TITLE: How to 19F MRI: Supplemental protocol
AUTHORS: Olga Maxouri, z.elkarghali, Mariah Daal, Sajjad Rostami, Ivonne Rodriguez, Leila Akkari, Mangala Srinivas, Rene Bernards, Regina Beets-Tan

[DESCRIPTION]
This protocol is intended as a general guideline for in vivo 19F MRI experiments  on a 7T Bruker system and ParaVision 6.0.1 software. Specific sequence parameters depend on the intended application and the imaging agent used.

[BEFORE_START]
NOTE: Since this is a general 19F MRI protocol, it is assumed the 19F MRI probe has already been administered to the animal.

[STEPS]
SECTION: Preparation of the instruments
1. Check whether the desired RF coil (e.g. a dual-tuned 1H/19F birdcage coil) and animal cradle are connected to the MRI.

SECTION: Preparation of the instruments
2. If inhalation anaesthetics are used, control the tubing towards the induction chamber and the animal cradle. Refill the isoflurane vaporiser if needed.
SECTION: Preparation of the instruments
3. Turn on the circulating warm water blanket system. 
SECTION: Preparation of the instruments
4. Place and secure the respiratory pillow on the cradle, where the thorax of the mouse will be.
SECTION: Preparation of the instruments
5. Place the absorbing tissue on the animal bed. Have the ophthalmic ointment ready.
6. In the control room, turn ON the system and start ParaVision 6.0.1. Select: File > New Study and fill in the required fields. Click “Create” followed by ”Exam”
SECTION: Animal preparation
7. Place the mouse cage in the flow hood. Open the valve of the vaporiser towards the induction box, and set the O2/air flow rate at 1 l/min. Move the mouse from the cage to the anaesthesia induction box. 
SECTION: Animal preparation
8. Set isoflurane at 2-3%. Assess the level of anaesthesia via the pedal reflex. Open the valve to the MRI and close the valve to the induction box.
SECTION: Animal preparation
9. Move the mouse to the animal bed, ensuring adequate contact with the respiratory pillow. Apply ophthalmic ointment and secure the animal with its teeth to the toothbar in the prone position. To monitor the animal's body temperature, insert the anal probe that is covered in the lubricated cover and tape the wire to the bed.
SECTION: Animal preparation
10. For quantitative 19F MRI, place a reference tube with a known concentration of 19F atoms alongside the mouse.
SECTION: Animal preparation
11. Cover the animal with the circulating warm water blanket and secure in place.
SECTION: Animal preparation
12. Place the 1H/19F volume coil over the animal bed. Check for any restriction of cables or tubing.
SECTION: Animal preparation
13.   Wobble adjustment of the RF coil
 
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     
Tune and match the RF coil to the correct resonance frequency and impedance, respectively. Back at the computer, open the adjustment platform on the examination card. Select “Wobble Adjustment” and click “Setup”. Choose the desired coil element (Supplementary figure 1a). The wobble curve should appear at the Acq/Reco display; if not, increase the sweep width. Go to the MRI room. Manually turn the tune/match capacitor rods on the RF coil and monitor the wobble curve on display at the monitor located in the back of the scanner. Adjust until the wobble curve is centred at 0 MHz and the vertex approximates the minimum (Supplementary figure 1b). Press "Stop" to finalize the wobble adjustment.

 
SECTION: Animal preparation
14. Repeat step 13 for the other coil element.
SECTION: Animal preparation
15. Insert the 1H/19F coil with the animal bed into the MRI, and position it at the isocenter of the magnet.
SECTION: Imaging of the animal
16. 
 
In the Palette > Explorer window, select Object (Mouse), Region (Abdomen) and Application (Anatomy). Drag the “1_Localizer” protocol to the scan queue and acquire the scan by pressing continue. Prior to imaging, the scanner system will perform several automated calibration steps.

Adjust the position of the animal or the geometry of the scan and repeat the scan as needed.
SECTION: Imaging of the animal
17. To acquire an anatomical 1H image, drag the desired protocol (e.g. T2_TurboRARE) into the scan queue and adjust the geometry (FOV, slices, slice thickness etc.). Store changes by clicking on "Apply". At the beginning of a study, the system performs iterative shimming, but additional scan shim adjustments may be required depending on the selected protocol. 
SECTION: Imaging of the animal
18. For this coil set-up the reference power calibrated using the 1H channel can be used on 19F (Supplementary figure 2). This value can be found at the setup card or at the adjustment platform. Note this number.

 
SECTION: Imaging of the animal
19. Acquire a non-localized 19F NMR spectrum using the Singlepulse sequence and following the next steps.
SECTION: Imaging of the animal
20. Go to Object (Mouse), Region (Head), Application (Spectroscopy) and drag the Singlepulse protocol to the scan queue. Adjust the flip angle to 90 degrees.
SECTION: Imaging of the animal
21. Change coil operation from 1H to 19F: go to the [System] tab →  Operation mode →  [F19] Tx/Rx Volume. Enter the adjustment platform and from the list select “Reference Power” > “Result”. Change the 19F reference power to the value obtained from the 1H calibration (step 18). Right click on “Reference Power” on the list and select “Save Adjustment Result” to use this value with different scans during a single study.
SECTION: Imaging of the animal
22. Go to “Palette” > “Explorer” > “Datasets”. To load the NMR spectrum, right click the Singlepulse sequence and select “Load in Topspin”.
SECTION: Imaging of the animal
23. Insert the following commands in Topspin sequential order:
“lb”, “ef”, “apk” and “pp”. For the “lb” command use a value of 10.
While the commands above are recommended in the Bruker manual (ParaVision 6.0.1 Operating Manual p.571), readers are also advised that it might be possible to achieve this by using the magnitude spectrum.
Supplementary figure 3 is an example of an acquired 19F NMR spectrum depicting the chemical shift offset of perfluoropolyether (PFPE)

 

SECTION: Imaging of the animal
24. In the adjustment platform go to “Basic frequency”>”Result” and enter the chemical shift offset of your peak of interest e.g. the major peak at -91 ppm. Right click on “basic frequency” to save the adjustment result.
SECTION: Imaging of the animal
25. Set up the 19F MRI protocol (e.g. T2_TurboRARE). Make sure to change coil operation from 1H to 19F. 
SECTION: Imaging of the animal
26. Insert imaging parameters appropriate to the relaxation properties of your 19F agent.
SECTION: Imaging of the animal
27. Copy the slice geometry from the 1H anatomical scan by dragging that protocol on top of the 19F protocol. Adjust matrix size to an attainable 19F resolution and start the scan.
SECTION: Imaging of the animal
28. After imaging export the images in DICOM (or other relevant format) for subsequent image analysis.