Introduction


ImageJ is a free, open-source scientific image processing application for Windows, MacOS and Linux. It has been continuously developed for over 20 years and has a user base of many thousands of scientists.


We recommend installing ImageJ using the Fiji (“ Fiji Is Just ImageJ”) package. Fiji consists of ImageJ bundled with some essential plugins and a user-friendly installer and updater.


Some of the functions discussed in this guide are included with Fiji but not with plain ImageJ. So in this guide, "ImageJ" means ImageJ within the Fiji package.


This guide will help you use ImageJ/Fiji with microscope images.


For more detailed tutorials, see More Help or contact us for one-on-one consultation.



Installing ImageJ using the Fiji package


Download Fiji. Select the version listed for your operating system (don't use "All Platforms").  Windows users, download the Windows (64-bit) version, unless your computer is very old.


Where to install Fiji

Install Fiji in a user directory (such as your Desktop or Documents folder), not Program Files or Applications. Otherwise, the automatic updater may not work.


Opening images


Usually, you can open an image using either of these methods:


  • Drag-and-drop it directly onto the ImageJ toolbar.
  • File > Open


If your image doesn't open correctly


Fiji has two ways to read commercial image formats (such as ND2 and CZI):  Bio-Formats and SCIFIO.


In most cases, Bio-Formats is recommended for microscope images.


To make sure Bio-Formats is used, Edit > Options > ImageJ2 and uncheck Use SCIFIO when opening files.


Bio-Formats Import Options



Bio-Formats is a plugin, included in the Fiji package, that interprets commercial image formats -- such as those from microscopes.


Not only does it read the image data, it also understands the scale information and lets you view details about the image acquisition. That means you do not have to convert your files into TIFF to use them in ImageJ -- in fact, it's better to keep them in the original format.


To deal with the many possible kinds of images, Bio-Formats displays Import Options when you open a file.


Usually, the default settings are ok. If you have problems, set the options as shown below.


Correcting colors with Bio-Formats


If your channel colors come out wrong, you can fix them when you open the image with Bio-Formats. (To change colors after opening the image, use the Channels Tool.)


Under Color options, choose Color mode: Custom.
Then, set the color scheme for each channel using the Red, Green, Blue component sliders as shown below.


The channels will be in the order they were acquired. Usually, this is in increasing order of wavelength (blue, green, red, far-red, transmitted).


In the example below, the first channel is blue and the last channel is magenta.


To make a channel gray (e.g. for phase-contrast or DIC), set all the sliders to 255.


Increasing memory to work with large files


Large data files (e.g. z stacks, tiled images, time-lapse videos) may cause ImageJ to run out of memory.


To increase the memory that ImageJ can use, Edit > Options > Memory and Threads.


Set Maximum Memory to 75% of your physical memory (RAM).



If you still run out of memory, open the file as a virtual stack by checking the box in Bio-Formats Import Options.


To find out how much physical memory you have:


Mac


Apple menu > About This Mac and look next to Memory.


Windows 7


  1. Click the Start button, right-click Computer, and then click Properties.
  2. In the System section, look next to Installed memory (RAM).


Viewing images


  • To zoom in on the image, mouse over the part of the image you want to blow up, and press the + (plus) key. Use the - (minus) key to zoom out. Hold Option or Alt to keep the window the same size while zooming.
  • To pan (move to a different part of the image when the image is larger than the window) hold the spacebar while dragging.


A stack is a sequence of images displayed in one window with a slider at the bottom. A confocal z series and a time-lapse movie are examples of stacks. Individual images in a stack are called slices.


A hyperstack is a dataset with multiple dimensions – e.g. z and time.


Step through the slices in each dimension by dragging the sliders in the window, or with the keyboard:


  • 1st dimension (uppermost slider): < > keys or arrow keys or mouse horizontal scrolling
  • 2nd dimension: hold down Control while doing the above
  • 3rd dimension: hold down Option or Alt while doing the above



Color and contrast


A composite image is a hyperstack with multiple color channels.


To analyze or adjust one channel at a time, select it with the slider at the bottom of the window.



Working with color using the Channels Tool


Use the Channels Tool (Image > Color > Channels Tool) to create a composite image, merge channels, turn channels on and off, change pseudocolor, or split channels into separate windows.



To change pseudocolor:


  1. Select the channel you would like to change, using the slider at the bottom of the window.
  2. In the Channels Tool, set the display mode to Color.
  3. Click More >> and select the color you want the channel to be.


Enhancing images with Brightness & Contrast


To balance colors or enhance faint features, set select each channel in turn and adjust it using B&C (Image > Adjust > Brightness/ Contrast). Do NOT click Apply, if you want to keep the original data.


Contrast can be deceptive

To compare brightness between images, set identical B&C levels.



Adding a scale bar


  1. Make sure scale information is shown in the image window. If you do not see it, find out the size of a pixel in your image and enter it using Analyze > Set Scale.



  1. Creating a scale bar:
    1. Analyze > Tools > Scale Bar.
    2. Specify how the scale bar should look and where it should be.
    3. Check Overlay, which saves the scale bar in a separate layer and does not alter your image data yet.
  2. Showing and hiding:
    1. To remove the scale bar temporarily, Image > Overlay > Hide Overlay.
    2. To bring it back, Image > Overlay > Show Overlay.
    3. To remove the scale bar permanently, Image > Overlay > Remove overlay.
  3. To export a copy of the image with the scale bar permanently "burned" on:
    1. Enhance colors and contrast as needed.
    2. In the Channels Tool, click More >> Convert to RGB. If you see an option for Keep Source, check the box.
    3. Image > Overlay > Flatten.
    4. Save the flattened image as a TIFF. Don't overwrite your original data!


Exporting to other applications


To convert an image for use in Photoshop or PowerPoint, or to email to someone who doesn't have ImageJ:


  1. Image > Color > Channels Tool and click More >> Convert to RGB.
  2. File > Save As... and select TIFF or whatever format you want.


To compare multiple images, set identical B&C levels before converting.


Data Warning

Converting to RGB changes pixel values. Don’t overwrite your original data, and don't analyze RGB images if they are not your raw data.


Finding commands


Press L to open the Command Launcher. Type what you’re looking for to see a list of commands. Select the function you want and click Run.


Citing ImageJ


Citations help support the scientists who are continually developing this free software.


If you use ImageJ or Fiji for a publication, please cite it using these guidelines .



More help


Please contact us with any questions. We are happy to help you learn how to use ImageJ or any of our other software packages to view and analyze your images.


You can also learn more at the following sites:





This work is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported License. {{