MRM Microcoil Performance Calibration and Usage Demonstrated on Medicago truncatula Roots at 22 T

NOTE: This protocol describes procedures for usage and evaluation of coil characteristics of a 1.5 mm inner diameter (ID) solenoid coil (Figure 1). The coil used to demonstrate the protocol is housed in a susceptibility-matched reservoir, but the protocol is equally applicable to unmatched coils. The protocol may be adapted to other sizes and different spectrometer setups.

1. Reference sample preparation

1. To prepare 100 mL of the sensitivity reference solution, dissolve 156.4 mg of CuSO₄ ∙ 5 H₂O into 80 mL of D₂O contained in a 100 mL GL45 flask. The copper sulfate reduces both T₁ and T₂ relaxation time, allowing for quicker measurements, while the D₂O prevents radiation damping and saturation effects. Manually stir until solids are completely dissolved.
   1. Adjust volume to 100 mL using deionized water for a final concentration of 1 g/L CuSO₄ (anhydrous, 6.3 mM). This concentration is sufficient to shorten T₁ and T₂ relaxation but not too high to be affected by precipitation. Seal the reference sample to prevent changing the ratio of H₂O: D₂O.
2. Optionally, connect the probe to a network analyzer, to test if the coil resonates at the desired resonance frequency. Perform an S₁₁ reflectance test to measure the frequency range achieved by tuning and for Q- factor measurements as described in detail by Haase et al.¹⁴. Connect the microcoil to the network analyzer using a co-axial cable. Use a BNC adapter cable if necessary.
   1. Set the center frequency on the network analyzer to the desired resonant frequency, depending on the intended magnetic field strength for which the coil is designed. Next, set the sweep width to 10 MHz. Adjust the variable capacitor on the microcoil assembly, if present, to the fine-tune the reflectance dip to the desired frequency.
   2. Record the reflectance level at the center frequency and the frequency f₁ and f₂ at the -7 dB level. Use these to calculate the Q-factor at the -7 dB level according to Haase et al.¹⁴

2. Sample preparation

1. If preparing a reference sample for coil calibration, transfer 1 mL of CuSO­₄ solution to a watch glass dish under a stereomicroscope.
2. If preparing a biological sample, transfer 1 mL of perfluorodecalin (PFD) into a watch glass under a stereomicroscope, which will be used to submerge the sample. PFD is used as it can fill air spaces in the specimen, without entering biological cells. It is also not observable by proton MRI. Immediately cover the watch glass with a Petri dish lid to prevent evaporative loss, before the PFD is needed.
   NOTE: PFD is highly volatile and a potent long-term greenhouse gas²¹. When its oxygen-dissolving properties and its low viscosity are not required, it may be substituted with Fomblin, a perfluoroether which also gives no observable ¹H signal, but which does not evaporate as quickly¹⁷.
3. Cut capillaries of suitable outer diameter to size, to fit inside the diameter of the microcoil holder (18 mm) and allow for repositioning (Figure 1C). Use a ceramic cutter to make an incision every 10-12 mm and break carefully on the incision point.
4. If preparing a reference sample, use tweezers and the stereomicroscope to bring a pre-cut capillary in contact with the surface of the CuSO₄ solution inside the watch glass, allowing capillary action to fill the capillary.
5. If preparing a biological sample, use tweezers and a stereomicroscope, to bring a pre-cut capillary in contact with the surface of the PFD inside the watch glass, allowing capillary action to fill the capillary fully. Release the capillary into the watch glass so that it becomes fully submerged.
   1. Carefully extract a five week old whole root system from its growth substrate, such as a perlite soil replacement. Clean the root sample meticulously of rhizosheath. Remove large soil particles using tweezers, and if smaller particles are present, remove them by washing the root system with distilled water. Photograph if needed for future reference. Select and excise a small section of fibrous root free of rhizosheath using a scalpel.
   2. For vacuum treatment, place the sample into a 1.5 mL tube containing a suitable fixative solution. Leave the tube cap off, and then seal the tube with parafilm to seal the opening of the tube. Then, punch holes in the film with a sharp tool to allow for ventilation of the tube.
   3. Place the sample tube in a vacuum chamber, seal the chamber, and connect a lab membrane vacuum pump to the chamber. Subject the sample to vacuum treatment for up to 30 minutes, to reduce the presence of air pockets within biological samples. Halt the vacuum treatment when no air bubbles are seen escaping the sample.
   4. While looking through a stereomicroscope, use tweezers to submerge the sample in the infiltration medium prepared previously. Wash the sample of potential debris.
   5. Insert the sample into the capillary using tweezers, while both the capillary and the sample are fully submerged to avoid the inclusion of air bubbles. Use a smaller capillary or syringe needle tip as a pushing rod (Figure 2B).
   6. Take the sample capillary from the medium watch glass, using tweezers. In the case of PFD, cover the Petri dish lid.
6. Shape the tissue paper into a fine point and use it to remove circa 1 mm of liquid from both ends of the capillary.
7. Melt a small volume of capillary wax using a wax pen. Apply wax on both sides. The wax will turn opaque when it solidifies. Take care to exclude air bubbles from the capillary (Figure 2C).
   NOTE: Avoid overheating wax or capillary as this may cause explosive boil off as well as cavitation pockets when the finished sample cools.
8. Afterwards, scrape off excess wax from the exterior of the capillary using a scalpel and wipe clean with fine tissue paper.

