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Applied Physical Sciences
Increase in signal-to-noise ratio of > 10,000 times in liquid-state NMR
Amersham Health Research and Development AB, Medeon, SE-205 12 Malmö, Sweden
Communicated by Albert W. Overhauser, Purdue University, West Lafayette, IN, June 20, 2003 (received for review April 16, 2003)
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Abstract |
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94 GHz. Subsequent to
polarization, the sample is dissolved by an injection system inside the DNP
magnet. The dissolution process effectively preserves the nuclear
polarization. The resulting hyperpolarized liquid sample can be transferred to
a high-resolution NMR spectrometer, where an enhanced NMR signal can be
acquired, or it may be used as an agent for in vivo imaging or
spectroscopy. In this article we describe the use of the method on aqueous
solutions of [13C]urea. Polarizations of 37% for 13C and
7.8% for 15N, respectively, were obtained after the dissolution.
These polarizations correspond to an enhancement of 44,400 for 13C
and 23,500 for 15N, respectively, compared with thermal equilibrium
at 9.4 T and room temperature. The method can be used generally for signal
enhancement and reduction of measurement time in liquid-state NMR and opens up
for a variety of in vitro and in vivo applications of
DNP-enhanced NMR.
However, despite significant technological advancements (increasing field strength and cooling of electronics), the application of NMR is limited by an intrinsically low sensitivity as compared with other analytical methods. Fundamentally, the low sensitivity originates from the low magnetic energy of nuclear spins compared with the thermal energy at room temperature. At a magnetic field strength of 1.5 T and room temperature, the 1H spins are polarized to only 5 ppm, and an improvement of 200,000 is thus theoretically possible. The two most sensitive nuclei are 1H and 19F, which have large magnetic moments and 100% abundance. But, even at the largest field strength available today (21 T), these nuclei are polarized to only 70 and 67 ppm, respectively. For other nuclei bearing lower magnetic moments (1/4 for 13C and 1/10 for 15N compared with 1H), the theoretical enhancement factor is proportionally greater. The weak nuclear polarization is generally compensated by a high concentration (i.e., a large number of nuclear spins). However, the sensitivity of several other nuclei is reduced further by the low natural abundance of the NMR-active isotope (1.1% for 13C and 0.36% for 15N).
A range of methods has been proposed to enhance the polarization of nuclear
spins (denoted hyperpolarization methods), e.g., optical pumping
(1),
para-hydrogen-induced polarization (PHIP)
(2,
3), and dynamic nuclear
polarization (DNP) (4,
5). These methods have the
potential to create nonthermal polarization close to unity. Optical pumping of
the noble gases 3He and 129Xe has been applied in MRI of
the lung. Hyperpolarized 129Xe has also been used extensively in
NMR spectroscopy both in vivo and in vitro
(6). The optical pumping
method, however, is limited to the polarization of some of the noble gas
isotopes. The PHIP effect involves the insertion of para-hydrogen
into a substrate molecule via catalytic hydrogenation. The spin order of the
resulting hydrogenated molecule can be converted into nuclear polarization by
various methods (4,
5). The PHIP method depends on
double or triple bonds in the substrate molecule. The third hyperpolarization
method, DNP, is based on polarizing the nuclear spins in the solid state. The
mechanism requires the presence of unpaired electrons, which are added to the
sample as, e.g., an organic free radical. In order for the DNP process to be
effective, the radical must be homogeneously distributed within the sample. To
achieve this in an aqueous sample, a glass-former, e.g., glycerol or glycol,
is added to prevent crystallization and to produce an amorphous solid after
cooling the sample. In the solid state, the high electron spin polarization is
in part transferred to the nuclear spins by microwave irradiation. It has been
demonstrated previously that the nuclear polarizations of 1H and
13C can be increased to almost 100% and 50%, respectively
(7,
8), by means of DNP at low
temperature. The method has mainly been applied to the production of polarized
targets for neutron-scattering experiments. The DNP method has also been used
to improve the sensitivity of NMR in the solid state
(9). An enhancement of 170
± 50 was observed for [1-13C]glycine at 9 T and 20 K, and in
a two-dimensional magic-angle spinning 13C-13C
correlation spectrum the observed enhancement was 17 for
[U-13C,15N]proline at 9 T and 100 K
(10). However, even with the
application of so-called magic-angle spinning, the spectral resolution in
these examples of DNP-enhanced solid-state NMR is not equivalent to that of
liquid-state NMR.
