This ImageJ macro, designed for use within Fiji (https://fiji.sc/), automates the stitching of multiple images into a single large image. It's particularly useful for images acquired using a microscope with a tiling function.

Important: This script requires Fiji, which is a distribution of ImageJ that includes many useful plugins, including the "Grid/Collection stitching" plugin used by this script. Plain ImageJ might not have all the required plugins. Download Fiji from https://fiji.sc/.

By using this script, you agree to cite it in any resulting publications, presentations, or other academic work.

If you use this script in your research and publish the results, please cite it as follows:

Wiegand, M. (2025). ImageJ Fiji Auto-Stitcher (Version 2.1) [Computer software]. 
GitHub. https://github.com/wiegandlmu/ImageJ-Fiji-Auto-Stitcher
Zenodo. https://doi.org/10.5281/zenodo.17273821

Recommended citation style for BibTeX:

@software{wiegand2025autostitcher,
  author = {Wiegand, Max},
  title = {ImageJ Fiji Auto-Stitcher},
  year = {2025},
  version = {2.1},
  url = {https://github.com/wiegandlmu/ImageJ-Fiji-Auto-Stitcher},
  note = {Computer software}
}

When you run the script for the first time, you will be asked to acknowledge this citation requirement.

Citing this work allows others to find and utilize this tool and acknowledges the effort put into its development.

• Flexible Filename Handling: Highly adaptable to various file and folder naming conventions through a user-friendly dialog
• Optimized Overlap: Uses a standard 20% overlap value that works well for most microscopy applications
• Optimized for Single Images: Streamlined for stitching individual 2D images (no Z-stacks)
• Sharp Stitching: Uses subpixel accuracy and optimized computation for precise overlap, resulting in sharp stitching results
• RGB Color Preservation: Automatically maintains color information in stitched images
• User-Friendly: Includes a configuration dialog and detailed instructions for users with no programming experience
• Automatic Grid Size Calculation: If needed, the script can calculate an appropriate grid size

• Fiji: Download and install Fiji from https://fiji.sc/

• Storage Format: Save images as TIFF (uncompressed or LZW-compressed)
• Tiling: Use the tiling function of your microscope to capture multiple images for stitching
• Folder Structure: The script expects images to be organized in subfolders within a main directory. Each subfolder should contain images belonging to one stitched image
• File Naming: You can customize the expected file naming in the dialog (see step 2)
• Overlap: The script uses a standard 20% overlap value
  • This works well for most microscopy applications
  • Ensure your microscope is configured to capture images with approximately 15-25% overlap
  • What is overlap? The percentage of the image area that overlaps with the neighboring image
  • Example: With 20% overlap, a 1000×1000 pixel image overlaps 200 pixels with its neighbor
  • If your microscope uses a significantly different overlap (e.g., 10% or 40%), you may need to modify the script (see "Advanced Settings" section)
• No Z-Stacks: This script is designed for single images only, not Z-stacks

1. Download the script:

   • If you are on the GitHub repository page, scroll up to the top
   • Click the green "Code" button
   • Select "Download ZIP"
   • Save the ZIP file to your computer and unzip it

2. Open the script in Fiji:

   • Open Fiji
   • Go to "Plugins" → "Macros" → "Run..."
   • Navigate to the unzipped folder and select the AutoStitch.ijm file
   • Click "Open"

3. Accept Citation Terms:

   • A dialog will appear asking you to acknowledge the citation requirement
   • Click "I Agree" to proceed