3. Mounting the sample

1. Place a microcoil underneath the stereomicroscope and insert the sample using tweezers while keeping the microcoil steady (Figure 2D).
2. Use a rod to center the sample in the microcoil, by sliding the capillary inside the solenoid coil.
3. Optionally, apply adhesive tape to fix the position of the capillary.
4. Inspect the capillary to ensure no air bubbles are visible inside the solenoid coil, to avoid MR signal destruction caused by susceptibility differences.
5. Attach the microcoil to the socket of the probe base, while keeping the microcoil upright (Figure 3A,3B).
6. Carefully slide the triple-axis gradient coils over the microcoil while matching the water-cooling connectors of the gradient to that of the probe base (Figure 3C). Turn the screw thread on the probe base to fix the gradient in place.
   NOTE: This step applies for a Micro5 probe only. In the case of other systems such as Micro2.5 or Biospect, the gradients are on a separate socket than the coil.

4. Determining coil characteristics

1. If the coil is tested for the first time, use the reference sample solution to create a homogeneous sample, which is useful for power calibration and B₁ homogeneity tests. Potential susceptibility problems due to the coil wires may be tested easily with this reference sample.
2. Insert the probe into the magnet and connect the necessary cables: RF transmit/receive cable, water-cooling lines, thermocouple cable and air cooling line.
3. Set the desired water-cooling temperature (recommended 298 K) for the water-cooling unit.
4. Set the target temperature (298 K) and the target gas flow (300 L/h). The gas flow might be different for a different coil design or sample volume. This applies only to systems with a temperature control system.
   NOTE: The next steps are only necessary when testing novel (home-built) coils.
5. Connect the probe using a 50 Ω co-axial cable to a network analyzer with a suitably wide sweep width (400 MHz), centered on the intended resonance frequency.
6. Observe the resonant modes by adjusting the variable matching and tuning capacitors that are present in the probe base.
7. Tune and match the resonant mode to the desired frequency.
8. Optionally, determine the coil quality factor (Q-factor) on the network analyzer. One method to obtain the quality factor is to use a coupling network and dividing the center frequency (f_c) by the width of the reflection dip at -7 dB (i.e., Q = f_c /(f₁ – f₂)¹⁴. Set f_c to the operating frequency of the magnet, while f₁ and f₂ are set to the -7 dB point left and right of f_c, respectively. Some network analyzers have Q-factor determination built-in.
9. Initiate a reflectance test on the scanner, usually called a wobble curve, and adjust the tuning and matching as necessary. It is recommended to set any tuning and matching capacitors to the midpoint of their range for new coils. Therefore, start with a high spectral sweep width. In some cases, it might be more convenient to tune and match the coil outside the magnet on a network analyzer.
10. Select a shim file for the largest volume coil of the imaging probe if it is available. If starting from a coil that has been used previously, use an available shim file. If both options are not available, start with all shim values set to 0.
11. Select the correct coil configuration for the microcoil if it is available in the imaging software (i.e., ParaVision). Otherwise, create a new coil configuration matching the specifications of the coil (e.g., single tuned or double-tuned) according to the manual of the system. Estimations for the safe limits for this solenoid microcoil used in this research with 1.5 mm inner diameter in size is 1 ms at 1 W peak power and 1 mW continuous power.
    CAUTION: The small capacitors (typically 1 mm in size) needed for microcoils are highly sensitive and easily damaged by high voltages. Automated pulse power determination might not function with non-standard coils, and too high powers could cause damage to the coil or other parts of the spectrometer. Therefore, manual adjustments are recommended.
12. Record a nutation curve for a new coil to obtain an indication of the correct RF-power for the coil (Figure 4). In case the safe limits for the coil are unknown, start with 10 µs at a low pulse power of 0.6 W and slowly increase the pulse lengths by 1 µs at a time until the signal appears.
    1. Using an FID-experiment in the absence of gradient encoding, vary the RF-pulse length systematically while keeping the pulse power constant. The ideal pulse length is the pulse length, where the signal intensity reaches the maximum. If testing a new coil, use a 10 µs pulse with a very low power first and start increasing the pulse power gradually.
       NOTE: In case the power is much higher than expected for the combination of coil characteristics and spectrometer, this is already an indication that the wrong resonant mode has been selected.
    2. For a coil with a homogeneous B₁-field, like a solenoid coil, determine the 180° pulse where the signal intensity decreases to zero²².
13. Set the determined 90° pulse power into the adjustment card of the created study. In ParaVision, the reference power adjustment card may be used to enter the hard pulse power.
14. Use a localizer scan with 3 slices, one slice in each of the three primary axes, to locate the position of the coil within the magnet. To do this, load a localizer scan from the default library of the spectrometer. Starting with a large field-of-view with no offset is recommended. Perform an automated receiver gain adjustment and manually start the measurement.
