A protocol to study biological tissue at high spatial resolution using ultra-high field magnetic resonance microscopy (MRM) using microcoils is presented. Step-by-step instructions are provided for characterizing the microcoils. Finally, optimization of imaging is demonstrated on plant roots.
This protocol describes a signal-to-noise ratio (SNR) calibration and sample preparation method for solenoidal microcoils combined with biological samples, designed for high-resolution magnetic resonance imaging (MRI), also referred to as MR microscopy (MRM). It may be used at pre-clinical MRI spectrometers, demonstrated on Medicago truncatula root samples. Microcoils increase sensitivity by matching the size of the RF resonator to the size of the sample of interest, thereby enabling higher image resolutions in a given data acquisition time. Due to the relatively simple design, solenoidal microcoils are straightforward and cheap to construct and can be easily adapted to the sample requirements. Systematically, we explain how to calibrate new or home-built microcoils, using a reference solution. The calibration steps include: pulse power determination using a nutation curve; estimation of RF-field homogeneity; and calculating a volume-normalized signal-to-noise ratio (SNR) using standard pulse sequences. Important steps in sample preparation for small biological samples are discussed, as well as possible mitigating factors such as magnetic susceptibility differences. The applications of an optimized solenoid coil are demonstrated by high-resolution (13 x 13 x 13 μm3, 2.2 pL) 3D imaging of a root sample.
Magnetic resonance imaging is a versatile tool to noninvasively image a wide variety of biological specimens, ranging from humans to single cells1,2,3. While MRI-scanners for medical imaging applications typically use magnets with a field strength of 1.5 T to 3 T, single-cell applications are imaged at much higher field strengths1,3,4. The study of specimens at resolutions below a hundred micrometers is referred to as magnetic resonance microscopy (MRM)5. However, MRM suffers from a low signal-to-noise ratio (SNR) compared to other available microscopy or imaging techniques (e.g., optical microscopy or CT). Several approaches can be pursued to optimize SNR6. One approach is to use a higher magnetic field strength, while a complementary approach is to optimize the signal detector for individual samples. For the latter, the dimensions of the detector should be adjusted to match the dimensions of the sample of interest. For small samples that are ≈0.5-2 mm in diameter (e.g., root tissues), microcoils are useful as the SNR is inversely proportional to the coil diameter6,7. Resolutions as high as 7.8 x 7.8 x 15 µm3 have been attained on animal cells using dedicated microcoils8. A variety of microcoil types exist, with planar and solenoid coils most commonly used depending on the application and tissue geometry9. Planar coils have high sensitivity close to their surface, which is useful for applications on thin slices. For example, a method designed specifically for imaging perfused tissue has been described for planar microcoils10. However, planar coils have a high falloff of sensitivity and no well-defined reference pulse power. Solenoid coils, being cylindrical, have a wider area of application and are more favored for thicker samples. Here, we describe the characteristics of the solenoid coil, a protocol to prepare samples for microcoil MRI, as well as the calibration of a solenoid microcoil (Figure 1A).
The solenoid coil consists of a conducting wire coiled, like a corkscrew, around a capillary holding the sample (Figure 1B). Microcoil assemblies can be constructed using only enameled copper wire, an assortment of capacitors, and a suitable base for soldering the components (Figure 1B). The major advantages are the simplicity and low cost, combined with good performance characteristics in terms of SNR per unit volume and B1 field homogeneity. The ease of construction enables fast iteration of coil designs and geometries. The specific requirements of solenoid microcoil design and probe characterization (i.e., the theory of electronics, workbench measurements, and spectrometer measurements for a variety of coil geometries) have been described extensively elsewhere7,11,12,13,14.
