold method select the number of iterations. 500 Define a correction factor (applies to all threshold methods). 0.99 For the Otsu Global and Iterative Threshold methods select a minimum allowed value. 0 For the Otsu Global and Iterative Threshold methods select a maximum allowed value. 1 For the Single Value method enter the theshhold value. 0.0018 Do you want to correct bleed through from another channel? No If yes. Enter the image you want to use for correction. OrigBlue If yes. Enter the bleeding ratio, which the image above will be multiplied to before subtracting. 0.065 Module #21: SubtractBackgroundPelkmans revision - 1 What did you call the image to be corrected? CorrGreen What do you want to call the corrected image? SubsGreen Select an a method for calculating the background value. Single Value For the Iterative Threshold method select the number of iterations. 500 Define a correction factor (applies to all threshold methods). 0.99 For the Otsu Global and Iterative Threshold methods select a minimum allowed value. 0 For the Otsu Global and Iterative Threshold methods select a maximum allowed value. 1 For the Single Value method enter the theshhold value. 0.0018 Do you want to correct bleed through from another channel? No If yes. Enter the image you want to use for correction. OrigBlue If yes. Enter the bleeding ratio, which the image above will be multiplied to before subtracting. 0.065 Module #22: SubtractBackgroundPelkmans revision - 1 What did you call the image to be corrected? CorrRed What do you want to call the corrected image? SubsRed Select an a method for calculating the background value. Single Value For the Iterative Threshold method select the number of iterations. 500 Define a correction factor (applies to all threshold methods). 0.99 For the Otsu Global and Iterative Threshold methods select a minimum allowed value. 0 For the Otsu Global and Iterative Threshold methods select a maximum allowed value. 1 For the Single Value method enter the theshhold value. 0.0018 Do you want to correct bleed through from another channel? Yes If yes. Enter the image you want to use for correction. CorrBlue If yes. Enter the bleeding ratio, which the image above will be multiplied to before subtracting. 0.065 Module #23: SubtractBackgroundPelkmans revision - 1 What did you call the image to be corrected? CorrFarRed What do you want to call the corrected image? SubsFarRed Select an a method for calculating the background value. Single Value For the Iterative Threshold method select the number of iterations. 500 Define a correction factor (applies to all threshold methods). 0.99 For the Otsu Global and Iterative Threshold methods select a minimum allowed value. 0 For the Otsu Global and Iterative Threshold methods select a maximum allowed value. 1 For the Single Value method enter the theshhold value. 0.0018 Do you want to correct bleed through from another channel? No If yes. Enter the image you want to use for correction. OrigBlue If yes. Enter the bleeding ratio, which the image above will be multiplied to before subtracting. 0.065 Module #24: MeasureObjectIntensity revision - 2 What did you call the greyscale images you want to measure? CorrBlue What did you call the objects that you want to measure? Nuclei Type "Do not use" in unused boxes. Cells Cytoplasm NuclearPeriphery ShrunkenNuclei Do not use Module #25: MeasureObjectIntensity revision - 2 What did you call the greyscale images you want to measure? CorrGreen What did you call the objects that you want to measure? Nuclei Type "Do not use" in unused boxes. Cells Cytoplasm NuclearPeriphery ShrunkenNuclei Do not use Module #26: MeasureObjectIntensity revision - 2 What did you call the greyscale images you want to measure? CorrRed What did you call the objects that you want to measure? Nuclei Type "Do not use" in unused boxes. Cells Cytoplasm NuclearPeriphery ShrunkenNuclei Do not use Module #27: MeasureObjectIntensity revision - 2 What did you call the greyscale images you want to measure? CorrFarRed What did you call the objects that you want to measure? Nuclei Type "Do not use" in unused boxes. Cells Cytoplasm NuclearPeriphery ShrunkenNuclei Do not use Module #28: MeasureObjectIntensity revision - 2 What did you call the greyscale images you want to measure? SubsBlue What did you call the objects that you want to