Stitching Micro-Manager Images

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. This is installed by default in the Fiji distribution. The major disadvantage to this program is that it will not stitch images larger than 2.14 gigapixels.

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 BFcorrector 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. Depending on the orientation of your camera relative to your stage you may need to select “Invert X Coordinates” or “Invert Y Coordinates”.
5. Otherwise, the default options usually work well.
6. Hit OK, and you should have your stitched image in a few minutes.
7. This works for single channel and multicolor images, and should work for Z-stacks as well, although I haven’t tested it as extensively for Z-stacks.

Information about other stitching options is available on the NIC wiki.