A solenoid coil can be built by keeping in mind design rules for the desired dimensions according to the guidelines described elsewhere15,16. In this specific case, a coil was used with an inner diameter of 1.5 mm, made from enameled copper wire, 0.4 mm in diameter, looped around a capillary of 1.5 mm outer diameter. This solenoid is held on a base plate on which a circuit is made, comprised of a tuning capacitor (2.5 pF), a variable matching capacitor (1.5-6 pF) as well as copper connecting wires (Figure 1A, 1C). The tuning capacitor is chosen to achieve the desired resonant frequency of 950 MHz, while the matching capacitor is chosen to achieve the maximum signal transmission at an impedance of 50 Ohm. The larger capacitor is variable to allow for finer adjustment. In regular operation, tuning and matching are performed using capacitors in the probe base. The assembled microcoil needs to be mounted on a probe so that it can be inserted into the magnet. An additional holder may be required, depending on the system. Here we use a 22.3 T magnet combination with a Bruker Console Avance III HD in combination with a Micro5 probe. In this case, we used a modified support insert equipped with the necessary connections to connect to the 1H channel of the probe (Figure 1A).
The susceptibility-matched design of the coil includes a reservoir with perfluorinated liquid to reduce susceptibility mismatches, arising from the copper coil being in close proximity to the sample17. A reservoir was made from a plastic syringe to enclose the coil and filled with fomblin. As the perfluorinated liquid needs to enclose the coil, the available diameter for a sample is reduced to an outer diameter of 1 mm. For ease of sample changing, the sample was prepared in a capillary with an outer diameter of 1 mm and an inner diameter of 700 µm. The necessary tools for sample preparation are shown in Figure 2A.
Basic experimental MR parameters are highly dependent on the hardware of the system used, including gradient system, field strength, and console. Several parameters can be used to describe the system performance, of which 90° pulse length and power, B1-homogeneity and SNR per unit volume (SNR/mm3), are the most practically relevant. SNR/mm3 is useful to compare the performance of different coils on the same system18. While hardware differences across systems may exist, the uniform application of a benchmarking protocol also facilitates the comparison of system performance.
This protocol focuses on calibration and sample preparation. The stepwise characterization of the performance of solenoid microcoils is shown: calibrating the 90° pulse length or power; assessing the RF- field homogeneity; and calculating SNR per unit volume (SNR/mm3). A standardized spin-echo measurement using a phantom is described to facilitate a comparison of coil designs, which allows for the optimization of distinct applications. Phantom and biological specimen sample preparations, specific for microcoils, are described. The protocol may be implemented on any suitable narrow-bore (≤60 mm) vertical magnet equipped with a commercially available microimaging system. For other systems, it can serve as a guideline and can be used with some adjustments.
Biological specimen preparation for MRI measurements is usually not very extensive since the specimen is imaged as intact as possible. However, air spaces in biological tissue can cause image artifacts due to differences in magnetic susceptibility19. The effect increases with increasing magnetic field strength20. Thus, air spaces should be avoided at high field strengths, and this might require the immersion of the sample in a fluid to avoid air around the tissue and the removal of air spaces within the tissue structures. Specifically, when microcoils are employed, excision of the desired sample tissue might be required, followed by submerging it in a suitable fluid. This is followed by insertion of the sample into a pre-cut capillary, and finally sealing the capillary with capillary wax. Using wax as a sealant instead of glue, flame-sealing or alternatives, means that the sample may be easily extracted. This procedure is demonstrated on the root of Medicago truncatula, a small leguminous plant. An advantage of this protocol is the potential for subsequent co-registration of MRI data with optical microscopy, since the sample is not destroyed during the MRI measurement.
The presented protocol is suitable for high spatial resolution in situ measurements, and more elaborate designs could allow for imaging in vivo samples, where challenges related to life support systems would need to be addressed.
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
2. Sample preparation
3. Mounting the sample
4. Determining coil characteristics
5. High-resolution imaging
6. Recovering samples for further study or storage
Coil Characterization
Upon successful tuning and matching of a coil, its performance may be characterized by the coil Q-factor, 90° reference pulse, and SNR/mm3. For the 1.5 mm ID susceptibility-matched solenoid coil demonstrated here, the measured Q-factor(unloaded) was 244, compared to 561 for a 5 mm birdcage coil.
