Digital Image Processing and Analysis with ImageJ

Sean R. Gallagher1

1 UVP, LLC, Upland, California
Publication Name:  Current Protocols Essential Laboratory Techniques
Unit Number:  Appendix 3C
DOI:  10.1002/9780470089941.eta03cs9
Online Posting Date:  October, 2014
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ImageJ is a freely available, cross‐platform (e.g., Windows, Mac, Linux) image processing and analysis program developed by the NIH. In addition to being readily available for no cost, ImageJ is supported by a wide range of constantly evolving user‐created functionalities to address a remarkable range of applications, complementing commercial software that typically comes with imaging instruments such as digital gel‐imaging systems or microscopy workstations. New processing/analysis macros and plug‐ins are routinely added to the support site, and are frequently validated via refereed publications. With the continued improvements and growth of fluorescence‐based applications, ImageJ continues to be a mainstay in the laboratory. ImageJ has extensive support materials available online, its base code is regularly updated, and a survey of Medline references indicates that it is one of the most widely used image‐analysis packages available today. © 2014 by John Wiley & Sons, Inc.

Keywords: computer graphics; image enhancement; image interpretation; imaging; microscopy; software; electrophoresis; gel analysis; fluorescence‐based imaging; digital imaging; protein blotting

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Table of Contents

  • Overview and Principles
  • Strategic Planning
  • Protocols
  • Basic Protocol 1: Visualization and Quantitation with ImageJ
  • Alternate Protocol 1: Quantitation of Bands with Area Selection
  • Support Protocol 1: Using Pseudo Colors to Differentiate Two Separate Fluorescent Signals on a Single Protein Blot
  • Understanding Results
  • Troubleshooting
  • Selected Plug‐Ins for ImageJ
  • Figures
  • Tables
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Basic Protocol 1: Visualization and Quantitation with ImageJ

  • Windows‐based computer (note that MacOS X and Linux are also supported)
  • Network and Internet connection (along with any needed passwords)
  • Dual monitors, one as the work area and the other as the marquee for the menu area (alternatively, use a larger monitor >24 in.)
  • Image acquired from digital imaging system such as a gel electrophoresis imager or a digital imaging microscopy workstation
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Literature Cited

Literature Cited
  Burger, W. and Burge, M. 2008. Digital Image Processing: An Algorithmic Introduction Using Java. Springer, New York.
  Burger, W. and Burge, M. 2009a. Principles of Digital Image Processing: Fundamental Techniques. Springer, London.
  Burger, W. and Burge, M. 2009b. Principles of Digital Image Processing: Core Algorithms. Springer, London.
  Collins, T.J. 2007. ImageJ for microscopy. BioTechniques 43:25‐30.
  Ferreira, T.A. and Rasband, W. 2012. The ImageJ User Guide Version 1.46r.‐guide.pdf.
Key References
  Ferreira and Rasband, 2012. See above.
  Recently updated, this is an excellent overview and instruction to the use of ImageJ.
  Pérez, J.M.M. and Pascau, J. 2013. Image Processing with ImageJ. Packt Publishing Ltd.
  Manual available as online book and from gives detail on a full range of 2‐D, 3‐D, and 4‐D applications of image analysis by ImageJ.
Internet Resources
  The main ImageJ site at the NIH, visited over10,000,000 times to date. Contains documentation and links on ImageJ.
  The ImageJ Wiki is a wealth of information in the user community, with HowTos, plug‐ins, video tutorials, etc.
  One of several video tutorials for ImageJ.
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