Open Source Software for Bone Histomorphometry


Bone histomorphometry is a powerful technique that allows the quantification of bone metabolism at the cellular level by analysing sections of bone stained using specialist techniques to visualise bone cells. Although the technique us very powerful, analysing the images by hand is extremely time consuming and very variable between operators. Several attempts have been made in the past to produce software that will automate bone histomorphometry. However, many of these are now obsolete and based on hardware and software that is no longer available. Very good commercial packages are available; however, they are relatively expensive and may need specific imaging hardware.

The open source image analysis package ImageJ developed at the NIH by Dr. W. Rasband is one of the leading software packages for image analysis, especially in microscopy. ImageJ is written in java, and this allows the software to run on any operating system which supports java, and currently includes Windows, MacOSX and Linux. Although ImageJ is highly functional, its user interface can be confusing for non-expert users. However, the ImageJ core package and the plugins can be used as imaging libraries in standalone programs. We have used this approach to develop java-based programs for semi-automated bone histomorphometry. These programs are open source and free to use. They have been extensively tested under Windows. In principle they should run under any operating system that supports Java, however, this has not been tested. The programs require Java (at least version 7, to be installed on the PC to run. The programs are optimised for multi-threaded operation and will benefit from modern multi-core processors. Memory use is fairly high for the calcein double label analysis, and a minimum of 8GB of RAM is recommended, but for large high resolution images 16GB may be required.

More information about the software

We developed three programs:

  1. TrapHisto for analysing TRAcP stained osteoclasts and bone resorption parameters 
  2. OsteoidHisto for analysing osteoid parameters 
  3. CalceinHisto for analysing calcein double labels and bone formation parameters 

The download page has links for a zip file containing all three programs, a quick start guide and sample images (Link). A demo movie for the TrapHisto program is available as well (although this was made using a pre-release version of the software), and movies for the other two programs are under development. The programs were developed for use in our own labs and are therefore optimised for use with our staining protocols, although other labs have successfully used these programs as well. Please see the sample preparation section for more details.

For further information, please contact Rob van ‘t Hof (