TITLE: MicroCT Scanning of Pig Vagus Nerves 
AUTHORS: Nicole  Thompson, Svetlana Mastitskaya, Enrico Ravagli, Kirill  Aristovich, David Holder

[DESCRIPTION]
This protocol describes the scanner setup, the parameters required and the reconstruction of microCT scans for imaging pig vagus nerves and the fascicles within them.

[STEPS]
SECTION: Nerve sample preparation
1. See protocol Nerve Sample Preparation for MicroCT Scanning Protocol for nerve sample preparation.
Ensure the cranial ends are down and the caudal ends are up (upside down) in the holder. 
SECTION: MicroCT scanner setup and parameters
2. A Nikon XT H 225 microCT scanner (Nikon Metrology, Tring, UK) was used in this protocol. 
SECTION: MicroCT scanner setup and parameters
3. Home the scanning stage. 
SECTION: MicroCT scanner setup and parameters
5. Change and ensure the target is set to molybdenum. 
SECTION: MicroCT scanner setup and parameters
10. Set the scanning parameters of the first scan to the following: 
35 kVp energy, 120 µA current, 7 W power, an exposure of 0.25 fps and 4 seconds, and a resolution with isotropic voxel size of 7 µm. 
SECTION: MicroCT scanner setup and parameters
11. Adjust the focus accordingly for the sharpest image, using the display screen to visualise.
SECTION: MicroCT scanner setup and parameters
4. Condition the X-ray filament at 200 kVp for 10 minutes.
SECTION: MicroCT scanner setup and parameters
6. Place the nerve sample within the machine and secure it to the stage. 
SECTION: MicroCT scanner setup and parameters
12. Create a reference with the following parameters: 
2 images, 64 frames. This should require 8 mins 55 seconds time. 
SECTION: MicroCT scanner setup and parameters
13. In the reconstruction options, do not crop the image bounding box. This can be done afterwards and is better for the stitching process down the line if all scans are not cropped. 
SECTION: MicroCT scanner setup and parameters
9. Start a program scan setup (When scanning a long nerve sample, despite it being cut into 4 cm lengths, at a desired resolution of 7 µm with a smaller field of view, multiple scans are required to cover the length of the nerve samples that have been wrapped around a sponge. Four (overlapping) scans are usually required.)
SECTION: MicroCT scanner setup and parameters
14. Choose optimised projections (around 3186) and save the scanning setup.
SECTION: MicroCT scanner setup and parameters
15. Add another scan to the program - it should replicate the previously setup parameters.
SECTION: MicroCT scanner setup and parameters
16. Shift the Y-axis by 12.74mm (or calculate your level of overlap desired and shift accordinigly) by moving the nerve sample down.
SECTION: MicroCT scanner setup and parameters
7. Move the stage to visualise the bottom of the nerve and ensure, by rotating the sample, that the sample is completely in the field of view at every angle.
SECTION: MicroCT scanner setup and parameters
8. Take note of the Y-axis co-ordinates (this will allow for calculation of the overlap and shift between the multiple scans).
SECTION: MicroCT scanner setup and parameters
17. Use the same reference that was just created and save the scan setup. 
SECTION: MicroCT scanner setup and parameters
18. Do this again for another two scans (or however many are required to visualise the full nerve).
SECTION: MicroCT scanner setup and parameters
19. Save the entire program and start the scan. This will be ~14 hours so it is advised to start it for an overnight scan.
SECTION: Reconstruction
20. Open the xteckt file in CT Pro 3D software (Nikon's software for reconstructing CT data generated by Nikon Metrology, Tring, UK). 
SECTION: Reconstruction
21. Calculate the centre of rotation manually with dual slice selection.
SECTION: Reconstruction
22. Perform a beam hardening correction with a preset of 2 and coefficient of 0.0. 
SECTION: Reconstruction
23. Crop the image if desired but ensure the nerve is within the cropped region throughout the length by toggling between the visualisation windows and plane directions.
SECTION: Reconstruction
24. Reconstruct as 16-bit volumes and triple TIFF 16-bit image stack files allowing for subsequent image analysis and segmentation in various software.