Introduction to Fiji

Introduction

Fiji is a distribution of the free, open source image analysis package ImageJ, Fiji stands for Fiji Is Just ImageJ. Fiji comes with a large collection of plugins already installed. It also has a built-in auto update system to make sure you always have the current versions of the plugins. Fiji has a biology slant to most of the plugins provided but they can be used for analysis of a range of samples.

Any publication that uses Fiji should cite the original Fiji paper

Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, Preibisch S, Rueden C, Saalfeld S, Schmid B, Tinevez JY, White DJ, Hartenstein V, Eliceiri K, Tomancak P and Cardona A (2012). Fiji: an open-source platform for biological-image analysis. Nat Methods Jun 28;9()7);676-82 (https://doi.org/10.1038/nmeth.2019)

Additional plugins can be added easily through the updater. Note that plugins normally have their own papers that should be referenced when they are used.

Where to get Fiji

Fiji can be downloaded from the Fiji website located here

http://fiji.sc/

This site also contains a comprehensive wiki site of what the various plugins do and some tutorials on doing simple analysis

Configuration

Several aspects of Fiji need to be configured correctly for it to perform optimally.

Memory and Processor Threads

By default, Fiji allocates a maximum amount of system memory to itself. This value can be increased if required. Also, the amount of simultaneous threads that can be ran at the same time (limited by processor cores) can be adjusted.

1. Go to Edit 🡪 Options 🡪 Memory and Threads

2. Set the amount of memory to what your system can handle. Don’t allocate all your system memory to Fiji, usually around 70% is a good amount. Also set the number of threads to the number of processor cores you have, including Hyper Threaded cores. 

There are two other options available to tick. Keep multiple undo buffers allows multi step undo. This is generally not recommended as very few functions in Fiji respond to the undo command. Run garbage collector on status bar click allows freeing up of memory within Fiji by clicking on the bottom of the Fiji bar.

1. Fiji will then tell you that the changes will take effect after you restart and what to do if something goes wrong

NOTE: On 32bit systems the maximum memory cannot be set higher than 1600MB. If you set it higher you will receive a warning. If you choose to ignore the warning you will have to delete the ImageJ.cfg file from the Fiji directory before you are able to restart the program.

Proxy Settings

Depending on where you are connecting to the Internet you may need to have a proxy server configured to be able to access updates etc. If you are unsure about this check with your IT department.

1. Go to Edit 🡪 Options 🡪 Proxy Settings

2. Enter the address and port for your proxy server (in this example proxy.letmeout.edu.au port 8080). Alternatively you can tick the Use system proxy settings box if you have a proxy server configured in your base system already. 

NOTE: This only works with a raw proxy server address, it will not work with auto-configuration proxy scripts. 

Updates

Fiji has the ability to automatically update the installed plugins and core files.

1. Go to Help 🡪 Update

2. The updater will then check what needs to be updated and provide you with a list 

3. Press the Apply Changes button to start downloading the updates. Pressing the advanced button when downloading begins will give you more detail of what is happening 

4. When the downloading is complete Fiji will ask you to restart to apply the changes

Adding an Additional Update Site

There is a large range of plugins and features that can be added to Fiji. A lot of these can be automatically added and kept up to date using the Update Sites function. 

NOTE: The addition plugins are needed to complete all the examples in this manual. The list required is on the next page.

1. In the Update Fiji window from above click the Manage update sites button at the bottom left

2. Select the update sites you want to add by ticking the relevant boxes. To be able to work through this manual and examples you will need to add the following update sites to make sure all the relevant plugins are installed

• 3D ImageJ Suite

• BAR

• BIG-EPFL

• CLIJ (CLIJ, CLIJ2 and assistants)

• CSBDeep

• Ilastik

• ImageScience

• Labkit

• Morphology

• StarDist

3. Once all required sites have been ticked press the close button and any required updates will be added to the list. Press Apply Changes to add these new plugins. 

4. As before fiji will need to be restarted to install the updates

Manually Installing Plugins

Not all plugins are available through the update sites. These plugins have to be downloaded from the internet and manually installed. 

For this example we will install the Nearest Neighbour Distances plugin by Yuxiong Mao as it will be used in analysis later.

The plugin file can be downloaded from https://icme.hpc.msstate.edu/mediawiki/index.php/Nearest_Neighbor_Distances_Calculation_with_ImageJ.html 

There is also a copy in Plugins\Nearest Neighbour Distances folder within the Fiji Manual Resources download.

1. Make sure Fiji is closed and open the folder Fiji is installed in. For Mac users right click (command click) the Fiji icon in your apps folder and choose Show Package Contents

2. Copy the NND_.class file that you have downloaded or from the folder mentioned above into the plugins folder

3. Restart Fiji and there should now be an Nnd entry in the plugins list 

Updating or Changing the Core ImageJ Install

The ImageJ core installed with Fiji is usually a little behind the current release of ImageJ. There are times when you may want to run a newer ImageJ core (for example when certain functionality has been added) before it becomes part of the standard Fiji install.

To update the core ImageJ in Fiji go to Help 🡪 Update ImageJ

In the resulting window it will show what version of ImageJ you are currently using and a drop down with choices to change imageJ to a newer (or older) version. 

NOTE: Updating ImageJ to a newer version could cause conflict issues with some plugins or Fiji components. You will also get a message when running the updater that some versions don’t match or things have been installed manually 

Monitoring and Clearing up Excess Memory Usage

As Fiji is used memory is taken up by Java. Even though you may not have any images open it may be using the bulk of the available resources.

Monitoring Memory

To monitor the currently used memory and the number of opened images go to Plugins 🡪 Tools 🡪 Monitor Memory

This will open the memory monitor that will show the amount of RAM currently being used (top left number), the percentage of the total available RAM that represents (value in brackets) and the number of open images (top right number). 

As more images are opened the available memory will fill up and the number of open images number will increase

When all open images are closed the memory is not always automatically given back to the system. In the example below it can be seen that there are no images open yet still 424MB of memory is being used.

To release the memory back to the system either click on the Memory window or click on the bottom of the main Fiji task bar 

Finding Commands

There are many commands available within Fiji and it can be hard to keep track of where they are all located. To locate a command (assuming you know the name of it) go to Plugins 🡪 Utilities 🡪 Find Commands… 

You can then search for a command and see where it is located in the menu structure or double click the entry to run the command. 

The Search Bar

Recent updates to Fiji have added a search bar at the bottom right of the Fiji toolbar.  You can type a command into this bar and it will display where the command is as well as information from the ImageJ Wiki and forums.