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ImageJ for microscopy
Tony J. Collins
McMaster Biophotonics Facility, McMaster University, Hamilton, ON, Canada
BioTechniques, Vol. 43, No. S1, July 2007, pp. S25–S30
Full Text (PDF)


ImageJ will celebrate its tenth anniversary in September of this year. These past 10 years have seen the Java-based open-source software mature into an invaluable laboratory tool. In addition to its impressive functionality, this cutting-edge image-processing tool has an indispensable support community of enthusiasts on the ImageJ mailing list.

Wayne Rasband is the core author of ImageJ; after developing the Macintosh-based National Institutes of Health (NIH) Image for 10 years, he made the brave decision of starting afresh with ImageJ using the Java programming language. By shifting to Java, Rasband liberated the software from an individual operating system. To run ImageJ, a given system needs only the operating system-specific Java runtime environment. Java runtime environments (JRE) are freely available, either from Sun or bundled with platform-specific installations of ImageJ ( With JRE available for most operating systems, ImageJ is platform-independent, running on Macintosh, Windows, Linux, and even a PDA operating system. The new 64-bit operating systems and their JRE have happily broken the long-held 1.7 Gb memory limit for Java applications. One of the downsides of the Java heritage is an interface that may feel a little unfamiliar. However, a few steps into ImageJ, and this minor inconvenience is forgotten.

While Rasband is the author of the core program, an extensive group of additional developers has written and made available a growing arsenal of short add-on programs to provide additional functionality to the core program. These additional files are either written in Java (the plugins) or in ImageJ's macro programming language (macros). Once saved to the ImageJ plugins folder, these functions are loaded on start-up and can be accessed via menu commands like any other core function.

400+ Plugins

Freely available for individual download, the 400+ plugins contribute to the success of ImageJ, but can also be overwhelming by their sheer magnitude. Where to start? One at a time?

The long list of plugins reflects ImageJ's usage throughout a range of fields in science and engineering; it is used in medical imaging, microscopy, the material sciences, not to mention biological light microscopy. A review of the breadth of ImageJ's role in image processing and analysis was published in July 2004 in BioTechniques. This range of applications is reflected in the plugins available. As such, not all are suited for use in microscopy, and some need to be finessed. Needless to say, collecting and maintaining the add-on files that could benefit a given research program would be prohibitively time-consuming and arduous.

MBF ImageJ

The ImageJ for Microscopy bundle and accompanying manual was developed to manage this wide-ranging array of plugins. Initially collated from the ImageJ home page to help the Laboratory of Molecular Signaling in Babraham Institute (UK), I developed it further at the Wright Cell Imaging Facility (TWRI, Canada); here it was released as WCIF ImageJ. When I recently joined the McMaster Biophotonics Facility (MBF; at McMaster University, Hamilton, Canada, I was encouraged to maintain this ImageJ for Microscopy bundle.

Here the package was resurrected as MBF ImageJ, containing all the plugins that I have found useful. The plugins are organized in submenus, and the bundle is described in an extensively illustrated online manual (which evolved from the original lab instructions). Users of the bundle are encouraged to cite the original authors of the plugins, who have been kind enough to make the results of their work freely available. The online manual provides links to original plugins and authors’ pages. In the following discussion, I describe plugins included in the MBF ImageJ bundle (these are freely available on an individual basis elsewhere).

The bundle comes in two forms. The first is a one-stop solution for Windows users. This includes a setup file that installs all of the required files. The second version is for non-Windows users. Here, the appropriate version of ImageJ from the ImageJ homepage must be installed followed by a download of the plugins only file (which must be unzipped to a user's ImageJ folder). Each of these approaches will match the installed ImageJ to the version described in the online MBF_ImageJ manual.

File Formats

ImageJ supports a wide number of standard image file formats, including the recent implementation 48-bit color composite imageJ support. The ability of ImageJ to open a wide variety of proprietary image formats has long been an important feature. Not only is the image data imported, the extra metadata is typically imported as well. This may include useful information, such as exposure settings and laser powers, but also essential settings such as pixel size, acquisition rate, and z-step—all required for proper interpretation of the data.

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