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. Include any preprocessing steps such as background subtraction, normalization, or artifact removal.]

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Atlas Selection and Configuration

Registration of histological sections to a reference atlas was performed using the Aligning Big Brains and Atlas (ABBA) plugin (version 0.10.9-SNAPSHOT) [1], integrated within the Fiji image processing platform [2]. ABBA leverages BigDataViewer [5] for visualization and BigWarp [6] for manual 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 1.393° along the X-axis and 0.888° along the Y-axis. These values were automatically set by DeepSlice [4]. For registration, three atlas channels were available: channel 0 (Nissl), channel 1 (Ara), and channel 2 (Label Borders). Channels 0 and 1 were used for alignment.

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Section Registration Workflow

A total of 97 serial sections were registered to the atlas, spanning positions from 2.055 mm to 10.086 mm along the atlas axis. All sections were spaced at approximately 83.7 μm intervals, with inter-slice distances ranging from 83.641 μm to 83.678 μm.

Each section underwent a multi-step registration process:

1. Initial Alignment: DeepSlice was used for automated positioning and initial affine transformation, correcting for atlas slicing angle.
2. Affine Registration: Elastix [7] was employed for 2D affine registration, mapping atlas channels 0 and 1 to the corresponding section channels.
3. Nonlinear Registration: Elastix was again used for 2D spline registration, mapping atlas channels 0 and 1 to the corresponding section channels. [PLACEHOLDER: Specify the number of control points used for spline registration, if applicable.]

All slices were processed identically using the following steps:

• Step 1: DeepSlice Affine (Atlas // Section)
• Step 2: DeepSlice Affine (Atlas // Section)
• Step 3: Elastix 2D Affine (Atlas Ch[0,1] // Section Ch[0,1])
• Step 4: Elastix 2D Spline (Atlas Ch[0,1] // Section Ch[0,1])

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Post-Processing

Registered sections were exported for subsequent analysis in QuPath [8].

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References

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

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