Method Article

EasyFiji: A Graphical Interface for User-Friendly Fluorescence Image Processing in Fiji

DOI:

10.3791/69441

⸱

February 20th, 2026

 , 
1Center for Bioimage Informatics, St. Jude Children's Research Hospital, 2Cell and Tissue Imaging Center, St. Jude Children's Research Hospital

In This Article

Summary

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EasyFiji is a graphical user interface plugin for Fiji (ImageJ) that provides a curated suite of fluorescence image visualization and processing tools frequently utilized by life scientists.

Abstract

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Fiji (Fiji Is Just ImageJ) is an extensive and extensible open-source image processing package widely employed by the bioimage analysis community. However, for non-computational life scientists, manually interacting with Fiji's many capabilities requires learning and navigating a deeply layered menu system. Moreover, some commands' default behaviors are non-ideal for fluorescence image display and processing. To increase efficiency for life scientists working with fluorescence microscopy images, we have developed EasyFiji, a curated graphical user interface (GUI) plugin for Fiji. Each EasyFiji command is executed via tooltip-enhanced buttons and sliders and always exhibits channel-specific execution, as required for fluorescence images. Commands that alter pixel intensities are also single-click undoable to enable more interactive processing and can be automatically recorded and saved as text for record keeping. An image info panel displays settings crucial for image interpretation. New image rendering and bleach correction functions are also provided. The plugin is freely available on GitHub and as a Fiji Update Site. This protocol outlines the steps to use EasyFiji's interface for visualization and processing of fluorescence microscopy images.

Introduction

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Fiji (Fiji Is Just ImageJ) is an extensive and extensible open-source image processing package widely employed by the bioimage analysis community1,2. Fiji's large code base of general-purpose algorithms, along with its macro language and plugin interface, can be conveniently leveraged by computational analysts to provide a solution for almost any image processing or analysis problem. However, for life science users with little computational experience, FIJI's >1,100 commands spread across a deeply layered dropdown menu system can be dauntingly complex to navigate and utilize effectively. Several ap....

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Protocol

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This protocol describes how to install and use EasyFiji.

1. To install EasyFiji…

  1. Download the latest version of Fiji (>1.54g) appropriate for the operating system and install it in a folder to which the user has read/write access (see the Table of Materials for a link to the Fiji download site).
  2. Launch Fiji and navigate to Help | Update… in the menu bar.
  3. In the Updater window, click Manage Update sites. Select EasyFiji from the list, and click Apply and Close. EasyFiji will be....

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Results

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Pseudo-colored renderings of multi-channel fluorescence images can be used to illustrate the spatial or intensity relationships between signals; however, native Fiji offers only an RGB composite rendering technique that inherently confounds coloration and brightness at the perceptual level. To illustrate this issue, Figure 2 uses a two-channel image of N-Myc and RNA Polymerase II (RNAPolII). In Figure 2A, each channel is displayed separately as grayscale so that.......

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Discussion

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Fiji is an open-source image processing and analysis software widely popular amongst image analysts. However, its complex menu interface and at times unintuitive behaviors relative to the display and processing of fluorescence images present challenges for non-computational life scientists. EasyFiji offers life scientists a curated set of thematically organized and tooltip-enhanced buttons and sliders that consistently exhibit channel-specific behaviors, as required when working with fluorescence images. All processing c.......

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Disclosures

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The authors declare that they have no competing financial interests or other conflicts of interest.

Acknowledgements

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We thank members of St. Jude's Cell and Tissue Imaging Center and the Center for BioImage Informatics for comments on the manuscript. Biological images were graciously provided by: Figure 2: Melissa Marzahn and Tanja Mittag; Figure 3: Peng Wei and James Morgan; Figure 4A: Aaron Pitre; Figure 4B: Aaron Pitre and Woo Jung Cho; Figure 5A: Helen Chen and Heather Mefford; Figure 5B: Sauradeep Sinha and Giedre Krenciute.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
EasyFiji GitHub SiteOpen Sourcehttps://github.com/stjude/EasyFiji
EasyFiji Image.sc forumOpen Sourcehttps://forum.image.sc/t/announcing-easyfiji-a-user-friendly-gui-plugin-for-fiji/117617
EasyFiji ImageJ.net siteOpen Sourcehttps://imagej.net/plugins/EasyFiji_plugin#quick-start
FIJI SoftwareOpen Sourcehttps://imagej.net/software/fiji/downloads
VLC Media PlayerOpen Sourcehttps://www.videolan.org/vlc/

References

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  1. Schindelin, J., et al. Fiji: an open-source platform for biological-image analysis. Nat Methods. 9 (7), 676-682 (2012).
  2. Rueden, C. T., et al. ImageJ2: ImageJ for the next generation of scientific image data. BMC Bioinformatics. 18 (1), 5....

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Tags

Fluorescence Image ProcessingFiji PluginGraphical User InterfaceBioimage AnalysisChannel AdjustmentBleach CorrectionMaximum Intensity ProjectionColocalization AnalysisImage MetadataGaussian Blur

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