The reference 90° pulse was 12 µs at a power level of 0.6 W; cf. 5 µs at 45 W for a 5 mm birdcage coil (Figure 4 and Figure 5). This equates to an RF pulse field strength (B1), using of 0.53 mT for the microcoil and 1.17 mT for the birdcage coil14 where y is the gyromagnetic ratio, while tau is the pulse duration. Since the pulse power levels (P) differ, coils may be compared in terms of transmit efficiency : 0.69 mT/W1/2 and 0.18 mT/W1/2 for the microcoil and birdcage respectively14. Comparing by a 90° pulse, the microcoil is found to be a factor ≈ 4 times more sensitive than the birdcage coil.
Effect of susceptibility matching
At ultra-high field strengths, sample and coil susceptibility become a dominant factor for image quality, as seen in Figure 7A,7B. Compared to a coil lacking a susceptibility matching fluid reservoir, the signal is retained longer and more homogeneously in a reference sample. However, due to the susceptibility reservoir, the maximum sample dimensions decrease with respect to the coil without the reservoir.
High-resolution imaging
A high resolution of 13 x 13 x 13 μm3 of a Medicago truncatula root specimen was attained in 20 hours and 23 minutes (Figure 8). Starting from the surface of the root, the root cortex is seen, along with some residual water on the outside of the root. Furthermore, the xylem is observed as a dark band enclosing the phloem. Some air pockets are observed as dark spots with complete signal loss.
Symbiotic root nodules of M. truncatula may also be imaged using this protocol (Figure 9). Using a slightly larger unmatched coil (length circa 3500 µm, inner diameter 1500 μm), images with a resolution of up to 16 x 16 x 16 μm3 were obtained in 33 minutes.
Figure 1: A solenoid microcoil. (A) The solenoid coil design consists of wire looped helically, typically wrapped around a capillary. The geometry of the wire, such as its thickness, diameter, number of windings, and wire spacing, influence the coil characteristics. (B) A home-built solenoid microcoil with a reservoir for susceptibility matching fluid (Fomblin). It consists of a 0.4 mm thick coated copper wire wound six times around capillary with an outer diameter of 1500 µm and a coil length of 3500 µm. The coil is submerged in a reservoir which is made from a syringe. Sample capillaries up to an outer diameter of 1000 µm can be inserted. Two capacitors are used, a 1.5 pF capacitor in series with the inductor and a second variable 1.5-6 pF capacitor is placed in parallel to the inductor. All components are soldered to a fiberglass board (yellow). It is mounted on a commercial holder (grey polymer) that is modified to support the reservoir. (C) Solenoid coil design components: 1. solenoid coil, 2. sample capillary, 3. 1.5 pF tuning capacitor, 4. variable matching capacitor, 5. fiberglass base plate, 6. copper wire leads. Please click here to view a larger version of this figure.
Figure 2: Sample preparation under a stereomicroscope. (A) Items needed for the preparation of microcoils. From left to right: 1. CuSO4 reference solution, 2. perfluorodecalin, 3. microcoil, 4. scalpel, 5. positive tension tweezers, 6. tweezers, 7. capillaries outer diameter = 1000 μm, 8. wax pen, 9. capillary wax, 10. nitrile gloves, 11. stereomicroscope, 12. watch glass with Petri dish cover, 13. plant material in growth substrate. Not shown: 2 mL syringe with ø 0.8 x 40 mm needle and fine tissue paper. (B) Close up of sample insertion into a capillary using tweezers, while both are kept submerged. (C) Sealing of the capillary using molten wax. (D) Insertion of the prepared capillary into the microcoil. Please click here to view a larger version of this figure.