measure? Nuclei Type "Do not use" in unused boxes. Cells Cytoplasm NuclearPeriphery ShrunkenNuclei Do not use Module #29: MeasureObjectIntensity revision - 2 What did you call the greyscale images you want to measure? SubsGreen What did you call the objects that you want to measure? Nuclei Type "Do not use" in unused boxes. Cells Cytoplasm NuclearPeriphery ShrunkenNuclei Do not use Module #30: MeasureObjectIntensity revision - 2 What did you call the greyscale images you want to measure? SubsRed What did you call the objects that you want to measure? Nuclei Type "Do not use" in unused boxes. Cells Cytoplasm NuclearPeriphery ShrunkenNuclei Do not use Module #31: MeasureObjectIntensity revision - 2 What did you call the greyscale images you want to measure? SubsFarRed What did you call the objects that you want to measure? Nuclei Type "Do not use" in unused boxes. Cells Cytoplasm NuclearPeriphery ShrunkenNuclei Do not use Module #32: MeasureTexture revision - 2 What did you call the greyscale images you want to measure? CorrBlue What did you call the objects that you want to measure? Nuclei Cells Cytoplasm NuclearPeriphery Do not use Do not use What is the scale of texture? 2 Module #33: MeasureTexture revision - 2 What did you call the greyscale images you want to measure? CorrBlue What did you call the objects that you want to measure? Nuclei Cells Cytoplasm NuclearPeriphery Do not use Do not use What is the scale of texture? 3 Module #34: MeasureTexture revision - 2 What did you call the greyscale images you want to measure? CorrRed What did you call the objects that you want to measure? Nuclei Cells Cytoplasm NuclearPeriphery Do not use Do not use What is the scale of texture? 3 Module #35: MeasureTexture revision - 2 What did you call the greyscale images you want to measure? CorrFarRed What did you call the objects that you want to measure? Nuclei Cells Cytoplasm NuclearPeriphery Do not use Do not use What is the scale of texture? 3 Module #36: MeasureTexture revision - 2 What did you call the greyscale images you want to measure? CorrGreen What did you call the objects that you want to measure? Nuclei Cells Cytoplasm NuclearPeriphery Do not use Do not use What is the scale of texture? 3 Module #37: MeasureTexture revision - 2 What did you call the greyscale images you want to measure? CorrGreen What did you call the objects that you want to measure? Nuclei Cells Cytoplasm NuclearPeriphery Do not use Do not use What is the scale of texture? 2 Module #38: MeasureTexture revision - 2 What did you call the greyscale images you want to measure? SubsGreen What did you call the objects that you want to measure? Nuclei Cells Cytoplasm NuclearPeriphery Do not use Do not use What is the scale of texture? 1 Module #39: MeasureTexture revision - 2 What did you call the greyscale images you want to measure? SubsGreen What did you call the objects that you want to measure? Nuclei Cells Cytoplasm NuclearPeriphery Do not use Do not use What is the scale of texture? 2 Module #40: MeasureTexture revision - 2 What did you call the greyscale images you want to measure? SubsGreen What did you call the objects that you want to measure? Nuclei Cells Cytoplasm NuclearPeriphery Do not use Do not use What is the scale of texture? 3 Module #41: MeasureTexture revision - 2 What did you call the greyscale images you want to measure? SubsGreen What did you call the objects that you want to measure? Nuclei Cells Cytoplasm NuclearPeriphery Do not use Do not use What is the scale of texture? 4 Module #42: MeasureTexture revision - 2 What did you call the greyscale images you want to measure? SubsGreen What did you call the objects that you want to measure? Nuclei Cells Cytoplasm NuclearPeriphery Do not use Do not use What is the scale of texture? 5 Module #43: MeasureTexture revision - 2 What did you call the greyscale images you want to measure? SubsGreen What did you call the objects that you want to measure? Nuclei Cells Cytoplasm NuclearPeriphery Do not use Do not use What is the scale of texture? 6 Module #44: 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? 5 What prefix should be used to name the batch files? Batch_ 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 (.) . 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 (.) . Note: This module must be the last one in the analysis pipeline. n/a