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While acquiring multiple positions in parallel, the microscope stage does not reposition perfectly. This may introduce some small shifts in consecutive frames at a given position. The aim of image registration is to align a series of frames taken from your raw data as it comes from the microscope. Put differently, the problem usually is to register the time points of the loaded raw image data, such that one of the existing channels is used for registration and all other channels get transformed accordingly.
Countless registration modules exist, and we encourage you to use any tool that works well for you. The ImageJ-plugin StackReg by Philippe Thévenaz and Michael Unser turned out to work perfectly fine, except that it cannot register image stacks with several colour channels. Therefore we made the necessary modifications.
We would kindly ask you to give credit to the original authors.
Installation of HyperStackReg
- Download the ImageJ/Fiji plugin here.
- As for Fiji plugins in general, simply copy the downloaded JAR file in the
pluginsfolder inside the Fiji folder. (On Mac systems you can look inside 'Fiji.app' by right clicking an selecting 'Show Package Contents'.
How to use HyperStackReg
- Open your data in Fiji, e.g. by using the menu File > Import > Image Sequence. If a dialog opens, keep default parameters and click ok.
- Afterwards, click the menu Image > HyperStacks > Stack to HyperStack . In the upcoming dialog, enter the number of colour channels and enter 1 for number of slices. Enter the initial number of slices divided by the number of colour channels as number of time points. E.g., for an initial number of 80 slices, this may look like this:
Afterwards, the image sequence should have to separate sliders allowing to scroll through time and channels.
- Navigate to any channel and time point and start the plugin by clicking Fijis Plugins > Registration > HyperStackReg menu entry. The chosen timepoint and channel will be used as initial reference for registration.
- If you have a virtual stack opened, the plugin will ask you for a place on your disk and save all registered slices to this folder.
- The checkbox 'sliding target', which is activated by default, causes the target frame to always be the last source frame. Otherwise all slices will be registered to the same, initially shown slice.
- Registration will only be performed on the channel index of the reference image. All other channels will be transformed according to the computed transformation on the initially selected reference channel.
Disclaimer: we did only test 'HyperStackReg' with 16bit multi-channel images. If you happen to find a bug or see that also other image types work well... please let us know!