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&#39; 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.

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
<ol>
	<li>To prepare 100 mL of the sensitivity reference solution, dissolve 156.4 mg of CuSO4 &#8729; 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.
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>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.
	<ol>
		<li>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.</li>
		<li>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</li>
	</ol>
	</li>
</ol>
2. Sample preparation
<ol>
	<li>If preparing a reference sample for coil calibration, transfer 1 mL of CuSO&#173;4 solution to a watch glass dish under a stereomicroscope.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.
	<ol>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>While looking through a stereomicroscope, use tweezers to submerge the sample in the infiltration medium prepared previously. Wash the sample of potential debris.</li>
		<li>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).</li>
		<li>Take the sample capillary from the medium watch glass, using tweezers. In the case of PFD, cover the Petri dish lid.</li>
	</ol>
	</li>
	<li>Shape the tissue paper into a fine point and use it to remove circa 1 mm of liquid from both ends of the capillary.</li>
	<li>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.</li>
	<li>Afterwards, scrape off excess wax from the exterior of the capillary using a scalpel and wipe clean with fine tissue paper.</li>
</ol>
3. Mounting the sample
<ol>
	<li>Place a microcoil underneath the stereomicroscope and insert the sample using tweezers while keeping the microcoil steady (Figure 2D).</li>
	<li>Use a rod to center the sample in the microcoil, by sliding the capillary inside the solenoid coil.</li>
	<li>Optionally, apply adhesive tape to fix the position of the capillary.</li>
	<li>Inspect the capillary to ensure no air bubbles are visible inside the solenoid coil, to avoid MR signal destruction caused by susceptibility differences.</li>
	<li>Attach the microcoil to the socket of the probe base, while keeping the microcoil upright (Figure 3A,3B).</li>
	<li>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.</li>
</ol>
4. Determining coil characteristics
<ol>
	<li>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.</li>
	<li>Insert the probe into the magnet and connect the necessary cables: RF transmit/receive cable, water-cooling lines, thermocouple cable and air cooling line.</li>
	<li>Set the desired water-cooling temperature (recommended 298 K) for the water-cooling unit.</li>
	<li>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.</li>
	<li>Connect the probe using a 50 &#937; co-axial cable to a network analyzer with a suitably wide sweep width (400 MHz), centered on the intended resonance frequency.</li>
	<li>Observe the resonant modes by adjusting the variable matching and tuning capacitors that are present in the probe base.</li>
	<li>Tune and match the resonant mode to the desired frequency.</li>
	<li>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 (fc) by the width of the reflection dip at -7 dB (i.e., Q = fc /(f1 - f2)14. Set fc to the operating frequency of the magnet, while f1 and f2 are set to the -7 dB point left and right of fc, respectively. Some network analyzers have Q-factor determination built-in.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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 &#181;s at a low pulse power of 0.6 W and slowly increase the pulse lengths by 1 &#181;s at a time until the signal appears.
	<ol>
		<li>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 &#181;s 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.</li>
		<li>For a coil with a homogeneous B1-field, like a solenoid coil, determine the 180&#176; pulse where the signal intensity decreases to zero22.</li>
	</ol>
	</li>
	<li>Set the determined 90&#176; 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.</li>
	<li>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.</li>
	<li>Alternatively, use a complementary way to find the correct 90&#176; 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&#176; 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).</li>
	<li>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.</li>
	<li>Calculate a volume-normalized SNR to allow for comparison of microcoil characteristics across different systems, adapted from the manufacturer&#39;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.
	<ol>
		<li>Determine the volume normalized SNR (SNR/mm3) in two steps. First, calculate the voxel volume (Vvoxel)(Eq. 1): 
		<img alt="Equation 1" src="/files/ftp_upload/61266/61266eq1.jpg" />&#160; &#160;(1) 
		NOTE: The units for Dx, Dy&#160;and Dslice are in mm. This calculation can likewise be performed for a series of slices.</li>
		<li>Select the regions of interest to determine the signal intensity (&#181;ROI) of the sample, and the signal intensity (&#181;noise&#173;) and standard deviation (&#963;noise&#173;) 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.</li>
		<li>Use the values to calculate a volume normalized SNR (Eq. 2): 
		<img alt="Equation 2" src="/files/ftp_upload/61266/61266eq2.jpg" />&#160; &#160;(2) 
		For the coil used here in combination with the reference solution, using Eq. 2 results in the following solution: 
		<img alt="Equation 3" src="/files/ftp_upload/61266/61266eq3.jpg" />&#160; &#160;(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.</li>
	</ol>
	</li>
	<li>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.</li>
</ol>
5. High-resolution imaging
<ol>
	<li>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&#176;, and receiver bandwidth 50 kHz.</li>
	<li>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.</li>
</ol>
6. Recovering samples for further study or storage
<ol>
	<li>Remove the sample capillary from the microcoil.</li>
	<li>Using tweezers, remove the wax plugs under a stereomicroscope.</li>
	<li>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.</li>
	<li>To prevent dehydration of the sample, store in a suitable medium for storage.</li>
</ol>