The purpose of this study was to develop a hyperpolarization method that would enable close-to-unity polarization of organic molecules in a liquid solution. We describe a dissolved-phase, DNP-enhanced NMR (DNP-NMR) method, in which the nuclear spins of a sample are hyperpolarized in the solid state and subsequently brought into a liquid solution after rapid dissolution. With this method, we show that it is possible to bring the polarized, cold solid sample into solution while preserving its nuclear polarization. This will enable the DNP-NMR method to be used in a wide range of different in vivo and in vitro applications and to take advantage of the narrow NMR lines in solution, leading to improved sensitivity and spectral resolution. We demonstrate the method by showing examples of enhanced polarization of 13C and 15N in the solid and liquid states of urea. In the article by Golman et al. (11), examples of [13C]urea as a marker for in vivo imaging are shown.
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Materials and Methods |
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DNP insert. The DNP insert is placed in the cold bore of
the magnet (Fig. 1). The DNP
insert consists of a central fiberglass tube (18-mm inner diameter) to guide
the sample when inserted. The sample is guided to the center of the magnet and
ends in a cylindrical metal container (38-mm diameter and 50-mm height) (part
7 in Fig. 1). The function of
the metal container is to confine the microwaves, which are guided to the
container through waveguides from the exterior. On the VTI top flange, a
50-µm Mylar foil is placed between a WR10 E-bend and a short piece of
straight WR10 waveguide going through the top flange. The Mylar foil serves as
a vacuum seal. The waveguide continues in a 27-mm-length transition to WR28, a
500-mm thin-walled stainless-steel WR28 piece, and finally in a section of
brass WR28. The lower-part brass WR28 couples the microwaves into the metal
container through a slit in the side of the cylinder. Inside the metal
container, a pair of saddle coils (16-mm diameter, 16-mm length) is placed to
measure the NMR signal. The saddle coil is connected to a coaxial transmission
line (UT85-SS-CuBe, Oxford Instruments, Tubney Woods, U.K.) and further to a
tuning-and-matching network. The tune-and-match circuitry consists of a
parallel variable capacitor and variable inductor and a series variable
capacitor. By adjusting the length of the transmission line and the three
variable impedances, the NMR circuitry is matched to 50 
at the
13C frequency of 35.89 MHz and the 1H frequency of 142.7
MHz. The 13C 90° flip angle is 100 µs with a transmit
power of 15 W. A pressure transducer (part 5 in
Fig. 1) measures the helium
vapor pressure below the lower radiation shield from outside the VTI through a
3-mm stainless-steel tube. The flow of helium into the VTI is regulated in a
servo loop. The level of helium is monitored by measuring the voltage across
three 100- Allen Bradley carbon resistors. A constant current heats the
resistors sequentially while the voltage across is measured. The voltage
profile is characteristic for helium gas phase and liquid phase. The first
resistor is placed 50 mm above magnetic center, and the two others are placed
above at 10-mm separation. Depending on the level of liquid helium, the needle
valve is opened or closed by a stepper motor, and the level of liquid helium
can be maintained between the lower and middle resistors.
Pumps. A 200-m3/h roots pump and
40-m3/h rotary vane backing pump (Leybold) (part 2 in
Fig. 1) is connected to a 50-mm
port on the side of the VTI. The pumping system is capable of sustaining a
vapor pressure of 0.8 mbar (1 bar = 100 kPa) (corresponding to a bath
temperature of 1.2 K) with the microwaves on. The vapor pressure is used as a
measure of temperature. The pumps can be closed off by a motorized butterfly
valve.
