Animal Brain Collection Project

# 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