4. Configuration Dialog:

   • A dialog box will appear. Fill in the following parameters:
     • Subfolder prefix: The prefix of your subfolder names (e.g., "XY" if your subfolders are named "XY01", "XY02", etc.)
     • Filename pattern: The pattern of your image filenames
       • Use {xy} as a placeholder for the subfolder number
       • Use {i} to represent the sequential image number
       • Example: H2_Image_XY{xy}_0000{i}_CH4 might represent files named H2_Image_XY01_00001_CH4.tif, H2_Image_XY01_00002_CH4.tif, etc.
     • File extension: The extension of your image files (e.g., ".tif")
     • Grid Size: Specify the grid size (e.g., "3x3") or use "0x0" to let the script automatically calculate the grid size
       • Automatic calculation (0x0): The script calculates a square grid based on the number of images
         • Example: 9 images → 3×3 grid, 16 images → 4×4 grid, 10 images → 4×4 grid (with 6 empty positions)
         • ⚠️ Important: Automatic calculation assumes a square or nearly-square grid arrangement
         • If your images are arranged in a rectangular grid (e.g., 2×6), you must enter the grid size manually
       • Manual entry: Recommended if you know your exact grid dimensions (e.g., "2x6", "3x4")
   • Click "OK"

5. Select Main Folder:

   • A new window will appear
   • Navigate to the main folder and select it (the folder containing the subfolders with the images)

6. Let the script do its job

7. Finding the stitched image:

   • The stitched image will be saved as stitched_result.tif inside each processed subfolder
   • The stitched image will also be displayed in Fiji

This usually means the script can't find the subfolders or images. Double-check:

• The parameters entered in the dialog, especially the "Subfolder prefix" and "Filename pattern"
• That your subfolders actually start with the specified prefix
• That your files match the pattern

• The script uses a standard 20% overlap value that works for most applications
• If your microscope uses a significantly different overlap:
  • Check your microscope settings to confirm the actual overlap percentage
  • Modify the tileOverlap variable in the script (see "Advanced Settings" section below)
  • Try values between 15-25%

• This should not happen with version 2.0+
• If it does, the script automatically converts to RGB before saving

• The script uses the "Save computation time (but use more RAM)" option for best results
• If you have limited RAM, you can modify the script to use "Save memory (but be slower)"

If needed, you can directly adjust the script settings.

To make changes to the script:

1. Open the AutoStitch.ijm file in a text editor (e.g., TextEdit on Mac, Notepad++ on Windows)
2. Make the desired changes
3. Save the file
4. Run the modified script in Fiji

Available parameters:

By default, the script uses 20% overlap. To change this:

// Fixed overlap at 20% (standard value for most applications)
tileOverlap = "20";

Change "20" to your desired overlap percentage (e.g., "15" or "25").

These are usually fine and should not be changed unless needed:

• fusionMethod: Method for blending overlapping regions (default: "[Linear Blending]")
• regThreshold: Regression threshold for the stitching algorithm (default: 0.30)
• maxAvgThreshold: Maximum/average displacement threshold (default: 2.50)
• absThreshold: Absolute displacement threshold (default: 3.50)
• computation_parameters: Set to "[Save computation time (but use more RAM)]" for best results
• imageOutput: How the output should be handled (default: "[Fuse and display]")
• outputName: The desired name for the stitched output image (default: "stitched_result.tif")

Find further information on how to adjust the script here: https://imagej.net/plugins/image-stitching

• ✅ Simplified user interface by removing overlap setting from dialog
• ✅ Set standard overlap to 20% (optimal for most microscopy applications)
• ✅ Added advanced settings section in README for custom overlap values
• ✅ Improved citation format with BibTeX support

• ✅ Fixed broken/blurry stitching results by optimizing computation parameters
• ✅ Added subpixel accuracy for sharper tile transitions
• ✅ Fixed black and white output by adding RGB conversion
• ✅ Added automatic overlap computation
• ✅ Improved user interface with flexible configuration dialog
• ✅ Added citation requirement dialog
• ✅ Simplified code and removed unnecessary functions
• ✅ Better console output for debugging

This project is licensed under the MIT License - see the LICENSE file for details.

• This script uses the "Grid/Collection stitching" plugin in Fiji which is based on the publication: Preibisch, S., Saalfeld, S., & Tomancak, P. (2009). Globally optimal stitching of tiled 3D microscopic image acquisitions. Bioinformatics, 25(11), 1463–1465. https://imagej.net/plugins/image-stitching
• Thanks to all contributors of the ImageJ and Fiji community

Remember: If you use this script in your research, please cite it! Your citation helps others discover this tool and supports continued development.