    NOTE: If the sample is exactly in the center of the gradient system, the localizer scan will show the sample. If the coil or sample is not centered in the image slices or missing, the localizer scan needs to be adjusted, in which case step 4.12 needs to be performed again.
15. Alternatively, use a complementary way to find the correct 90° pulse based on image evaluation. Once an approximate pulse power is found using the nutation curve, adjust the pulse powers gradually to check the image for B₁-field homogeneity. For some coils with an inhomogeneous B₁ field, the 90° pulse power determined using the nutation curve may be overestimated, which leads to overtipping in the desired sweet spot of the coil. In this case, reduce the reference pulse power and check the new images against the previous images (Figure 5).
16. Manually shim the magnetic field based on the FID signal. A recommended order for initial shimming is Z-Z²-Z-X-Y-Z-Z²-Z-XY-XZ-YZ-Z. In the case of a solenoid, the main symmetry-axis is in the XY-plane. Therefore, shims in different directions may result in a stronger correction of the B₀ homogeneity for this coil configuration. Higher-order shims have little effect and may be ignored.
17. Calculate a volume-normalized SNR to allow for comparison of microcoil characteristics across different systems, adapted from the manufacturer's protocol¹⁸. For the microcoils used here, we used a spin-echo sequence with the following parameters: field-of-view (FOV) 6 mm x 6 mm, repetition time (TR) 1000 ms, echo time (TE) 7 ms, Matrix 256 x 256 and slice thickness = 0.5 mm. Adjust the slice thickness until the receiver gain is unitary. Next, adjust the number of slices so that slices extend beyond the region of B₁-field homogeneity. Record the images without signal averaging, if possible.
    1. Determine the volume normalized SNR (SNR/mm³) in two steps. First, calculate the voxel volume (V_voxel)(Eq. 1):
          (1)
       NOTE: The units for D_x, D_y and D_slice are in mm. This calculation can likewise be performed for a series of slices.
    2. Select the regions of interest to determine the signal intensity (µ_ROI) of the sample, and the signal intensity (µ_noise­) and standard deviation (σ_noise­) for a region outside the sample (i.e., the noise). The mean signal is taken from the center of the image, while the noise signal is calculated from the corner patches (Figure 6). Either the spectrometer control software or general-purpose image processing software may be used for these calculations. Use a single repetition if possible, to maintain comparability between different coils.
    3. Use the values to calculate a volume normalized SNR (Eq. 2):
          (2)
       For the coil used here in combination with the reference solution, using Eq. 2 results in the following solution:
          (3)
       NOTE: When comparing the SNR of coils at different magnetic field strengths, the relaxation properties of the phantom need to be measured²³, unless a very long repetition time and very short echo time are used.
18. Check for susceptibility problems due to magnetic field inhomogeneities: load and run a multiple gradient-echo (MGE) sequence (Figure 7). Magnetic field inhomogeneities due to susceptibility differences are visible in the images with longer echo times, as the gradient echo does not refocus spins, which dephase due to static field inhomogeneities. This way, inhomogeneities in the sample may be visualized (due to air spaces in the sample), as well as B₀ field inhomogeneities introduced by the coil material. Use the following parameters, to be adjusted depending on the specifications of the spectrometer and coil used: TR 200 ms, TE 3.5 ms with 48 echoes spaced 3.5 ms apart, flip angle 30 degrees. Matrix size 128 x 128.
    NOTE: If multiple (potential) resonant modes or reflection dips were observed in the resonance (wobble) curve, repeat the above steps for each resonant mode to determine the most sensitive one. Depending on the microcoil, different parts of the microcoil assembly may be prone to unintended resonance modes.

5. High-resolution imaging

1. Run a 3D-FLASH experiment with the following parameters: TR 70 ms, TE 2.5 ms, matrix size of 128 x 64 x 64, FOV 1.6 x 0.8 x 0.8 mm, flip angle 30°, and receiver bandwidth 50 kHz.
2. Derive the pulse powers from the reference pulse power determined earlier; this is automatic in most imaging software. Determine the receiver gain using automatic adjustments. Adjust the FOV if necessary, covering the whole object in both phase-encoding directions to avoid aliasing. Run a gradient duty cycle simulation, if available on the system, to verify that the duty cycle of the experiment stays within the specifications of the gradient coils.
   NOTE: These parameters are specific to the coil used for demonstration; it is important to optimize to the local system specifics.

6. Recovering samples for further study or storage

1. Remove the sample capillary from the microcoil.
2. Using tweezers, remove the wax plugs under a stereomicroscope.
3. Use a syringe to wash the sample out of the capillary with a solution of choice. Alternatively, use a glass pusher rod to eject the sample.
4. To prevent dehydration of the sample, store in a suitable medium for storage.