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D., Edner, C., Smirnoff, N., Love, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perfluorodecalin+enhances+in+vivo+confocal+microscopy+resolution+of+Arabidopsis+thaliana+mesophyll.">Perfluorodecalin enhances in vivo confocal microscopy resolution of Arabidopsis thaliana mesophyll.</a> New Phytologist. 186 (4), 1018-1025 (2010).</li><li>van Schadewijk, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+Resonance+Microscopy+at+Cellular+Resolution+and+Localised+Spectroscopy+of+Medicago+truncatula+at+22.3+Tesla.">Magnetic Resonance Microscopy at Cellular Resolution and Localised Spectroscopy of Medicago truncatula at 22.3 Tesla.</a> Scientific Reports. 10 (1), 971 (2020).</li></ol>

The authors have nothing to disclose.

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.&#160;

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 &#8776;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 &#181;m3 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 &#181;m. 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&#176; 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&#176; 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 (&#8804;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.

<table border="0" cellpadding="0" cellspacing="0" style="width:1012px;" width="1012">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Reference solution preparation</td>
			<td style="width:138px;"></td>
			<td style="width:119px;"></td>
			<td style="width:529px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">CuSO4</td>
			<td style="width:138px;">Sigma-aldrich</td>
			<td>469130</td>
			<td style="width:529px;">Crystalline powder for creating reference solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">D2O</td>
			<td style="width:138px;">Sigma-aldrich</td>
			<td>151882</td>
			<td style="width:529px;">Liquid used to prepare reference sample</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Weigh Scale</td>
			<td style="width:138px;">Sartorius</td>
			<td>PRACTUM513-1S</td>
			<td style="width:529px;">Scale for weighing compounds</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Sample preparation</td>
			<td style="width:138px;"></td>
			<td style="width:119px;"></td>
			<td style="width:529px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Capillary 1000 &#956;m (Outer diameter)</td>
			<td style="width:138px;">Hilbenberg GmbH</td>
			<td>1408410</td>
			<td style="width:529px;">Sample capillaries</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Capillary wax</td>
			<td style="width:138px;">Hampton Research</td>
			<td>HR4-328</td>
			<td style="width:529px;">Solid wax used to seal samples</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Disposable Scalpel</td>
			<td style="width:138px;">Swann-Morton</td>
			<td>No. 11</td>
			<td style="width:529px;">Used to excise samples</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Perfluorodecalin</td>
			<td style="width:138px;">Sigma-aldrich</td>
			<td>P9900</td>
			<td style="width:529px;">Liquid used for submerging sample</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Stereo Microscope</td>
			<td style="width:138px;">Olympus</td>
			<td style="width:119px;">SZ40</td>
			<td style="width:529px;">Tabletop binocular microscope</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Syringe</td>
			<td style="width:138px;">Generic</td>
			<td style="width:119px;">-</td>
			<td style="width:529px;">Used to apply PFD and manipulate the sample</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Vacuum Pump</td>
			<td style="width:138px;">Vacuubrand</td>
			<td>MZ2C</td>
			<td style="width:529px;">Two-stage membrane vacuumpump used for removing air pockets from samples.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Wax pen</td>
			<td style="width:138px;">Hampton Research</td>
			<td>HR4-342</td>
			<td style="width:529px;">Handheld wax pen used to melt and apply capillary wax to samples</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Imaging Hardware</td>
			<td style="width:138px;"></td>
			<td style="width:119px;"></td>
			<td style="width:529px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">22.3 T Magnet</td>
			<td style="width:138px;">Bruker GmbH</td>
			<td style="width:119px;">950 US2</td>
			<td style="width:529px;">Narrowbore superconducting magnet</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Air cooler</td>
			<td style="width:138px;">Bruker GmbH</td>
			<td style="width:119px;">-</td>
			<td>Used to regulate probe temperature</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Console</td>
			<td style="width:138px;">Bruker GmbH</td>
			<td style="width:119px;">Avance III HD</td>
			<td style="width:529px;">Controls operation of the spectrometer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Micro5 gradient coils</td>
			<td style="width:138px;">Bruker GmbH</td>
			<td style="width:119px;">Mic5</td>
			<td style="width:529px;">Removable gradient coils mount on the Micro5 probe body</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Micro5 Probe body</td>
			<td style="width:138px;">Bruker GmbH</td>
			<td style="width:119px;">Mic5</td>
			<td style="width:529px;">Holds microcoils and gradient coils</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">RF microcoil</td>
			<td style="width:138px;">Home-built</td>
			<td style="width:119px;">-</td>
			<td style="width:529px;">contains Fomblin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Vector Network Analyzer</td>
			<td>Copper Mountain Technologies</td>
			<td style="width:119px;">TR1300/1</td>
			<td>Used to perform S11 reflectance test, frequency range 300kHz to 1.3 GHz</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Water cooler</td>
			<td style="width:138px;">Bruker GmbH</td>
			<td style="width:119px;">BCU-20</td>
			<td>Open loop watercooling to dissipate heat from gradient coil operation.</td>
		</tr>
	</tbody>
</table>

