Stitching Images Acquired in Micro-Manager

There are a number of programs available for stitching images acquired in Micro-Manager, or stitching images more generally. For images acquired from Micro-Manager, the simplest option is to use the Grid/Collection Stitching Plugin (Preibisch, Saalfeld, and Tomancak 2009). This is installed by default in the FIJI distribution. The major disadvantage to this program that I am aware of is that it will not stitch images larger than 2 gigapixels. This corresponds to about 400 2048×2048 images. The exact number depends on the overlap between images and the image size.


Acquiring a grid of images in Micro-Manager

The easiest way to do this is by using the Create Grid option in the Multi-Dimensional acquisition GUI. To access it:

  1. Open up Multi-Dimensional Acquisition in Micro-Manager.
  2. Click the Edit position list button.
  3. Click the Create Grid button.
  4. Move to the left edge of your sample and click the left-most set button.
  5. Repeat this procedure for the other three edges of your sample.
  6. Set the overlap (5-20% seems to work well).
  7. Acquire your data (make sure to save in Image stack format).
  8. You may want to use the MultiChannelShading plugin to flat-field correct your images.

Stitching images using the Grid/Collection Stitching plugin in Fiji

  1. The Grid/Collection Stitching Plugin is located in Plugins → Stitching → Grid/Collection Stitching.
  2. In the first dialog box, select Type: “Positions from file” and Order: “Defined by image metadata”
  3. In the second dialog box select the first position in your grid for “Multi series file”.
  4. Do you need to check one of the invert Coordinates boxes?
  • For data acquired on high-speedspeed microscope, check “Invert Y Coordinates”.
  • For data acquired on the spinning disk confocal, check “Invert X Coordinates” and “Invert Y Coordinates”.
  • For data acquired on the CSU-W1 spinning disk confocal, check “Invert X Coordinates”.
  • For data acquired on the light sheet microscope, check “Invert X Coordinates”.
  • For data acquired on the Weill CSU-W1/SoRa, you DO NOT need to invert coordinats
  1. Otherwise, the default options usually work well.
  2. Hit OK, and you should have your stitched image in a few minutes.

This works for single channel and multicolor Z-stack images.

Other Stitching Options

For images bigger than 2 gigapixels, or if this doesn’t work for you for some other reason, a number of other stitching programs have been published. I’ve listed a few here:

  • Microsoft Image Composite Editor (ICE). This was developed for stitching panoramas acquired on digital SLR, but has a motion model (Planar Motion 1) for images acquired by X-Y translation. It has no limit to the size of the image that can be stitched, but cannot stitch 3D images or data other than grayscale or RGB images. For large 2D stitching projects, however, it works well. To use it, you will want to save your images as one image per position. You will want to know the number of images acquired in the X and Y directions. Use “New Structured Panorama” to load your images, specify the order they were acquired in, select “Planar Motion 1” for the camera motion, and it will do the rest.
  • TrakEM2, part of Fiji, is supposed to do stitching. I have not tested it extensively.
  • Terastitcher can stitch 2D and 3D images and has no size limitations for stitching, however it cannot save stitched images larger than 2 gigapixels. I have not tested it extensively. (Bria and Iannello 2012)
  • iStitch, a plugin for Vaa3D. I have not tested it.
  • XuvTools does 3D stitching but not 2D stitching. No size limitations on stitching. I have not tested it. (EMMENLAUER et al. 2009)
  • Photoshop has an image stitching option, as do several other image manipulation programs. I haven’t tried any of these.


Bria, Alessandro, and Giulio Iannello. 2012. TeraStitcher - A tool for fast automatic 3D-stitching of teravoxel-sized microscopy images. BMC Bioinformatics 13, no. 1 (November 27).

EMMENLAUER, M., O. RONNEBERGER, A. PONTI, P. SCHWARB, A. GRIFFA, A. FILIPPI, R. NITSCHKE, W. DRIEVER, and H. BURKHARDT. 2009. XuvTools: free, fast and reliable stitching of large 3D datasets. Journal of Microscopy 233, no. 1 (January): 42–60.

Preibisch, S., S. Saalfeld, and P. Tomancak. 2009. Globally optimal stitching of tiled 3D micrscopic image acquisitions. Bioinformatics 25, no. 11 (April 3): 1463–1465.