Microwave generator. The microwave source (ELVA-1, St. Petersburg, Russia) (part 4 in Fig. 1) has a maximum output power of 200 mW at 94 GHz, a tuning range of 500 MHz, and a frequency-modulation bandwidth of 10 kHz. The output level can be attenuated continuously up to 60 dB by an analogue solid-state attenuator. The frequency is controlled via an analogue tuning input. Arbitrary waveforms for frequency modulation can be supplied via an additional analogue input.
Software. The polarizer is controlled by a LABVIEW program running on a personal computer controller. The VTI pressure is read through a RS232 connection. The analogue and digital signals for controlling the microwave frequency, microwave power, and the Allen Bradley resistors are generated or sampled by a PXI-6052E sampling card (National Instruments, Austin, TX).
Sample Handling and Dissolution. Preparation of the sample. Urea (99% 13C-labeled, 505 mg, 8.27 mmol) was dissolved in glycerol (1.26 g) to give a nearly saturated solution [29% urea (wt/wt)] with a volume of 1.38 ml. The trityl radical (Tris{8-carboxyl-2,2,6,6-tetra[2-(1-hydroxyethyl)]-benzo(1,2-d:4,5-d')bis(1,3)dithiole-4-yl}methyl sodium salt) (12) was added to reach concentrations of either 15 or 20 mM. A portion of the solution (40-50 mg) was dispensed as droplets into liquid nitrogen and transferred to the sample container (part 9 in Fig. 1) as frozen pellets. Lower molecular weight samples were prepared in a similar way by reducing the amount of urea dissolved in a given amount of glycerol.
Loading the sample. The sample holder (part 8 in Fig. 1) is a Teflon tube designed to hold the sample container (part 9 in Fig. 1) in position in the magnetic field and subsequently to enable the elevation of the sample before dissolution. When loading the sample container, the sample holder and container are first cooled in a nitrogen bath. Subsequently, the frozen pellets are placed in the container via an opening in the sample holder, and the sample holder is lowered into the VTI. The sample holder is sealed with a rubber septum. The VTI is evacuated to 0.8 mbar to cool the sample to 1.2 K, and the microwave irradiation of the sample is started. During the polarization process, the sample is submerged in liquid helium at the center of the magnetic field (3.35 T).
Dissolution of the sample. Shortly before dissolution, the
microwave irradiation is stopped, the VTI is pressurized with helium gas, and
the sample is raised 10 cm from the magnetic center to leave the liquid
helium. At this position, the magnetic field is 3 T. A 10-ml syringe is
filled with 7 ml of boiling water and connected to an injection wand (part 10
in Fig. 1). The injection wand
is an assembly of two Teflon tubes running inside a carbon-fiber tube. The
lower part of the housing is open-ended, allowing for the insertion of the
sample container. The Teflon housing also contains a mixing chamber, ensuring
the necessary dilution of the sample before it leaves the magnetic field of
the polarizer. After inserting the wand into the sample holder, tightly
connecting to the sample container, the hot water is injected rapidly (<1
s) into the sample container through one of the tubes in the wand. The sample
is dissolved by the hot water and displaced via the second tube to a receiving
container.
Polarization Measurement. The polarization in the solid state is quantified by measuring the NMR signal with a Varian (Palo Alto, CA) INOVA400 console. The free induction decay is acquired after a 5-µs pulse (4.5°). The first three points of the free induction decay (15 µs) are omitted before a weighted (2-kHz line broadening) Fourier transform, yielding the solid-state spectrum. The NMR signal intensity is measured by numerical integration of the NMR absorption line after third-order polynomial baseline correction. The nuclear polarization is quantified by comparing the integrals of the DNP-NMR spectrum and the thermal equilibrium solid-state spectrum.
The liquid-state NMR spectra are acquired on the same console and a 9.4-T high-resolution NMR magnet. The hyperpolarized liquid solution is transferred to a 5-mm NMR tube in a spinner and inserted into the 9.4-T magnet in <6 s. The liquid-state polarization is quantified by comparison with the thermal equilibrium liquid-state signal when the thermal equilibrium spectrum can be obtained in a single acquisition. Otherwise a concentrated reference sample of [13C]urea is used. The signal-to-noise ratio (SNR) is measured as peak height divided by the standard deviation of the noise in a 200-Hz spectral region after applying a 1-Hz exponential weight function.