mrm microcoil performance calibration and usage demonstrated on medicago truncatula roots at 22 t

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 &#956;m3, 2.2 pL) 3D imaging of a root sample.

Coil Characterization 
Upon successful tuning and matching of a coil, its performance may be characterized by the coil Q-factor, 90&#176; 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&#176; pulse was 12 &#181;s at a power level of 0.6 W; cf. 5 &#181;s at 45 W for a 5 mm birdcage coil (Figure 4 and Figure 5). This equates to an RF pulse field strength (B1), using <img alt="Equation 4" src="/files/ftp_upload/61266/61266eq4.jpg" /> 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 <img alt="Equation 5" src="/files/ftp_upload/61266/61266eq5.jpg" />: 0.69 mT/W1/2 and 0.18 mT/W1/2 for the microcoil and birdcage respectively14. Comparing by a 90&#176; pulse, the microcoil is found to be a factor &#8776; 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 &#956;m3 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 &#181;m, inner diameter 1500 &#956;m), images with a resolution of up to 16 x 16 x 16 &#956;m3 were obtained in 33 minutes.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61266/61266fig1.jpg" /> 
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 &#181;m and a coil length of 3500 &#181;m. The coil is submerged in a reservoir which is made from a syringe. Sample capillaries up to an outer diameter of 1000 &#181;m 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. &#160;(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. <a href="https://www.jove.com/files/ftp_upload/61266/61266fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61266/61266fig2.jpg" /> 
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 &#956;m, 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 &#248; 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. <a href="https://www.jove.com/files/ftp_upload/61266/61266fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61266/61266fig3.jpg" /> 
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. <a href="https://www.jove.com/files/ftp_upload/61266/61266fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61266/61266fig4.jpg" /> 
Figure 4: Nutation curve. A nutation curve is acquired to determine the reference pulse power. The reference pulse power (90&#176; 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 &#181;s each time. The maximum signal intensity indicates the 90&#176; pulse, around 12 &#181;s. The 180&#176; pulse may also be determined in this way using the minimum intensity. <a href="https://www.jove.com/files/ftp_upload/61266/61266fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/61266/61266fig5.jpg" /> 
Figure 5: Visual determination of 90&#176; 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 &#181;s pulse length. (B) 12 &#181;s pulse length, optimal for this coil. (C) 13 &#181;s pulse length. (D) 20 &#181;s 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. <a href="https://www.jove.com/files/ftp_upload/61266/61266fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/61266/61266fig6.jpg" /> 
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. <a href="https://www.jove.com/files/ftp_upload/61266/61266fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/61266/61266fig7.jpg" /> 
Figure 7: RF homogeneity evaluated by gradient echo imaging. A multiple gradient echo (MGE) sequence is used to evaluate RF (B&#173;1&#173; -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&#176;. Field of view was 5 x 5 mm, matrix 128 x 128, resolution 39 x 39 x 200 &#181;m. (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. <a href="https://www.jove.com/files/ftp_upload/61266/61266fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/61266/61266fig8.jpg" /> 
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 &#181;m3&#173;. 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 &#181;m3&#173;. 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. <a href="https://www.jove.com/files/ftp_upload/61266/61266fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/61266/61266fig9.jpg" /> 
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 &#181;m3&#173;. 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 &#181;m3&#173;. Matrix size was 128 x 64 x 64 and field of view 2 x 1 x 1 mm. Receiver bandwidth 50 kHz. <a href="https://www.jove.com/files/ftp_upload/61266/61266fig9large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about MRM Microcoil Performance Calibration and Usage Demonstrated on Medicago truncatula Roots at 22 T at JoVE.com

