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What are the best practices and tips for using ImageJ to stitch large and complex microscopy datasets?

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Image stitching is a technique that combines multiple images into a larger and more detailed one. It is especially useful for microscopy, where the field of view is often limited and the resolution is high. In this article, you will learn the advantages of image stitching for microscopy, and some best practices and tips for using ImageJ, a popular and free software for image processing and analysis.

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Ensure proper overlap:

Starting with a 30% overlap in grid mode is key for successful image stitching. It provides a good balance between enough data for seamless merging and manageable file sizes.
Background subtraction:

Before stitching, subtracting background noise using a reference image greatly enhances the quality. This step ensures more uniform image intensity, which is crucial for accurate stitching.

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1 What is ImageJ?

ImageJ is an open-source, Java-based program that can handle a wide range of image formats and operations. It has a graphical user interface, a macro language, and a plugin system that allows users to extend its functionality. ImageJ is widely used in the scientific community, especially in the fields of biology and medicine, for tasks such as segmentation, measurement, filtering, and visualization.

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Erica Erickson, PhD

Materials Science Writer | Bruker Nano Surfaces & Metrology
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ImageJ is also an oft-used resource in Materials Science. Overall, the capabilities and flexibility of ImageJ cannot be overstated. It is an incredibly powerful open-source tool that is free, accessible, and easy to find help for.

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Laureano Gómez

Líder Nacional en Clasificación Arancelaria , Especialista en Bioingeniería , Ciencias de Materiales, Unidades Funcionales ; Consultoría , Asesoría y Capacitación Técnica Especializada para la Industrias.
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It was very helpful to me, IT WAS analyzed IF THE ISQ EXTENSION opens OTHER PROGRAMS LIKE Matlab, Ansys, comsol, Abacus. BUT IT WAS NOT possible. LATER WE WORKED WITH THE ImageJ PROGRAM WHICH is a public domain Java image processing program inspired by NIH Image for Macintosh. THESE FILES WERE MANAGED TO BE EXECUTED BUT IT WAS NOT POSSIBLE TO MANIPULATE THE IMAGES. FINALLY, WE CHOSE TO use THE 3D Slicer SOFTWARE, which is a free and open source software package for image analysis and scientific visualization where it is possible to meet our proposed objectives.

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2 Why use ImageJ for image stitching?

Image stitching can be a difficult task for microscopy images due to their large size, high resolution, complex shapes, and varying contrast and illumination. Fortunately, ImageJ offers several advantages for image stitching. It can process multiple types of images, including 2D, 3D, color, grayscale, and time-lapse. There are also plugins available such as Grid/Collection Stitching, Stitching, and BigStitcher that can help with the process. Additionally, ImageJ can handle images that are too large to fit in memory by using virtual stacks and memory mapping. It also has the ability to align images based on different features like intensity, phase correlation, or landmarks. Moreover, it is capable of correcting for distortions such as rotation, translation, scaling, and perspective.

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Erica Erickson, PhD

Materials Science Writer | Bruker Nano Surfaces & Metrology
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Background subtraction can also be a useful tool for stitching. If the images to be stitched have uneven intensity distribution (typically darker/shadowed around the edges), that can be easily subtracted from all images in ImageJ. Just collect an image with the same conditions on a plain sample (e.g., white balance card, piece of paper, or blank area of the real sample). Then, use batch processing to subtract that image from all of the images to be stitched.

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Yasir Al-Saffar

Quality Engineering Team Leader at Boston Scientific
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Another reason image stitching can be a difficult task for microscopy images is shape and focus distortions at the edges of the image caused by lenses.

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Laureano Gómez

Líder Nacional en Clasificación Arancelaria , Especialista en Bioingeniería , Ciencias de Materiales, Unidades Funcionales ; Consultoría , Asesoría y Capacitación Técnica Especializada para la Industrias.
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As I mentioned, it is a good option and moreover it is open source, it is excellent for making cuts and being able to meet the desired objectives. In my case it was estimating bone breakage.

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3 How to prepare images for image stitching?

Before stitching images, it is important to prepare them properly to ensure the best quality and accuracy. Check the format and metadata of the images, and convert them if necessary. ImageJ supports many formats such as TIFF, JPEG, PNG, DICOM, and FITS, but some formats may lose information or quality during conversion. Additionally, some metadata like pixel size, calibration, and color mode may be essential for image stitching. Cropping the images to remove unwanted areas like borders or artifacts can reduce file size and computation time while enhancing contrast and brightness and reducing noise and blur can make the images more clear and consistent. Lastly, renaming the images in a logical order like row-column or x-y-z can help image stitching plugins recognize the relative position and orientation of the images.

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Erica Erickson, PhD

Materials Science Writer | Bruker Nano Surfaces & Metrology
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Reminder that almost all of this can be done with batch processing! Especially for cropping, this is best done in a batch so that each image has the same dimensions.

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4 How to choose an image stitching plugin?

When selecting an image stitching plugin for ImageJ, there are several factors to consider. First, the type and number of images should be taken into account, as some plugins can only stitch 2D images while others can handle 3D or time-lapse images. Additionally, the method and mode of stitching should be considered, as some plugins offer intensity-based, feature-based, or landmark-based stitching in pairwise, sequential, or grid modes. Finally, the output and options of stitching should be taken into account, as some plugins can output stitched images in different formats (e.g., TIFF, HDF5) and offer options such as fusion, blending, adjustment, or cropping.

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Sourav Patnaik

"There is never enough data". Isn't it?
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For stitching, I have found that images with 30% overlap in grid mode to be a good starting point for confocal and light microscopy images.

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5 How to use an image stitching plugin?

The exact steps to use an image stitching plugin may vary, but generally, you should open the images or folder containing them in ImageJ. Then, select the plugin from the Plugins menu or toolbar, configure the parameters and options, and run the plugin. Depending on the size and number of images, this may take some time. Afterward, check the result and save it if satisfied; if not, adjust the parameters and options and run the plugin again.

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6 How to optimize and troubleshoot image stitching?

Image stitching can sometimes produce unsatisfactory results, such as gaps, overlaps, misalignments, or artifacts. To optimize and troubleshoot image stitching, it's important to use high-quality images with sufficient overlap and contrast to improve feature detection and matching. Additionally, you should use appropriate parameters and options for the plugin, as well as additional tools or plugins to correct or refine the result. Alternative plugins or methods can also be used to compare or combine the results to evaluate, validate, or improve the stitching.

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What are the best practices and tips for using ImageJ to stitch large and complex microscopy datasets?

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1 What is ImageJ?

2 Why use ImageJ for image stitching?

3 How to prepare images for image stitching?

4 How to choose an image stitching plugin?

5 How to use an image stitching plugin?

6 How to optimize and troubleshoot image stitching?

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