Multi‐Session Roi Registration
Note: This functionality is only available on the branch x/issue/030-create-tutorial
One of the most important things you can do in order to get good matches for rois across multiple sessions is to spend some extra minutes at the microscope before starting a recording session to optimize the x-, y- and z-position of the objective so that the FOV you record is as close as possible to a reference FOV. Have a good average image from the reference session open on the recording PC and use it to optimise the objective position.
Strongly expressing cells or highly active cells can be good landmarks, but small blood vessels can also work well if there is a good amount of background fluorescence. If you have the option to run a small test-scan and create an average image on the microscope, that is very helpful. If there is brain motion and the average image is blurry, consider to also install software so that you can run motion correction before creating an average image.
Lastly, brain tissue is not static, so as time passes from the reference session, some parts of the FOV might not match well with the reference FOV any longer.
Prerequisites: You need a set of motion corrected image stacks from a set of sessions taken at roughly the same FOV. You also need a set of Rois for the reference session.
In this step, a set of rois will be migrated from a reference session (typically the first recording session for a FOV) to successive sessions. Select a set of sessions in the overview table and find the "Migrate Rois to Fovs" method under Processing - Roi Registration. The method will use average images of the motion corrected stacks and align them to each other. The quality of the result will depend on how well the FOV locations are matching. If there are big x-y offsets, or z-displacements this might be a problem.
When the average images have been aligned, the pixel shifts are used to compute the optimal position of the reference rois in each of the successive sessions. A new set of rois are created for each session and saved to a data variable called RoiArrayLongitudinal.
The method will also open the results in the imviewer so that you can visually evaluate if the new roi positions were estimated well.
In this step, it is possible to manually edit rois (add, remove and reshape). Rois that are added or removed in one FOV will automatically be added or removed from other FOVs. Got to the menu, and find "Edit Rois".
This will open the RoiManager plus an FOV switcher window. You can use the switcher to jump between FOVs of different sessions. NB: Change you make will be saved whenever you press to select FOVs.
This step is necessary in order to run the last step. It will create roi thumbnail images for each of the rois across all sessions, and also compute some simple statistics.
This method is still under development!