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

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.

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

This protocol describes a signal-to-noise ratio (SNR) calibration and sample preparation method for solenoidal microcoils combined with biological ...

mrm-microcoil-performance-calibration-usage-demonstrated-on-medicago

Research

JoVE Journal

Biology

5.4K Views.  Leiden University. 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.

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

Loading Drosophila Nerve Terminals with Calcium Indicators

Calcium plays many roles in the nervous system but none more impressive than as the trigger for neurotransmitter release, and none more profound than as the messenger essential for the synaptic plasticity that supports learning and memory. To further elucidate the molecular underpinnings of Ca2+-dependent synaptic mechanisms, a model system is required that is both genetically malleable and physiologically accessible. Drosophila melanogaster provides such a model. In this system, genetically-encoded fluorescent indicators are available to detect Ca2+ changes in nerve terminals. However, these indicators have limited sensitivity to Ca2+ and often show a non-linear response. Synthetic fluorescent indicators are better suited for measuring the rapid Ca2+ changes associated with nerve activity. Here we demonstrate a technique for loading dextran-conjugated synthetic Ca2+ indicators into live nerve terminals in Drosophila larvae. Particular emphasis is placed on those aspects of the protocol most critical to the technique's success, such as how to avoid static electricity discharges along the isolated nerves, maintaining the health of the preparation during extended loading periods, and ensuring axon survival by providing Ca2+ to promote sealing of severed axon endings. Low affinity dextran-conjugated Ca2+-indicators, such as fluo-4 and rhod, are available which show a high signal-to-noise ratio while minimally disrupting presynaptic Ca2+ dynamics. Dextran-conjugation helps prevent Ca2+ indicators being sequestered into organelles such as mitochondria. The loading technique can be applied equally to larvae, embryos and adults.

Calcium is a ubiquitous messenger in the nervous system, essential for triggering neurotransmitter release and changes in synaptic strength. Here we demonstrate a technique for loading Ca2+-indicators into Drosophila nerve terminals. We also demonstrate fabrication of the required apparatus and emphasize points critical for the technique's success.

Calcium plays many roles in the nervous system but none more impressive than as the trigger for neurotransmitter release, and none more profound than as ...

Synthesis and Calibration of Phosphorescent Nanoprobes for Oxygen Imaging in Biological Systems

Oxygen measurement by phosphorescence quenching [1, 2] consists of the following steps: 1) the probe is delivered into the medium of interest (e.g. blood or interstitial fluid); 2) the object is illuminated with light of appropriate wavelength in order to excite the probe into its triplet state; 3) the emitted phosphorescence is collected, and its time course is analyzed to yield the phosphorescence lifetime, which is converted into the oxygen concentration (or partial pressure, pO2). The probe must not interact with the biological environment and in some cases to be 4) excreted from the medium upon the measurement completion. Each of these steps imposes requirements on the molecular design of the phosphorescent probes, which constitute the only invasive component of the measurement protocol. Here we review the design of dendritic phosphorescent nanosensors for oxygen measurements in biological systems. The probes consist of Pt or Pd porphyrin-based polyarylglycine (AG) dendrimers, modified peripherally with polyethylene glycol (PEG's) residues. For effective two-photon excitation, termini of the dendrimers may be modified with two-photon antenna chromophores, which capture the excitation energy and channel it to the triplet cores of the probes via intramolecular FRET (F&ouml;rster Resonance Energy Transfer). We describe the key photophysical properties of the probes and present detailed calibration protocols.

