Getting started with Fiji and ImageJ for microscopy

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 Is Just ImageJ”) package. Fiji consists of ImageJ bundled with Fijisome 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 or for one-on-one consultation.More Help contact us

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 version, unless your computer is old.Windows (64-bit) very

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, and .Edit > Options > ImageJ2 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.

Where to install Fiji

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

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 opening the image, use the .)after Channels Tool

Under , choose .Color options 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 to 75% of your physical memory (RAM).Maximum Memory

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

To find out how much physical memory you have:

Mac

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

Windows 7

Click the button, right-click , and then click .Start Computer PropertiesIn the section, look next to .System 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 or to keep the window the same size while zooming.- Option AltTo pan (move to a different part of the image when the image is larger than the window) hold the spacebarwhile dragging.

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

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

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

1st dimension (uppermost slider): keys or keys or mouse horizontal scrolling< > arrow2nd dimension: hold down while doing the aboveControl3rd dimension: hold down or while doing the aboveOption Alt

Color and contrast

A image is a hyperstack with multiple color channels.composite

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

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To change pseudocolor:

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

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 > ). Do NOT click , if you want to keep the original data.Adjust > Brightness/ Contrast Apply

Contrast can be deceptive

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

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Adding a scale bar

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

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Creating a scale bar:Analyze > Tools > Scale Bar.Specify how the scale bar should look and where it should be.Check , which saves the scale bar in a separate layer and does not alter your image data yet.Overlay

Showing and hiding:To remove the scale bar , .temporarily Image > Overlay > Hide OverlayTo bring it back, .Image > Overlay > Show OverlayTo remove the scale bar , .permanently Image > Overlay > Remove overlay

To export a copy of the image with the scale bar permanently "burned" on:Enhance colors and contrast as needed.In the Channels Tool, click . If you see an option for , More >> Convert to RGB Keep Sourcecheck the box.Image > Overlay > Flatten.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:

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

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

Finding commands

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.

Press to open the Command Launcher. Type what you’re looking for to see a list of commands. Select the Lfunction 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 with any questions. We are happy to help you learn how to use ImageJ or any of our other contact ussoftware packages to view and analyze your images.

You can also learn more at the following sites:

Analyzing fluorescence microscopy images with ImageJ by Peter BankheadGetting started, , and other pages on the ImageJ websiteUser guides

Forum and : search for similar issues, or ask the user community for helpmailing list

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