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Results |
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pol, was 4,900 s. After turning off the microwaves,
T1 was measured to be 28,200 s. A maximum 13C
polarization of 42% was obtained in the solid state for 15 mM radical
concentration, microwave frequency of 93.952 GHz, microwave power of 100 mW
(source output level), and temperature of 1.1 K. After increasing the radical
concentration to 20 mM, the maximum achieved polarization decreased to 26%
(Fig. 3), and the build-up time
constant and relaxation time were shortened to 2,755 and 15,800 s,
respectively. The error on the single exponential data fit was <2% in all
cases. The variation in results obtained from identical sample preparations
was <10%.
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The dependence of the 13C polarization on the microwave frequency is shown in Fig. 3. The DNP spectrum showed a positive and a negative enhancement peak of almost equal intensity: +26% at 93.942 GHz and -22% at 94.005 GHz. The separation of the two peaks was 63 MHz.
The 13C NMR line width (full width at half-maximum) in the solid state was 6 kHz (2-kHz line broadening), and the resonance frequency was 35.89 MHz.
Liquid-State Polarization. To quantify the gain in nuclear polarization in the liquid state, the solid sample was dissolved according to the described method and transferred to the 9.4-T magnet within 6 s for NMR acquisition. In this experiment, unlabeled (natural abundance 13C) urea was used. A 13C spectrum of the unlabeled urea sample was acquired and compared with the thermal equilibrium spectrum (Fig. 4). The thermal equilibrium spectrum was obtained after 65 h of averaging (232,128 transients) at Ernst-angle conditions (repetition time of 1 s and pulse angle of 13.5°). The 13C polarization of the hyperpolarized sample was 20%. The SNR of the hyperpolarized spectrum was 4,592, and the SNR of the thermally polarized sample was 7.
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An elegant, simple, and accurate method to measure the 13C polarization is to acquire a 15N spectrum and compare the integral of the doublet peak (J-coupled to the 13C-enriched carbon). The polarization of 13C is the normalized difference of the two integrals (Fig. 5). In this manner, a polarization of 37% was obtained for 13C. From the same spectrum the 15N polarization was determined to 7.8%.
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Discussion |
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10% relaxation), it can be
concluded that very little polarization is lost during dissolution. On the
microscopic scale, the dissolution process is yet to be characterized.
However, assuming that dipolar relaxation to solvent protons is the dominant
T1 relaxation mechanism, we estimate that the minimum
T1 of the carbonyl carbon ranges between 0.5 and 1.0 s at
3.35 T (during the viscous phase of the dissolution). Thus, the dissolution
process must be fast on this time scale. Several parameters such as the nature
and temperature of the dissolution solvent may influence the degree of
relaxation during the dissolution. When comparing samples having 15 and 20 mM radical concentration, the largest solid- and liquid-state polarizations were observed at the lower radical concentration; however, this is at the expense of a prolonged polarization time. The radical concentration was not decreased further in this study, because the polarization after 1 h of microwave irradiation, which constitutes our intended operating time scale, was better at 20 mM. The signal enhancement was found to be independent of the microwave power level in the interval of 50-200 mW (data not shown). Below 50 mW, the enhancement was found to decrease. We believe that this optimum for the microwave power is due to heating of the sample by resonant absorption of the electron spins.
The DNP spectrum appears to be dominated by the thermal mixing effect (13). The positive and negative enhancement peaks are separated by 63 MHz (Fig. 3). A dominant contribution from the solid effect would appear as peaks separated by twice the nuclear Larmor frequency, i.e., 72 MHz. However, a contribution from the solid effect cannot be excluded. The positive and negative enhancement peaks are asymmetric, which is explained by an asymmetry of the EPR spectrum. We have tested frequency modulation of the microwave source. This has previously been reported to increase the polarization and decrease the polarization rate (5). For the investigated system, however, we have not observed any benefit from frequency modulation. The high polarization of 42% in the solid state is due to the favorable EPR line width of the trityl radical. Furthermore, the trityl radical is unique in terms of water solubility and stability.