We present principles of oxygen measurements by phosphorescence quenching and review design of porphyrin-based dendritic nanosensors for oxygen imaging in biological systems.

Oxygen measurement by phosphorescence quenching [1, 2] consists of the following steps: 1) the probe is delivered into the medium of interest (e.g. blood ...

Direct Restart of a Replication Fork Stalled by a Head-On RNA Polymerase

In vivo studies suggest that replication forks are arrested due to encounters with head-on transcription complexes. Yet, the fate of the replisome and RNA polymerase (RNAP) following a head-on collision is unknown. Here, we find that the E. coli replisome stalls upon collision with a head-on transcription complex, but instead of collapsing, the replication fork remains highly stable and eventually resumes elongation after displacing the RNAP from DNA. We also find that the transcription-repair coupling factor, Mfd, promotes direct restart of the fork following the collision by facilitating displacement of the RNAP. These findings demonstrate the intrinsic stability of the replication apparatus and a novel role for the transcription-coupled repair pathway in promoting replication past a RNAP block.

The fate of the replisome following a collision with a head-on RNA polymerase (RNAP) is unknown. We find that the replisome stalls upon collision with a head-on RNAP, but resumes elongation after displacing the RNAP from DNA. Mfd promotes replication restart by facilitating displacement of the RNAP after the collision.

In vivo studies suggest that replication forks are arrested due to encounters with head-on transcription complexes. Yet, the fate of the replisome and RNA ...

Swimming Performance Assessment in Fishes

Swimming performance tests of fish have been integral to studies of muscle energetics, swimming mechanics, gas exchange, cardiac physiology, disease, pollution, hypoxia and temperature. This paper describes a flexible protocol to assess fish swimming performance using equipment in which water velocity can be controlled. The protocol involves one to several stepped increases in flow speed that are intended to cause fish to fatigue. Step speeds and their duration can be set to capture swimming abilities of different physiological and ecological relevance. Most frequently step size is set to determine critical swimming velocity (Ucrit), which is intended to capture maximum sustained swimming ability. Traditionally this test has consisted of approximately ten steps each of 20 min duration. However, steps of shorter duration (e.g. 1 min) are increasingly being utilized to capture acceleration ability or burst swimming performance. Regardless of step size, swimming tests can be repeated over time to gauge individual variation and recovery ability. Endpoints related to swimming such as measures of metabolic rate, fin use, ventilation rate, and of behavior, such as the distance between schooling fish, are often included before, during and after swimming tests. Given the diversity of fish species, the number of unexplored research questions, and the importance of many species to global ecology and economic health, studies of fish swimming performance will remain popular and invaluable for the foreseeable future.

The lives of the majority of fish are predicated on swimming. This protocol describes techniques for capturing a range of swimming modes available to individual and schooling fish, and includes metrics associated with swimming physiology and behaviour.

Swimming performance tests of fish have been integral to studies of muscle energetics, swimming mechanics, gas exchange, cardiac physiology, disease, ...

Generation of Composite Plants in Medicago truncatula used for Nodulation Assays

Similar to Agrobacterium tumerfaciens, Agrobacterium rhizogenes can transfer foreign DNAs into plant cells based on the autonomous root-inducing (Ri) plasmid. A. rhizogenes can cause hairy root formation on plant tissues and form composite plants after transformation. On these composite plants, some of the regenerated roots are transgenic, carrying the wild type T-DNA and the engineered binary vector; while the shoots are still non-transgenic, serving to provide energy and growth support. These hairy root composite plants will not produce transgenic seeds, but there are a number of important features that make these composite plants very useful in plant research. First, with a broad host range,A. rhizogenes can transform many plant species, especially dicots, allowing genetic engineering in a variety of species. Second, A. rhizogenes infect tissues and explants directly; no tissue cultures prior to transformation is necessary to obtain composite plants, making them ideal for transforming recalcitrant plant species. Moreover, transgenic root tissues can be generated in a matter of weeks. For Medicago truncatula, we can obtain transgenic roots in as short as three weeks, faster than normal floral dip Arabidopsis transformation. Overall, the hairy root composite plant technology is a versatile and useful tool to study gene functions and root related-phenotypes. Here we demonstrate how hairy root composite plants can be used to study plant-rhizobium interactions and nodulation in the difficult-to-transform species M. truncatula.

