![]() Helpful for unexpected images: Empty wells, images with dramatic artifacts, etc.Set safety limits on automatic threshold to guards against false positives.Multiplication factor applied to threshold.Background, RobustBackground: Good for images in which most of the image is comprised of background.Otsu: Default - Good for readily identifiable foreground / background.Method: Pick the method that provides the best results.What is the best threshold value for dividing the intensity histogram into foreground and background pixels… Frequency Or here? Pixel values.Definition: Division of the image into background and foreground Here?.Many options for thresholding, cut and join methods, etc.Step 3: Cut and join objects to “improve” their shape.Step 2: Identify objects as regions brighter than the threshold.Step 1: Distinguish the foreground from the background by picking a good threshold.Once the images are loaded, how do you find objects of interest?.Can use text matching to define the difference between images in a set All images stained for GFP have the text Channel1- in the name Assign each image a meaningful name name for downstream reference Same for DNA images (Channel2-).Multiple channels at multiple locations. ![]() Multiple channels at one imaging location.Related how? Depending on the imaging device, one file may represent.Loads an “image set” which is a group of related images, in preparation for further processing.Data Tools: Measurement exploration, measurement output.Measurement: Collection of measurements from objects of interest. ![]() Object processing: Identification, modification of objects of interest.Image processing: Often used for pre-processing prior to object identification.File processing: Image input, file output.Clicking on a different module updates the settings view.Settings panel: View and change settings for each module.Usually these should be separate folders Input folder: Contains images to be analyzed Output folder: Contains the output file plus exported data and images.Folder panel: Change default input and output directories.Image Tool (also displayed by clicking on image).The figure window has additional menu options.File panel: Displays files in default image folder Load pipeline by double-clicking on it View images by double-clicking on the filename.Modules executed in order from top to bottom Module help Add or remove modules Change module position.Pipeline panel: Displays modules in pipeline.Determining which measure(s) are most useful to identify interesting samples.Deciding what compartments to identify and how to identify them.For image-based assays, the basic objective is always to.Support data analysis based on individual cells.Philosophy: Measure everything, ask questions later.Goal: Provide powerful image analysis methods with a user-friendly interface.Software available for Windows, Mac and Linux Image Analysis & Quantification Image-centric Data Analysis.CPU: Xeon E5-2643 v2 3.Getting Started with CellProfiler Mark-Anthony Bray, Ph.D Imaging Platform, Broad Institute Cambridge, Massachusetts, USA.“csif-Imaris” (Imaris, Zen blue, Matlab, NVivo).CPU: Xeon Silver 4116 2.1GHz (2 Processors).WS4 aka CSIF-LLS-analysis (remote/in-person)(Microvolution, 3i LLS processing, CellProfiler4, Cellpose2, QuPath, Napari, Imaris).WS3 aka “csif-spcimage”: csif-spcimage(remote/in-person) (Microvolution, Zen black 2.3/zen blue 2.6, Huygens, Matlab, Imaris, CellProfiler4, CellProfiler-Analyst, Trackmate, Cellpose2, QuPath, Napari, NVivo).CPU: Intel Xeon Gold 6244 CPU RAM: 192 GB. ![]()
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