We have investigated molecules other than urea and obtained similar
results. For example, the substance
(C1-hydroxymethyl-1[13C]cyclopropyl)methanol
(14) with long 13C
T1 was polarized to 20%. The described DNP-NMR method
is capable of enhancing the NMR signal of most 13C and
15N and in our experience only limited by the relaxation during the
transfer of the hyperpolarized sample. The generality of the DNP-NMR method
makes it unique compared with other hyperpolarization techniques.
The concentration of the paramagnetic agent, which is essential for
polarization, is 0.2-0.3 mM in the final solution. However, in the
particular example of urea, >99% of the trityl radical can be removed from
the aqueous solution by filtration through a short anion-exchange column. Such
a filtration will eliminate the very small relaxation effects of the radical
and render the solution more suitable for in vivo use.
The polarizer is capable of reaching a base temperature of 1.2 K of the
pumped 4He bath in the presence of continuous microwave
irradiation. This temperature is obtained relatively easily. Elevating the
temperature (by decreasing the pumping speed) will not reduce the helium
consumption considerably but will have impact on the polarization and the
polarization rate. In our experience the DNP enhancement factor is independent
of temperature in the range of 1-2 K, and therefore the nuclear polarization
is inversely proportional to the temperature. The polarization rate is
typically changing with temperature to a power of more than 1. This means that
the polarization time may be reduced significantly if SNR can be killed in the
individual experiment. Conversely, a further reduction of the temperature
(3He or dilution refrigerator) or an increase of the magnetic field
should further increase the polarization at the expense of prolonged
polarization build-up time.
The strong signal enhancement can be translated into a tremendous reduction in signal averaging time. The conventional NMR spectrum of Fig. 4 was obtained after 65 h of signal averaging. The improvement in SNR from the averaging is estimated to 43 times when corrected for the pulse angle, repetition time, and T1 (the Ernst-angle conditions). Still, the SNR of the enhanced spectrum is 656 times higher (ratio of the measured SNR). Thus, signal averaging for 6562 x 65 h (3,200 years) would have been required to reach the same SNR without enhancement, although a T1-shortening agent could be used to shorten this duration.
The 15N polarization enhancement was on the same order as for 13C in the experiments. However, no attempts were made to optimize the conditions for DNP of 15N. It therefore is likely that the 15N polarization enhancement can be increased further.
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Possible Applications of DNP-NMR |
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TopAbstractMaterials and MethodsResultsDiscussionPossible Applications of DNP-NMRConclusionReferences |
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DNP-NMR for in vitro use requires a high-resolution NMR spectrometer (magnet and console) to be added to or combined with the DNP polarizer. In the described experimental setup, the dissolved sample was transported from the DNP polarizer to a high-resolution magnet. This transfer is currently performed in 6 s, thus limiting the analysis of short T1 nuclei. For high-resolution NMR applications, it is more practical to dissolve the sample and perform the spectroscopic measurements within the DNP polarizer. This requires an NMR coil arrangement in close proximity to the dissolution chamber but extends the range of molecules and nuclei that can be investigated by the method.
In human whole-body imaging, the large concentration of water protons allows anatomical images of high quality (i.e., with good contrast and high temporal resolution) to be obtained. However, limited information of physiological relevance is contained in the images. With DNP hyperpolarization, it may be possible to image nuclei other than protons, thus enabling a range of new applications based on imaging agents at millimolar concentrations. For instance, the strong signal may enable MR angiography with significantly better temporal and spatial resolution than possible today (as outlined in ref. 11).
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Conclusion |
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10,000-fold enhancement of the nuclear
polarization. |
Footnotes |
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* To whom correspondence should be addressed. E-mail: jan.henrik.ardenkjaerlarsen{at}amersham.com.
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