Generation of Composite Plants in Medicago truncatula used for Nodulation Assays

We demonstrate how hairy root composite plants can be used to study plant-rhizobium interactions and nodulation in the difficult-to-transform species Medicago truncatula.

Similar to Agrobacterium tumerfaciens, Agrobacterium rhizogenes can transfer foreign DNAs into plant cells based on the autonomous root-inducing (Ri) ...

Quantifying Agonist Activity at G Protein-coupled Receptors

When an agonist activates a population of G protein-coupled receptors (GPCRs), it elicits a signaling pathway that culminates in the response of the cell or tissue. This process can be analyzed at the level of a single receptor, a population of receptors, or a downstream response. Here we describe how to analyze the downstream response to obtain an estimate of the agonist affinity constant for the active state of single receptors.
Receptors behave as quantal switches that alternate between active and inactive states (Figure 1). The active state interacts with specific G proteins or other signaling partners. In the absence of ligands, the inactive state predominates. The binding of agonist increases the probability that the receptor will switch into the active state because its affinity constant for the active state (Kb) is much greater than that for the inactive state (Ka). The summation of the random outputs of all of the receptors in the population yields a constant level of receptor activation in time. The reciprocal of the concentration of agonist eliciting half-maximal receptor activation is equivalent to the observed affinity constant (Kobs), and the fraction of agonist-receptor complexes in the active state is defined as efficacy (&#949;) (Figure 2).
Methods for analyzing the downstream responses of GPCRs have been developed that enable the estimation of the Kobs and relative efficacy of an agonist 1,2. In this report, we show how to modify this analysis to estimate the agonist Kb value relative to that of another agonist. For assays that exhibit constitutive activity, we show how to estimate Kb in absolute units of M-1.
Our method of analyzing agonist concentration-response curves 3,4 consists of global nonlinear regression using the operational model 5. We describe a procedure using the software application, Prism (GraphPad Software, Inc., San Diego, CA). The analysis yields an estimate of the product of Kobs and a parameter proportional to efficacy (&tau;). The estimate of &tau;Kobs of one agonist, divided by that of another, is a relative measure of Kb (RAi) 6. For any receptor exhibiting constitutive activity, it is possible to estimate a parameter proportional to the efficacy of the free receptor complex (&tau;sys). In this case, the Kb value of an agonist is equivalent to &tau;Kobs/&tau;sys 3.
Our method is useful for determining the selectivity of an agonist for receptor subtypes and for quantifying agonist-receptor signaling through different G proteins.

A method for estimating the affinity constant of an agonist for the active state (Kb) of a G protein-coupled receptor is described. The analysis provides absolute or relative measures of Kb depending on whether constitutive receptor activation is measurable. Our method applies to various responses downstream from receptor activation.

When an agonist activates a population of G protein-coupled receptors (GPCRs), it elicits a signaling pathway that culminates in the response of the cell ...

An Improved Method for Accurate and Rapid Measurement of Flight Performance in Drosophila

Drosophila has proven to be a useful model system for analysis of behavior, including flight. The initial flight tester involved dropping flies into an oil-coated graduated cylinder; landing height provided a measure of flight performance by assessing how far flies will fall before producing enough thrust to make contact with the wall of the cylinder. Here we describe an updated version of the flight tester with four major improvements. First, we added a &#34;drop tube&#34; to ensure that all flies enter the flight cylinder at a similar velocity between trials, eliminating variability between users. Second, we replaced the oil coating with removable plastic sheets coated in Tangle-Trap, an adhesive designed to capture live insects. Third, we use a longer cylinder to enable more accurate discrimination of flight ability. Fourth we use a digital camera and imaging software to automate the scoring of flight performance. These improvements allow for the rapid, quantitative assessment of flight behavior, useful for large datasets and large-scale genetic screens.

