Stitching Images

Introduction

There are some situations where you need to capture a large field of view but do not want to sacrifice resolution by using a low powered objective. Some microscopes have the ability to automatically create montage images by controlling the stage but if you only have a manual stage there is are two plugins in Fiji to either manually or automatically create a montage from pictures you capture.

Tips for Capturing Your Images

When you capture your images to create a montage make sure they overlap by about 10-15% so the plugin can have some common features to reference. If your sample is something like sparsely dispersed cells you may need to make the overlap higher to remove any potential errors in the final montage.

Manually Creating a Montage

You can assemble a montage manually, like a jigsaw, using the MosaicJ plugin. This works well for small numbers of images but can be very intensity for montages made up of many images.

1. Go to Plugins 🡪 MosaicJ 

2. In the MosaicJ window go to File 🡪 Open Image Sequence… 

3. Go to the folder Demo Images\Widefield\H and E and highlight the file H and E 01.tif and press Open. 

4. The four H and E images should be placed in the bottom of the MosaicJ window. 

5. Click on each image to get it to move to the main part of the window. 

6. Now drag the pieces around and assemble them like a jig saw. When a piece is selected it has a blue outline around it. To nudge a piece hold down the Ctrl key and use the Arrow keys. 

7. When you are happy with the montage go to File 🡪 Create Mosaic 

8. After some processing (progress bar at the top of the window) the completed montage will open in Fiji and MosaicJ will close. 

Automatically Creating a Montage – Brightfield

1. Go to Plugins 🡪 Stitching 🡪 Grid/Collection Stitching 

2. In the next window set Type to Unknown Position and press OK. 

NOTE: If you know the order (number of rows, columns etc.) the images were captured in you can choose the appropriate Type from the list to cut down on processing time.

3. Set the Directory to the Demo Images\Widefield\H and E folder 

4. Leave all the other settings at their default values and press OK. Press OK the window that opens up next.

You can watch the progress of the stitching in the Log window. 

5. Once finished the completed montage will be displayed. 

Automatically Creating a Montage – Fluorescence

Creating merges of fluorescent images works the same way but you will need to merge each of the channels separately and then overlay them at the end.

1. Use the automatic method above to merge the 4 channels of images in the Demo Images\Widefield\Fluorescent Montage folder 

2. Try to use the Merge Channels command to create a composite image. You will receive the following error message. 

3. To get around this error all we need to do is put the images into a stack. Go to Image 🡪 Stacks 🡪 Images to Stack… 

4. In the window that comes up make sure the Method: is set to Copy (Centre) and press OK. 

5. Now that you have a stack it needs to be converted to a composite image so that colours can be easily assigned to each channel. Go to Image 🡪 Colour 🡪 Make Composite. Select Composite as the Display Mode and press OK. 

6. You now have a composite stack that can be coloured and converted as needed.