Figure 3: The component of a micro-imaging probe. (A) Micro5 probe base, containing all necessary connections for water cooling, heating, temperature sensors, gradient power, RF (co-axial connector visible) and optionally probe identification (PICS). Underneath the probe base are knobs that allow for adjusting the variable tuning and matching capacitors, as well as retaining screws to hold the probe in place inside the spectrometer. (B) The home-built microcoil mounting atop the probe-base. Note the variable capacitors (white ceramic) mounted on the probe-base that allow for tuning and matching. (C) Integrated 3-axial gradient mounted on the probe base with water-cooling receptacles and gold-plated contacts for grounding the gradient. Please click here to view a larger version of this figure.
Figure 4: Nutation curve. A nutation curve is acquired to determine the reference pulse power. The reference pulse power (90° pulse) is defined as the combination of power and pulse length needed to generate a B1 field that flips all available magnetization in the z-direction to the transverse plane. A series of a pulse is recorded in the absence of gradient encoding. With each pulse, either pulse length or pulse power is incremented. Here the pulse power is set to 0.6 W, while the pulse length is incremented by 1 µs each time. The maximum signal intensity indicates the 90° pulse, around 12 µs. The 180° pulse may also be determined in this way using the minimum intensity. Please click here to view a larger version of this figure.
Figure 5: Visual determination of 90° pulse power. Once an approximate reference pulse power has been found using a nutation curve, it may be checked visually by varying the pulse length. Depending on the coil, the B1 field may be more or less sensitive to changes. (A) 11 µs pulse length. (B) 12 µs pulse length, optimal for this coil. (C) 13 µs pulse length. (D) 20 µs pulse length. If the pulse power is set too high, over-tipping may occur, thereby reducing image intensity in the center of the coil (arrowhead). The increased B1 field also increases the range of the coil, as can be observed in the width of the image. Please click here to view a larger version of this figure.
Figure 6: Region of Interest placement. The regions of interest (ROI) for the volume normalized SNR calculation can be seen. The mean sample intensity is taken from an ROI that falls within the reference solution sample. The mean noise and standard deviation are calculated from one or more ROI located in the corners of the image. Please click here to view a larger version of this figure.
Figure 7: RF homogeneity evaluated by gradient echo imaging. A multiple gradient echo (MGE) sequence is used to evaluate RF (B1 -Field) homogeneity using a series of gradient echoes. Basic parameters were: repetition time 200 ms, echo time 3.5 ms with the number of echoes 48, echo spacing 3.5 ms, 64 averages, acquisition time 27 m 18 s, flip angle 30°. Field of view was 5 x 5 mm, matrix 128 x 128, resolution 39 x 39 x 200 µm. (A) Susceptibility-matched coil. The susceptibility matching fluid (Fomblin) surrounding the RF coil reduces susceptibility effects due to the coil wire. Small air bubbles cause loss of signal as the echo time increases. (B) A coil (not susceptibility matched) with equal coil diameter. At longer echo times, increasing artifacts caused by B0 field inhomogeneity are observed. Please click here to view a larger version of this figure.
Figure 8: 3D imaging of a Medicago truncatula root section. (Top) FLASH image. Several features of the root section can be distinguished, including the epidermis (e), cortex (c), phloem (ph) and xylem (xy). Air pockets (a) in the root cause complete signal loss. Basic parameters were as follows: Repetition time 70 ms, echo time 2.5 ms, 256 averages, acquisition time 20 h 23 m. Resolution 13 x 13 x 13 µm3. Matrix size was 128 x 64 x 64 and field of view 1.6 x 0.8 x 0.8 mm. Receiver bandwidth 50 kHz. (Bottom) MSME image. Basic parameters were as follows: Repetition time 500 ms, echo time 5.2 ms, 28 averages, acquisition time 15 h 55 m. Resolution 13 x 13 x 13 µm3. Matrix size was 128 x 64 x 64 and field of view 1.6 x 0.8 x 0.8 mm. Receiver bandwidth 70 kHz. Please click here to view a larger version of this figure.
