1. Sample Preparation

• Brain tissues were fixed in 4% paraformaldehyde (PFA).
• Before MRI scanning, samples were immersed in a 0.2 mM gadolinium-containing 0.1 M phosphate-buffered saline (PBS PH: 7.4) for at least one week.
• During MRI measurements, samples were immersed in Fluorinert (Sumitomo 3M Limited, Tokyo, Japan) to minimize background signal.

2. MRI Scanners and Coil Selection

a. For Small to Medium-sized Brains

• Scanner: 7T MRI scanner (BioSpec 70/16, Bruker Biospin GmbH, Ettlingen, Germany)
• Gradient System: Actively shielded gradients with a maximum strength of 700 mT/m
• Coils:
  • Volume coils: φ 22 mm, 38 mm, 62 mm, and 72 mm (Bruker Biospin GmbH, Ettlingen, Germany)
  • Solenoid coils: φ 16 mm and 30 mm (Takashima Seisakusho, Tokyo, Japan) Coil selection was based on sample size.

b. For Large Brains (e.g., bamboo mouse)

• Scanner: 3T Philips MRI scanner (Ingenia, Philips, Netherlands)
• Coil: 32-channel head coil

3. Imaging Sequences and Parameters

a. 3D Anatomical Imaging (rapid acquisition with relaxation enhancement (RARE) sequence)

• Method: RARE
• Parameters:
  • Echo time (TE): 20 ms
  • Repetition time (TR): 300 ms
  • RARE factor: 4
  • Number of averages: 1–8 (adjusted to maintain signal-to-noise ratio >20)
  • Spatial resolution: 100–200 µm (depending on sample and scanner)

b. 3D DTI

• Method: Spin echo-based DTI
• Parameters:
  • TE: Minimum
  • TR: 200 ms
  • MPG gradient duration (δ): 3.5 ms
  • MPG gradient separation (Δ): 9.5 ms
  • b-value: 2000 s/mm²
  • Diffusion directions: 30
  • Number of 1 b0 images: 5
  • Number of averages: 1
  • Spatial resolution: 100–200 µm