An Improved Method for Accurate and Rapid Measurement of Flight Performance in Drosophila

Here we describe a method for rapid and accurate measurement of flight performance in Drosophila, enabling high-throughput screening.

Drosophila has proven to be a useful model system for analysis of behavior, including flight. The initial flight tester involved dropping flies into an ...

MALDI-Mass Spectrometric Imaging for the Investigation of Metabolites in Medicago truncatula Root Nodules

Most techniques used to study small molecules, such as pharmaceutical drugs or endogenous metabolites, employ tissue extracts which require the homogenization of the tissue of interest that could potentially cause changes in the metabolic pathways being studied1. Mass spectrometric imaging (MSI) is a powerful analytical tool that can provide spatial information of analytes within intact slices of biological tissue samples1-5. This technique has been used extensively to study various types of compounds including proteins, peptides, lipids, and small molecules such as endogenous metabolites. With matrix-assisted laser desorption/ionization (MALDI)-MSI, spatial distributions of multiple metabolites can be simultaneously detected. Herein, a method developed specifically for conducting untargeted metabolomics MSI experiments on legume roots and root nodules is presented which could reveal insights into the biological processes taking place. The method presented here shows a typical MSI workflow, from sample preparation to image acquisition, and focuses on the matrix application step, demonstrating several matrix application techniques that are useful for detecting small molecules. Once the MS images are generated, the analysis and identification of metabolites of interest is discussed and demonstrated. The standard workflow presented here can be easily modified for different tissue types, molecular species, and instrumentation.

MALDI-Mass Spectrometric Imaging for the Investigation of Metabolites in Medicago truncatula Root Nodules

Mass spectrometric imaging (MSI) is a powerful tool that can be used to discover and identify various chemical species in intact tissues, preserving the compounds in their native environments, which can provide new insights into biological processes. Herein a MSI method developed for the analysis of small molecules is described.&#160;

Most techniques used to study small molecules, such as pharmaceutical drugs or endogenous metabolites, employ tissue extracts which require the ...

Methods to Discover Alternative Promoter Usage and Transcriptional Regulation of Murine Bcrp1

Gene expression in different tissues is often controlled by alternative promoters that result in the synthesis of mRNA with unique &#8212; usually untranslated &#8212; first exons. Bcrp1 (Abcg2), the murine orthologue of the ABC transporter Breast Cancer Resistance Protein (BCRP, ABCG2), has at least four alternative promoters that are designated by the corresponding four alternative first exons produced: E1U, E1A, E1B, and E1C. Herein, in-silico protocols are presented to predict alternative promoter usage for Bcrp1. Furthermore, reporter assay methods are described to produce reporter constructs for alternative promoters and to determine the functionality of putative promoters upstream of the alternative first exons that are identified.

With the murine ABC transporter Bcrp1 (Abcg2) as an example, in-silico protocols are presented to detect alternative promoter usage in genes expressed in mouse tissues, and to evaluate the functionality of the alternative promoters identified using reporter assays.

Gene expression in different tissues is often controlled by alternative promoters that result in the synthesis of mRNA with unique &#8212; usually ...

Light Sheet Fluorescence Microscopy of Plant Roots Growing on the Surface of a Gel

One of the key questions in understanding plant development is how single cells behave in a larger context of the tissue. Therefore, it requires the observation of the whole organ with a high spatial- as well as temporal resolution over prolonged periods of time, which may cause photo-toxic effects. This protocol shows a plant sample preparation method for light-sheet microscopy, which is characterized by mounting the plant vertically on the surface of a gel. The plant is mounted in such a way that the roots are submerged in a liquid medium while the leaves remain in the air. In order to ensure photosynthetic activity of the plant, a custom-made lighting system illuminates the leaves. To keep the roots in darkness the water surface is covered with sheets of black plastic foil. This method allows long-term imaging of plant organ development in standardized conditions.

This protocol shows a plant sample preparation method for light-sheet microscopy. The setup is characterized by mounting the plant vertically on the surface of a gel and letting it grow in controlled bright conditions. This allows long-term observation of plant organ development in standardized conditions.

One of the key questions in understanding plant development is how single cells behave in a larger context of the tissue. Therefore, it requires the ...