ImageJ with Bio-Formats plugin opens raw data exactly as they appear in the original software with the benefit of preserving the underlying raw data.

With Hyperstack and Composite selected in the top left and bottom left of the import settings menu, the raw data will open properly. (Split Channels should NOT be checked.)

The Autoscale button is optional. It does not change the raw data but rescales each channel's display linearly.

The version of ImageJ running on my personal computer opens files from Zeiss Zen and Nikon Elements as they displayed originally (when you choose Hyperstack and Composite in the menu).

There are more recent versions of Bio-Formats that work with more formats, but this works for all the Zen and Elements files I've encountered at work.
* ImageJ 1.52
* Java 1.8
* Bio-Formats 5.1.10-DEV

Click here for a webpage primer on working with channels.

There are also ways of changing the colors and contrast uniformly across datasets.

Let's say you have a directory with 37 images that were taken exactly the same way and you want them all output as a montage of each channel with a merged image and a scalebar on each one in a format you could paste into PowerPoint. Instead of processing each one manually, why not use a macro that will process the entire directory for you? We can modify this macro to do automated tasks such as this as you need them.

Or let's say you have all nuclei in one channel stained with dapi (blue) or DRAQ5 (far-red) and you want to know the intensity of histones in the nuclei stained with Alexa488 (green) and in a different channel the protein levels for each nucleus immediately outside the nucleus stained with Cy3 (red). This may be automated.

Furthermore, let's say you need to manually outline different regions and have the counts described above reported separately per region. ImageJ can do this too and label them, such as reporting independently for "White Pulp", "Red Pulp", and "Marginal Zone".

ImageJ installation   (as of June 2018)


Download from

Install bundled with Java 1.8

Do not install in the Programs Files directory or Applications (on Mac) unless you are sure you have permissions to read/write/edit in this folder.

Install in a location where you have unlimited read/write access such as My Documents. More details on installation here:
If you have problems with this on a computer installed by your IT department, you may need to have them update your permissions.

I do not recommend this, but an alternative is to use Fiji which comes with a lot plugins. Get it at

When you run ImageJ the first time, follow the drop down menus to Edit > Options > Memory & Threads and set memory as you need.

When you run ImageJ the first time, follow the drop down menus to Help > Update ImageJ... to get the latest version. Scroll to the bottom of the versions list and choose Daily Build.

If you have problems installing on Mac, please look here.



You need to install the LOCI Bio-Formats so that you may open files saved in different formats. The file is named "bioformats_package.jar" and goes int he plugins folder in the ImageJ folder.

As of 15 April 2015 you may download the most recent version from
As of May 2016 you may find the most recent version at

Find bioformats daily build and download to the plugins folder within the ImageJ folder. (If you installed Fiji, you do not need to do this step; LOCI plugins are included in Fiji.)

If the firewall won't let you download from the open microscopy website, try downloading this version of the BioFormats plugin that works with ImageJ 1.50.



If you have a package of favorite macros, put them in the macros directory and rename the file "StartupMacros.txt". This way, they will automatically load at startup. I do this with the macros at so that a collection of my most used macros are always available.

Other plugins you may find useful are at is another great resource. We are (slowly) building a library of macros here too, ../index.html, but they aren't necessarily user friendly or well documented. Most need customization for individual datasets.




Want to know more about the history of ImageJ and how it is pioneering in the field of image analysis, way ahead of commercial products? Read this article in Nature Methods.