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

This protocol allows for characterization and usage of microcoils for ultra high field magnetic resonance imaging. MRI is a unique and noninvasive tool for exploring physiology, metabolism, and diffusion properties of biological specimen. Using high magnetic field strength and microcoils adapted to a sample of interest, images of up to cellular resolution can be obtained.

In this method, we describe step-by-step how to determine the characteristics of commercial or home built microcoils for imaging applications. We use biological specimens smaller than one millimeter in diameter and an ultra high field vertical bore NMR spectrometer. Using home-built microcoils, we can adjust the size of the radio frequency coil to the size of the sample.

In this research, we're using a small piece of plant root. This is useful because the coil sensitivity is proportional to the decreasing coil diameter. We therefore can obtain images with a higher signal to noise ratio for small samples.

Due to the small size and the fragile nature of these microcoils, it is important to establish some basic parameters such as for instance, the 90 degree pulse length and pulse power, the safe operating limits, and finally to calculate the coil sensitivity in a way that can be compared across different NMR systems. The solenoid microcoil consists of a wire coiled around a capillary and two capacitors:the tuning and the matching capacitor. The tuning capacitor is chosen to achieve the desired resonant frequency of 950 megahertz, while the matching capacitor has chosen to achieve the maximum signal transmission.

It's an impedance of 50 Ohm. The larger capacitor is variable to allow for finer adjustment. The coil sits on a base plate which is fixed to a modified socket.

Optionally, A reservoir for susceptibility matching fluid may be added to reduce susceptibility effects from the coil wire. Into a watch glass transfer one milliliter of perfluorodecalin, or PFD, 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 PFD with a Petri dish lid to prevent evaporation before it is needed. If preparing a reference sample use a copper sulfate solution instead.

Next, carefully extract a root system from its growth substrate. Excise a small section using a scalpel. For vacuum treatment, place the sample in an Eppendorf tube containing a fixative solution.

Then seal the tube with a filament and punch holes to allow for ventilation. Subject the sample to vacuum treatment. Air bubbles may be seen escaping the sample.

While looking through a stereo microscope use tweezers to submerge both sample and capillary in the solution prepared previously. Then insert the sample into the capillary using tweezers while both capillary and sample a fully submerged. Use a smaller capillary or a syringe needle tip as a pushing rod.

Shape tissue paper into a fine point and use it to remove around one millimeter of liquid from both ends of the capillary. 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. Afterwards, scrape off excess wax using a scalpel.

Insert the sample into the microcoil using tweezers, while keeping the microcoil steady. Use a rod to center the sample in the coil. If the coil is tested for the first time, use a copper sulfate reference sample for power calibration and determining the SNR and the B1 field homogeneity.

This protocol is demonstrated on a vertical bore 22.3 Tesla spectrometer equipped with a micro imaging probe with integrated gradient coils, capable of up to three Tesla per meter. Attach the microcoil to the probe base while keeping the microcoil upright. Then slide over the triple axis gradient coil.

Turn the screw thread on the probe base to fix the gradient in place. Insert the probe into the magnet and make the necessary connections. Initiate a wobble curve and adjust tuning and matching as necessary.

Starting with a high spectral sweep width is recommended. Be aware that multiple resonant modes may be present. SNR tests for each mode may be needed to determine the correct resonant mode.

Select the correct coil configuration for your microcoil. In case the safe limits for the coils are unknown, start with 10 microseconds at a low pulse power of 0.6 watt and slowly increase the pulse length by one microsecond at a time until a signal appears. Record a nutation curve for a new coil to obtain the correct pulse length and power for the 90 degree pulse.

In order to do so vary the pulse duration systematically while keeping the pulse power constant. In case of an inhomogeneous B1 field, the 90 degree pulse can be estimated from the lengths at which maximum signal intensity is obtained. Use a localizer scale with large field of view to locate the position of the coil within the magnet.

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 off-center, adjust the localizer scan. Once an approximate pulse power is found using the nutation curve, vary the pulse powers gradually for a series of images to check for the most homogenous image.

For some coils with an inhomogeneous B1 field, the determined 90 degree pulse may be overestimated leading to overtipping in the desired sweet spot of the coil. Manually shim the magnetic field based on the FID signal. Depending on the orientation of the microcoils shims with different orientation may result in a stronger correction of the B0 homogeneity.

Next, a volume normalized SNR must be calculated. First, calculate the voxel volume by multiplying the X and Y resolution times the slice thickness. The SNR is calculated by subtracting the mean noise from the mean signal and dividing it by the standard deviation of the noise multiplied by the voxel volume.

The mean signal is taken from the center of the image, while the noise signal is calculated from the corner patches. Run a multiple gradient echo sequence to check for potential susceptibility problems due to the coil wire and the sample itself. For submerged coils, the difference in susceptibility between the coil wires and their environment is greatly reduced.

High resolution imaging maybe attained with 3D flash experiments. Several root features may be distinguished such as the endodermis, cortex, and xylem, which are difficult to resolve using larger coils. Multislice, multiecho sequences may also be used to reduce the influence of susceptibility.

However, this comes at the cost of reduced sensitivity per unit of time. Root nodules of Medicago truncatula may also be imaged using this protocol. An isotropic resolution of 31 micrometers was obtained in four minutes, while an isotropic resolution of 16 micrometers was obtained in 33 minutes.

This allows various physiological aspects of small root nodules to be studied in detail. For successful sample preparation, it is important to completely submerge both sample and capillary in the liquid. This prevents the formation of air bubbles which negatively affect the image quality.

Because MR imaging is non-destructive, we can remove the biological specimen after the MR scan and use it for further study using for instance, optical microscopy. After watching this video you should have a basic understanding of microcoil characterization and operation for imaging applications. This may be applied to a wide variety of biological specimen.