Name: EasyFiji: A Graphical Interface for User-Friendly Fluorescence Image Processing in Fiji (Video) | JoVE Journal
Uploaded: 2026-02-20T15:00:00.000+00:00
Duration: 10 min 9 s
Description: Learn to use EasyFiji, a user-friendly graphical interface plugin for Fiji (ImageJ) that simplifies fluorescence image processing for life scientists. This method streamlines visualization and analysis workflows.

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We thank members of St. Jude&#39;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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<li>Mutterer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Custom+toolbars+and+mini+applications+with+Action+Bar%5BTitle%5D)+AND+%22Figshare%22%5BJournal%5D)">Custom toolbars and mini applications with Action Bar.</a> Figshare. 10 (m9), (2017).</li>
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<li>Taylor, A. B., Ioannou, M. S., Watanabe, T., Hahn, K., Chew, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Perceptually+accurate+display+of+two+greyscale+images+as+a+single+colour+image%5BTitle%5D)+AND+%22J+Microsc%22%5BJournal%5D)">Perceptually accurate display of two greyscale images as a single colour image.</a> J Microsc. 268 (1), 73-83 (2017).</li>
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<li>Dunn, K. W., Kamocka, M. M., McDonald, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+practical+guide+to+evaluating+colocalization+in+biological+microscopy%5BTitle%5D)+AND+%22Am+J+Physiol+Cell+Physiol%22%5BJournal%5D)">A practical guide to evaluating colocalization in biological microscopy.</a> Am J Physiol Cell Physiol. 300 (4), C723-C742 (2011).</li>
<li>Miura, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bleach+correction+ImageJ+plugin+for+compensating+the+photobleaching+of+time-lapse+sequences%5BTitle%5D)+AND+%22F1000Res%22%5BJournal%5D)">Bleach correction ImageJ plugin for compensating the photobleaching of time-lapse sequences.</a> F1000Res. 9, 1494(2020).</li>
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The authors declare that they have no competing financial interests or other conflicts of interest.

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...

Fiji (Fiji Is Just ImageJ) is an extensive and extensible open-source image processing package widely employed by the bioimage analysis community1,2. Fiji&#39;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&#39;s &#62;1,100 commands spread across a deeply layered dropdown menu system can be dauntingly complex to navigate and utilize effectively. Several approaches have been taken towards simplifying the manual use of Fiji. Fiji&#39;s search bar is an alternative to menu navigation, providing access to excellent help documentation, but only if the user knows the name of the algorithm they need. Fiji&#39;s toolbar buttons can be customized to provide rapid access to user-defined commands, but this procedure requires writing a macro code and foresight of which commands will be most useful. The ActionBar plugin provides a standalone graphical user interface (GUI) window with customizable and organizable arrays of buttons but again, coding and experience are required to populate the buttons effectively3. None of these solutions satisfy the needs of life scientists with little image processing experience.
Beyond user interface issues, many life scientists working with fluorescence microscopy images require channel-specific display and processing procedures. However, some commonly utilized Fiji commands are not channel-aware by default. For example, users may want to render pseudo-colored channels together in either an equally salient way, or to specifically highlight regions of similar intensity between channels, or to preserve the grayscale contrast of a morphological signal while also displaying fluorescence. In each of these use cases, Fiji&#39;s RGB composite rendering technique obscures the desired information at the perceptual level. When processing multi-channel images, native Fiji image filters (i.e., Process | Filters), either process only the active 2D bit plane or else all bit planes in the image window (i.e., the &#39;stack&#39; as defined by class ImageStack). The first behavior fails to process across the z- or t- dimension for a given channel, while the second behavior is almost invariably improper, since each channel contains a unique staining pattern and signal-to-noise ratio, necessitating the use of channel-specific processing parameters. Native Fiji&#39;s bleach correction commands (Image | Adjust | Bleach Correction) act incorrectly when applied to multi-channel fluorescence images, because again, they correct across the entire ImageStack, where channel data is interleaved, rather than correct across the z-or t-dimension within each channel individually.
To address these issues for life scientists working with fluorescence microscopy images, we have developed EasyFiji, a curated and guided graphical user interface plugin for Fiji. EasyFiji is a curated set of thematically organized commands that enable channel-aware rendering and processing. Four tabulated panels, Display, Process, Save, and Image Info, each contain topically related collections of tool-tip-enhanced buttons and sliders for command execution. Other conveniences include an undo functionality for interactive processing, a simple, plain-text action recorder that can automatically save pixel-modifying actions along with an image, and a formatted display of acquisition settings important for image interpretation. EasyFiji also offers new image rendering and bleach correction tools. EasyFiji does not support quantitative image analysis or batch-processing functions, as these procedures should only be performed with the help of an expert bioimage analyst. Although EasyFiji is concise by design, all native Fiji commands are always available through the native Fiji menu system. We believe that EasyFiji&#39;s audience-targeted design4 will increase the use of Fiji amongst life scientists. Here we present EasyFiji&#39;s design, implementation, and application to fluorescence microscopy images. The plugin is easy to install via a Fiji update site (https://imagej.github.io/list-of-update-sites/ and https://imagej.net/plugins/EasyFiji_plugin), while open-source code can be downloaded via GitHub; the plugin and source code are freely available (https://github.com/stjude/EasyFiji).&#160;

