Introduction to CellProfiler

Peter Sobolewski (he/him)

Systems Analyst, Imaging Applications

Research IT

https://cellprofiler.org/home

Plan

• Introduction to CellProfiler
• Overview of basic CellProfiler concepts
  • projects, pipelines, modules
• User Interface (UI) Walk-through
• Hands-on Segmentation Tutorial
• Hands-on Input Modules Tutorial

What is CellProfiler?

CellProfiler is an open-source image analysis program

Key feature:
CellProfiler has a graphical user interface (GUI) for assembling image processing and analysis workflows (called pipelines) using a sequence of modular analysis modules

A bit of background

• project started in 2003 by Anne Carpenter and Thouis Jones at MIT
• currently developed by the Beth Cimini lab at the Broad Institute
• >17000 publications citing CellProfiler
  • Many shared pipelines: https://cellprofiler.org/published-pipelines

Strengths of CellProfiler

• It was designed for batch image analysis using reproducible workflows—without scripting

• It includes advanced algorithms for image (pre)processing, segmentation, etc. as modular modules

• It includes in-app documentation for module parameters plus extensive help menu

• End-to-end workflows: images go in, data tables go out

Limitations

• Limited viewing capabilities:
  • single planes, single channels only
• No annotation tools
• Limited out-of-memory performance
  • lacking support for multiscale (“pyramidal”) images
  • lacking built-in tiling/chunking
• Limited OMERO support
• Problematic OME.TIFF handling

The CellProfiler concept

• Create a project that has an associated image file list of locations and an associated pipeline
• Use the GUI to add a sequence of modules to implement your analysis—these form a pipeline
  • Start with input images and end with measurements in a spreadsheet (.csv) or a MySQL database
• run in batches over large number of images
• analyze measurement data in the downstream tool of your choice (Python, R, Excel, etc.)

CellProfiler projects & pipelines

• projects contain a list of the locations of your image files plus an associated CellProfiler analysis pipeline
  • projects will also contain some cached computed results

• pipelines can be exported, but do not contain image locations—this is ideal for sharing or reuseing workflows

Typical pipeline

• start with setting up Input modules
• Add Image Processing modules to facillitate segmentation
• Add Object Processing modules to segment the image (identify objects) and manipulate objects
• Add Measurement modules to get quantified information you’re interested in
• Finish with Data Tools and File Processing to export data or images

CellProfiler UI

Keys to success

• ensure image inputs are properly set up (NamesAndTypes) and that they and intermediate step outputs have meaningful names
• use Test mode to see the outputs of individual modulues (pipeline steps) one-at-a-time
  • let’s you test parameters on a single image or any number of images (even a randomly selected one)
  • can run the whole pipeline or just individual modules

Getting more help

• Online manual: https://cellprofiler.org/manuals

• Extensive tutorials: https://tutorials.cellprofiler.org/

• Extensive examples: https://cellprofiler.org/examples

• Image.sc forum: https://forum.image.sc/tag/cellprofiler

Segmentation Tutorial

• Ensure you have the tutorial materials downloaded. If not, download the .zip file
• Inside the folder is a PDF walkthrough, if you want to follow along on your own

Import the starting pipeline

• Inside the folder are also two .cppipe files, which define the CellProfiler pipelines
  • segmentation_start.cppipe is our starting point, it handles the initial image importing and set up.
  • Drag-and-drop it onto the Pipeline Modules section (middle left pane) to import it.

Tip

The segmentation_final.cppipe is the final pipeline, which you can use to see the completed analysis pipeline!

Import the images

• Import the images by drag-and-dropping the images_Illum-corrected folder onto the central image list pane
• Click on Apply filters to the file list to filter out non-images

Tip

Double-click an image in the list to open and view it

Completed setup

Input Modules Tutorial

This tutorial will introduce you to Input Modules in CellProfiler: Images, Metadata, NamesAndTypes, and Groups. These modules are crucial for loading and organizing your input data so CellProfiler manages it properly in the analysis pipeline.

Getting started

• Ensure you have the tutorial materials downloaded. We will use a sub of the official CellProfiler Input Modules Tutorial materials. You can download the .zip file.
• There is a blog post walkthrough this tutorial if you want to follow along on your own.