Menu: Atlas
Atlas: align slices to Allen CCF mouse atlas
Select slices in the CCF atlas corresponding to each slice.
This brings up a new interface displaying a slice in the CCF atlas:
Steps to choose CCF slices:
Tilting the axis chooses the orientation at which the brain was sliced. This is set once at the beginning, and changing the tilt after setting slices clears all currently set slices.
Suggested process for choosing tilt:
- First set left/right tilt, then up/down tilt
- For left/right, find a slice with clear asymmetry (e.g. posterior hippocampus)
- For up/down, find slice with clear dorsal/ventral landmarks (e.g. anterior hippocampus and ventral fiber tracts)
- After setting up/down tilt, re-check/fine-tune left/right tilt
Controls for changing slices: the scroll wheel (+shift = faster) moves the atlas slices (in-out of plane), and the histology slice buttons change the histology slice:
Change the tilt of the atlas left/right and up/down to match the slicing orientation
For at least 2 histology slices with clear and unique landmarks, set the atlas slices
Once at least 2 slices are set, the rest can be set automatically with the "Interpolate" button. This assumes equal slice spacing between set slices. If you have non-continuous slices, you can set the start/end of each slice section, then interpolate within sections.
After interpolating, you can always manually set a new slice to fine-tune if necessary. You can also clear single or all slices with the Clear and Clear all buttons.
You can also get a ballpark check of slice accuracy with the Quick Align button, which performs and displays a quick alignment between the currently selected atlas/histology slice:
Once all slices are set, press the Save button. If not all slices are set, this will not allow saving. This will save all atlas slice positions, and also run a first-pass of auto-alignment from atlas slices to histology slices:
Automatic alignment is usually done after choosing atlas slices, but can be run (or re-run) from this menu item.
Automatic alignment is done by estimating an affine transformation from the atlas slice to a grayscale version of the histology image. The two images need to be roughly similar for this to work, so will likely not work on a partial slice, or with a fluorescence image in which most of the anatomical slice features are not visible.
To improve fit: This alignment relies on finding a threshold to detect the slice from the background, which is done using the currently set color limits. If the slice and background are too similar (e.g. a brightfield channel is included), try adjusting the brightness until the slice is more clearly distinct from the background:
Manual alignment is done by selecting corresponding points on the histology and atlas slices. This is done by nonlinear fitting, and overwrites the automatic alignment.
This can be done on any subset of slices. To manually align: select this menu item, then click corresponding points on each slice to adjust the fit:
A minimum of 3 matched points are required. You can select multiple points on a slice at a time, as long as those are clicked in order on the other slice (e.g. you can click 3 points on the histology slice, and then the 3 corresponding points on the atlas slice).