Figure 9: 3D imaging of a Medicago truncatula root nodule. (Top) Low-resolution image. Basic parameters were as follows: Repetition time 60 ms, echo time 2.3 ms, 4 averages, acquisition time 4 m. Resolution 31 x 31 x 31 µm3. Matrix size was 64 x 32 x 32 and field of view 2 x 1 x 1 mm. Receiver bandwidth 50 kHz. (Bottom) High-resolution image. Basic parameters were as follows: Repetition time 60 ms, echo time 2.3 ms, 8 averages, acquisition time 33 m. Resolution 16 x 16 x 16 µm3. Matrix size was 128 x 64 x 64 and field of view 2 x 1 x 1 mm. Receiver bandwidth 50 kHz. Please click here to view a larger version of this figure.
This protocol is best suited to biological samples, as many materials and geological samples have significantly shorter T2 relaxation times, which cannot be imaged by the sequences used here. Even some biological tissues, which exhibit high sample magnetic susceptibility heterogeneity, can be difficult to image at ultra-high field as the effects are correlated to the field strength24. The protocol is not only useful for new coils but may also aid in troubleshooting and diagnosis of potential problems. When testing new or unknown samples, this protocol can be performed beforehand on the reference solution to verify that the experimental setup is functioning according to specifications. This aids in troubleshooting since the spectrometer can be excluded as a source of artifacts and malfunctions. In addition, this sets the tuning and matching capacitors on the probe to values typical for the microcoil.
When no signal is recorded upon the first experiment, the field of view of the localizer scan can be enlarged to check if the sample is seen. Next, recheck if the coil is tuned correctly and attempt another localizer scan. It is possible that the coil exhibits additional unintended resonant modes, in which case the correct one needs to be determined. If still no image can be obtained, remove the sample to check its position within the microcoil assembly and verify that the sample is intact (i.e., no air bubbles or leaks in the seals are present). Lastly, a sample may be prepared with water instead of PFD. In case the sample gives little detectable signal in the localizer scan, the surrounding water in the capillary can still be detected.
As microcoils are ideally very close to the sample, the magnetic susceptibility differences between the air and the wire can cause additional signal loss, as seen in Figure 7B. Potential artifacts include spatial mismapping and anomalous signal intensity variation. Especially gradient-echo type pulse sequences are affected by this non-uniform signal loss. For this reason, we presented a susceptibility-matched coil, by submerging the wire in fluorinert liquid (Fomblin or FC-43). The B1 estimation method included in this protocol can help determine whether the B1 susceptibility differences warrant the inclusion of susceptibility matching strategies in the design of the coil assembly. An alternative approach for constructing a susceptibility matched coil is to use susceptibility-matched wire25. Furthermore, only susceptibility issues due to the coil are addressed with this approach. Susceptibility mismatches inside the sample (e.g., due to air spaces) remain challenging.
Air pockets or bubbles pose an experimental challenge that causes extensive signal loss, caused by susceptibility differences at the interface of the air and the fluid or specimen19 (Figure 5A). A critical aspect of successful sample preparation is the submersion of both sample and capillary. However, even small bubbles can cause signal losses, especially for gradient echo type sequences. Mobile air bubbles can migrate through the capillary until they are in contact with the sample. Some of these effects can be alleviated by slightly tilting the capillary so that one end is higher than the other. Tilting ensures potential air bubbles are held in place at the higher end, without disturbing the sample. It is also important to check that the capillary wax forms a good seal, as dehydration can cause large air bubbles to form.
For the air spaces inside the sample, PFD was used to fill up the intercellular air spaces while not penetrating the cell membranes26. However, even with this approach, we were not able to remove all air spaces. Additionally, this approach means that we need an additional agent, which is usually not preferred due to the desire to study a system as noninvasively as possible.
