Image Stack Alignment

Introduction

When taking large confocal stacks of unstable specimens it is possible for the sample to move and result in slices that do not align. This can be corrected with a plugin found in Fiji.

It is also possible to apply these methods to serial sections from either histology or transmission electron microscopy. Additionally is can be used to correct drift in a live imaging data set as well.

Auto Alignment of Confocal Data

1. Open Fish – MP Stack.tif from the Demo Images\Confocal\Fish folder 

2. If you play through the stack you will notice, especially between slices 70 and 100, that the images shift to the left of the frame. First duplicate the stack as the plugin will overwrite the original data. 

To correct the alignment go to Plugins 🡪 Registration 🡪 StackReg 

3. In the next window set the Transformation type to Rigid Body and press OK. 

NOTE: There are four selections for the type of transformation.
Translation: Will move planes in X and Y
Rigid Body: Will move planes in X and Y as well as rotate
Scaled Rotation: Same as rigid body but will scale/zoom planes as well
Affine: Same as scaled rotation but can also deform images into trapezoid shapes

1. The stack will process to align. When it is finished play through the stack and notice that the drift that was present before is now gone.

Auto Alignment of Serial Sections

Sometimes it is necessary to align serial sections (either histology or TEM). In this example we will align a TEM stack but the same principals hold true for a histology stack.

1. Open TEM Stack.tif from the Demo image\TEM folder 

2. If you play through the stack you will notice that the slices do not align, but that there are common features between them that could be aligned. First do the same as above, duplicate the stack and use StackReg with Rigid Body transformation.

3. When you play through the stack you will notice that towards the end it wanders off a bit. Try again but this time with the Scaled Rotation transformation selected.

4. The result will be a little better but the data will be a bit distorted as the scaled rotation transformation will change the zoom level of some slices.

5. Try again with the Affine transformation selected. This probably gives the best visual alignment but notice that the last few slices have been heavily warped by the algorithm. So while looking quite good, this data has been totally destroyed.

6. To achieve a better alignment without introducing artefacts it may be necessary to use a different plugin. Duplicate the stack again and go to Plugins 🡪 Registration 🡪 Linear  Stack Alignment with SIFT. This plugin is more computer intensive and will take longer to run but can give much more accurate alignment of a stack. 

7. Leave the settings as default in the window that opens up but change the Expected Transformation method to Translation. Press OK. 

8. The result should be a fairly well aligned stack without any introduced artefacts.

Auto Alignment Bonus Fun

Open Rough Ride.tif from the Demo Images\Alignment Fun folder and try using the Linear STack ALignment with SIFT module to get a smoother ride.