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Jacqueline Ross, jacqui.ross@auckland.ac.nz

MICROSCOPY NEW ZEALAND INC. CONFERENCE WORKSHOP 2017

Stitching images using Fiji 31 January 2017

Example 1 – Manual stitching with MosaicJ

1. Go to Plugins – Stitching – MosaicJ; 2. The Mosaic grid canvas will open up as below;

3. In the MosaicJ window, go to File – Open and open the first image from each folder, e.g. top001.tif, middle001.tif, bottom001.tif;

4. The images will be loaded into a filmstrip at the bottom of the MosaicJ window. Double-click on each image to add it into the MosaicJ grid area.

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5. Click and drag the images until you have them in approximately the correct positions;

6. Go to Scale – Magnify to display the images at greater resolution;

7. Click on the top image and go to Object – Maximise Contrast;

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8. Now you can move the image around until it is in the correct location. Do the same with the bottom image.

9. If necessary, use the Nudge controls under the Edit menu to make small adjustments;

10. When you are satisfied with the position of the tiles, go to Object – Reset Contrast for each image. 11. Then go to File – Create Mosaic to complete the blending process into a single image;

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Example 2 - Pairwise stitching – automatic stitching of selected images

1. Open the first image in each folder as before. 2. Go to Plugins - Stitching – Pairwise Stitching. Select the first two images and click OK;

3. Then select from the options below. For a transmitted light image, Minimum intensity or Linear Blending is likely to give the best results. The rest of the default settings usually work well so you may not need to change them. Make your selection and click OK.

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Minimum Intensity Linear blending

4. Then repeat the Pairwise Stitching using the stitched image as the First image (reference) and the bottom image as the Second image;

5. Click OK. Select the Fusion method for the stitching. It’s best to use the same method you used for the first pair. You can also provide a name for the Fused mage;

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6. Click OK to create the tiled image;

Example 3 - Gridwise collection stitching

Note that all images must be contained in a single directory.

1. Go to Plugins – Stitching – Grid/Collection stitching. 2. The window below will appear;

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3. If you know how the images have been acquired, e.g. order and direction, then you can select the correct options from the two drop-down boxes shown below;

4. Otherwise, select Unknown position as shown below and click OK.

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5. The window below will appear. Select the Directory where the files are located (Single images for stitching). A configuration file will be created with details of the stitching result (Output Text file name). You can select a name for the file. Choose the Fusion method you want to use and accept the default values and options as below. Note that you can choose to have the tiled image saved directly to disk (drop-down box under Image output).

6. Click OK to start the process. If you selected Confirm files, then a window will appear as below.

7. Click OK to proceed.

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Example 4 - Using a Macro to stitch multiple images from a time-series

1. Open the Macro called Stitch_BMT.ijm;

2. Click Run. The macro will then create a folder called Tiled and fuse all of the image sets. The fused images will be saved into the Tiled folder.