The cylindrical shape of capillaries means that perfusion setups should be viable, especially for tissues vulnerable to decay, such as biopsies or studying processes in living root material. Two steps could realize a perfusion setup. First, connecting a medium feed tube as well as a drain tube at either side of the capillary would be sufficient to create a chemostat. Second, the addition of an indentation in the sample capillary could hold the sample in place against the direction of flow. This is analogous to a protocol published for planar microcoils10.
The noninvasive nature of MR imaging, combined with the inert liquid used in this protocol (PFD or Fomblin) means after completion of experiments, samples may be removed from their capillaries for further study. Combinations include optical or electron microscopy and other destructive imaging techniques. We have recently demonstrated a combination with optical microscopy on Medicago truncatula root nodules27.
We have demonstrated a method for imaging plant material using dedicated microcoils on an ultra-high field NMR spectrometer. Relatively large sample volumes can be studied at high resolution with good RF homogeneity. Furthermore, spectroscopic imaging can be performed at higher resolutions than otherwise feasible. Adapting microcoil design to samples is facilitated by an efficient method to determine coil performance characteristics. The solenoid coil approach may also be readily applied to other samples than plants, including animal tissue.
The authors have nothing to disclose.
Experiments at the 950 MHz instrument were supported by uNMR-NL, an NWO-funded National Roadmap Large-Scale Facility of the Netherlands (project 184.032.207). R.S. was supported by the BioSolarCells consortium project U2.3. J.R.K. was supported by the Netherlands' Magnetic Resonance Research School (NMARRS) graduate school [022.005.029]. We thank Defeng Shen and Ton Bisseling for providing the Medicago truncatula samples. We further thank Klaartje Houben, Marie Renault, and Johan van der Zwan for technical support at the uNMR-NL facility. We would also like to thank Volker Lehmann, Henny Janssen, and Pieter de Waard for technical help. We express our gratitude to Frank Vergeldt, John Philippi, and Karthick B. Sai Sankar Gupta for their advice. Lastly, we thank Jessica de Ruiter for providing the voice-over to the video.
Reference solution preparation | |||
CuSO4 | Sigma-aldrich | 469130 | Crystalline powder for creating reference solution |
D2O | Sigma-aldrich | 151882 | Liquid used to prepare reference sample |
Weigh Scale | Sartorius | PRACTUM513-1S | Scale for weighing compounds |
Sample preparation | |||
Capillary 1000 μm (Outer diameter) | Hilbenberg GmbH | 1408410 | Sample capillaries |
Capillary wax | Hampton Research | HR4-328 | Solid wax used to seal samples |
Disposable Scalpel | Swann-Morton | No. 11 | Used to excise samples |
Perfluorodecalin | Sigma-aldrich | P9900 | Liquid used for submerging sample |
Stereo Microscope | Olympus | SZ40 | Tabletop binocular microscope |
Syringe | Generic | – | Used to apply PFD and manipulate the sample |
Vacuum Pump | Vacuubrand | MZ2C | Two-stage membrane vacuumpump used for removing air pockets from samples. |
Wax pen | Hampton Research | HR4-342 | Handheld wax pen used to melt and apply capillary wax to samples |
Imaging Hardware | |||
22.3 T Magnet | Bruker GmbH | 950 US2 | Narrowbore superconducting magnet |
Air cooler | Bruker GmbH | – | Used to regulate probe temperature |
Console | Bruker GmbH | Avance III HD | Controls operation of the spectrometer |
Micro5 gradient coils | Bruker GmbH | Mic5 | Removable gradient coils mount on the Micro5 probe body |
Micro5 Probe body | Bruker GmbH | Mic5 | Holds microcoils and gradient coils |
RF microcoil | Home-built | – | contains Fomblin |
Vector Network Analyzer | Copper Mountain Technologies | TR1300/1 | Used to perform S11 reflectance test, frequency range 300kHz to 1.3 GHz |
Water cooler | Bruker GmbH | BCU-20 | Open loop watercooling to dissipate heat from gradient coil operation. |