<table><tbody><tr><td>EasyFiji GitHub Site</td><td>Open Source</td><td></td><td>https://github.com/stjude/EasyFiji</td></tr><tr><td>EasyFiji Image.sc forum</td><td>Open Source</td><td></td><td>https://forum.image.sc/t/announcing-easyfiji-a-user-friendly-gui-plugin-for-fiji/117617</td></tr><tr><td>EasyFiji ImageJ.net site</td><td>Open Source</td><td></td><td>https://imagej.net/plugins/EasyFiji_plugin#quick-start</td></tr><tr><td>FIJI Software</td><td>Open Source</td><td></td><td>https://imagej.net/software/fiji/downloads</td></tr><tr><td>VLC Media Player</td><td>Open Source</td><td></td><td>https://www.videolan.org/vlc/</td></tr></tbody></table>

easyfiji: a graphical interface for user-friendly fluorescence image processing in fiji

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

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.

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...

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.

Fiji (Fiji Is Just ImageJ) is an extensive and extensible open-source image processing package widely employed by the bioimage analysis community. ...

If you ever use Fiji, you know how powerful it is and how quickly you can get lost in all these menus and settings. EasyFiji takes the stress out of image processing with simple buttons, sliders, and smart tools that just work. It's Fiji but friendly.The recommended way to install EasyFiji is through the Fiji update site. Just click the help, update, click manage update site and type EasyFiji. Remember to click this and click apply and close.Apply changes, then restart EasyFiji. Advanced users could also choose to download the JAR files from the GitHub webpage. For the color channel adjustment, we can open any multi-channel images, and using the channel color tab, we can choose different lookup tables here.We can choose red, green, blue, extra, or we can choose the black one to disable the specific channel. We can use all channel to show all the channel colors, or we can use each channel to display only a specific channel. For the gain sliders, we can make the images brighter.If the image is too dark, we can make this brighter. And for the offset one, we can set the minimal intensity on the image. So we can adjust all the channels'color or only specific channels.For the channel view, open the image of interest. This one is a multi-channel images. So each individual channel have different intensities and a location.For the Colocalization button, it will create the RGB images by using two channels. It will highlight regions where both channel have similar intensities by displaying in those pixels in yellow. The FFMerge button also combines two channels into a RGB display but in a different way.Here, each channel remains equally visible. This make overlapping regions easier to interpret without losing channel balance. The FGMerge button overlays one or more channels onto a green scale channel.It is useful when combining with green scale images like DIC or EM.channels are preserved by the green scale channel size, overall contrast can give you a much better image quality. Here is another example images using FGMerge. For the stack views, the EasyFiji plugin provides function like maximum intensity projection, orthogonal slice views, and the kymograph.Image processing tab. Open an image, click the record button to start recording command. The smooth function will apply a Gaussian blur, and the denoise function will apply a Median filter, and the sharpen button will apply 2D unsharper mask, and subject background will apply the rolling ball signal, rolling ball algorithm to remove background.And applied look up table will change the, will apply the gain and offset as specified in the display panel. Here we could see, like, after applying this processing command, we could see, like, the image quality gets much better. Here I will show example of using Local Correction button to apply a correction factor so that the medium intensity of each frame becomes local to the value of the fit curve.Local and global correction can be applied sequentially to the same dataset. Here it shows the intensity profile before and after bleach correction. We could see that the signal has been corrected.Here we show different saving options. The save panel is use-case orientated to a non-expert in selecting the correct file format for their use. We can save the images as a raw dataset.It will contain all the image's informations. We can save as JPEG for presentation, or we can save the images as PNJ format. And we can also save the image stack as movies.The image info panel, this panel display vendor-specific acquisition metadata in a human readable way for images acquired thus. After this video, you should have a good understanding of how to use this plugin to visualize and processing biomedical images. Our purpose to develop EasyFiji is to make image analysis easier, faster, and more accessible for life scientists.Whether you are charting contrast, applying filters, or exploring image metadata, EasyFiji is here to make your work with Fiji smoother and more enjoyable.

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Learn to use EasyFiji, a user-friendly graphical interface plugin for Fiji (ImageJ) that simplifies fluorescence image processing for life scientists. This method streamlines visualization and analysis workflows.

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

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