Methods: Registration of Histological Sections to 3D Atlas

Image Acquisition and Preprocessing

[PLACEHOLDER: Describe image acquisition protocol, including microscope/scanner used, magnification, resolution, number of channels, and staining methods employed]

Atlas Selection and Configuration

Registration of histological sections to a reference atlas was performed using the Aligning Big Brains and Atlas (ABBA) plugin (version 0.11.0) [1] running within the Fiji image processing platform [2]. ABBA utilizes BigDataViewer [5] for visualization and BigWarp [6] for visualization of spline transformations. The 'allen_mouse_10um_java' atlas at 10 μm resolution [3] was used as the reference template. [PLACEHOLDER: Specify the anatomical orientation of atlas sectioning - coronal, sagittal, or horizontal]. The atlas orientation was adjusted to better match the sectioning plane of the experimental tissue, with a rotation of 02.468 degrees along the X-axis and 00.091 degrees along the Y-axis. These values were set by DeepSlice. For registration purposes, two atlas channels were utilized: channel 0 (Nissl) and channel 1 (Ara).

Section Registration Workflow

A total of 25 serial sections were registered to the atlas, spanning positions from 02.186 mm to 10.132 mm along the atlas axis. The sections were spaced at varying intervals (see Table 1 for details). All sections were processed identically with the following registration steps:

1. DeepSlice Affine transformation (twice for improved accuracy) [4].
2. Elastix 2D Affine registration with channel mapping (atlas Ch0,1 → section Ch0,1).
3. Elastix 2D Spline registration with channel mapping (atlas Ch0,1 → section Ch0,1).

Post-Processing

Registered sections were exported for subsequent analysis in QuPath [7].

References

[1] ABBA paper: https://doi.org/10.1016/j.celrep.2025.115876
[2] Fiji paper: https://doi.org/10.1038/nmeth.2019
[3] Allen Mouse CCF: https://doi.org/10.1016/j.cell.2020.04.007
[4] DeepSlice paper: https://doi.org/10.1038/s41467-023-41645-4
[5] BigDataViewer paper: https://doi.org/10.1038/nmeth.3392
[6] BigWarp paper: https://doi.org/10.1109/ISBI.2016.7493463
[7] QuPath paper: https://doi.org/10.1038/s41598-017-17204-5
[8] Elastix paper: https://doi.org/10.1109/TMI.2009.2035616

Table 1: Section Positions and Intervals

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