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Imaging
Towards Single Biomolecule Imaging via Optical Nanoscale Magnetic Resonance Imaging Alberto Boretti,* Lorenzo Rosa, and Stefania Castelletto
Nuclear
magnetic resonance (NMR) spectroscopy is a physical marvel in which electromagnetic radiation is charged and discharged by nuclei in a magnetic field. In conventional NMR, the specific nuclei resonance frequency depends on the strength of the magnetic field and the magnetic properties of the isotope of the atoms. NMR is routinely utilized in clinical tests by converting nuclear spectroscopy in magnetic resonance imaging (MRI) and providing 3D, noninvasive biological imaging. While this technique has revolutionized biomedical science, measuring the magnetic resonance spectrum of single biomolecules is still an intangible aspiration, due to MRI resolution being limited to tens of micrometers. MRI and NMR have, however, recently greatly advanced, with many breakthroughs in nano-NMR and nano-MRI spurred by using spin sensors based on an atomic impurities in diamond. These techniques rely on magnetic dipole–dipole interactions rather than inductive detection. Here, novel nano-MRI methods based on nitrogen vacancy centers in diamond are highlighted, that provide a solution to the imaging of single biomolecules with nanoscale resolution in-vivo and in ambient conditions.
Prof. A. Boretti Department of Mechanical and Aerospace Engineering (MAE) Benjamin M. Statler College of Engineering and Mineral Resources West Virginia University (WVU), PO Box 6106 325 Engineering Sciences Building, Morgantown, WV 26506, USA E-mail: [email protected] and [email protected] Dr. L. Rosa, Prof. S. Castelletto Swinburne University of Technology Centre for Micro-Photonics (H74), PO Box 218 Hawthorn, VIC 3122, Australia Dr. L. Rosa University of Parma Department of Information Engineering Viale G.P. Usberti 181/A, 43124 Parma, Italy Prof. S. Castelletto School of Aerospace Mechanical and Manufacturing Engineering RMIT University, PO Box 71 Bundoora, VIC 3083, Australia DOI: 10.1002/smll.201500764 small 2014, 11, No. 34, 4229–4236
1. Introduction The study of spin polarization by nuclear magnetic resonance (NMR) has been, for a long time, an integral part of scientific fields ranging from medicine to materials science to semiconductor electronics. It was subsequently applied to sensing magnetic fields in now-conventional NMR with the opportunity to obtain a 3D nuclear spin image using magnetic resonance imaging (MRI) inductive detection techniques, based on a high magnetic-field gradient. These clinical methods rely on substantial high-field superconducting magnets, utilized to build the polarization of a number of nuclear spins of the order of 1015. This allows a reduction of the noise from naturally dephasing spins in the large probed volume. The sensitivity of this inductive detection depends on the first time derivative of the magnetic flux, scaling with the detected frequency, which increases with the strength of the applied magnetic field. This leads to extensive magnets with high cost, limited transportability of NMR/MRI instrumentation, and a resolution at best of tens of micrometers in present research
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using magnetic resonance microscopy. This resolution is insufficient for imaging at the molecular scale.[1] A key engineering challenge in imaging and sensing in biomedical applications is tracking processes that occur across different scales from micrometers to nanometers. The understanding of processes at the molecular level important to assess biological functions requires MRI with nanometric sensitivity. Thorough modifications to the structure and electron configuration of individual biomolecules such as proteins or DNA could be tracked by using nano-MRI. Another application of nano-MRI is in imaging the dynamics of molecular engineering. To move away from high magnetic fields, either hyperpolarization techniques and/or more sensitive detection methods are required. Novel approaches based on optical probes acting as atomic size spin sensors that can function at the nanoscale could provide a revolutionary paradigm for magnetic resonance microscopy at the molecular level. The idea behind atomic size optical spin probes was proposed many decades ago by Kastler.[2] It consists of using the energy-level transitions corresponding to an optical emission of the ground state of paramagnetic atoms. The detecting atoms in a specific spin level are excited and then polarized by an optical field.[3,4] Optical spin detection methods were established with the discovery of optically detected magnetic resonance (ODMR),[3] attractive for the ability to precisely detect local small magnetic fields due to electrons or small ensembles of nuclear spins. These methods relied on the tracking of spin echoes and the free precession of electrons from initially phosphorescent molecules, excited optically in their higherenergy triplet state in a zero magnetic field. The typical decay type of their spins was determined by observing the modulation of their phosphorescence intensity during the application of a specific microwave field sequence such as Hanh-Echo. Once these systems are subject to other spins, a change in their dephasing time occurs. This allows the quantification of the presence of small magnetic fields or the concentration of nuclear spins with, in principle, a very high resolution. The problem that initially hampered these methods/systems from entering more applied NMR/MRI protocols was the need for a cryogenic operation temperature and the intrinsic short dephasing time of the chosen sensors, in the range of microseconds. Long dephasing times in the range of seconds for sensing electron/nuclear spins are in fact needed to increase the resolution of these techniques. A nitrogen vacancy (NV) is an extrinsic defect available in diamond. It consists of a nitrogen atom substituting a carbon in the lattice close to a vacant carbon site.[5,6] The NV has been extensively studied and provides a solution to some of the original problems related to MRI at the nanoscale. The major concept underlying the use of NVs in diamond in nano-MRI relies on sensing only a small number of sampled spins. These spins are detected and imaged by using their intrinsic statistical polarization or spin–spin coupling between the sampled spins and the atomic size NV sensor. This sensor is positioned in very close proximity to the target spins, without resorting to the application of high magnetic fields. NV is a photostable fluorescent atomic-size spin probe and can be used as an NMR sensor even in nanodiamonds (60 µs were also observed via universal dynamical decoupling protocols in nano diamonds.[12] This result is, however, not sufficient for application in nanoMRI with the required resolution due to a lack of highquality, single-digit-sized nanodiamonds. In this concept article, we will highlight the key findings of the recent developments of nanoscale NMR and MRI based on NV sensors, where the high sensitivity of NV magnetometry methods have been applied to detect and image nuclear and electron spins. We will follow the processes that led to several relevant breakthroughs in this field, focusing on the different methodologies adopted.
2. Optical Nanoscale Nuclear Magnetic Resonance Engineering NV spins for high-sensitivity magnetometry was the first important milestone to achieve present nano-MRI
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Figure 1. a) NV center atomistic illustration and NV electronic levels showing the ground and excited states with spins 0; ±1 (NV total spin S = 1) with the NV optical transition (637 nm) and the microwave transition (2.78 GHz in zero magnetic field). The transition into the singlet state is dark (no photoluminescence) and is spin selective, and is therefore responsible for the photoluminescent modulation of the defect if excited using a microwave in the presence of a small magnetic field B. b) Sequence to establish the optical spin polarization and the read-out (using a 532 nm laser pulse), and the microwave pulse time sequence to realize AC magnetometry, using a Hanh-echo or spin-echo sequence made of π/2–π–π/2 microwave pulses. c) Microwave sequence to realize universal dynamical decoupling to prolong the coherence time of a NV spin. This sequence consists of replacing single refocusing π pulses in the spin-echo sequence with a train of nπ–π pulses. d) Illustration of a confocal modality to sense a small ensemble of nuclear spins at a nanometric distance from a NV center sensor operating in AC magnetic field modality, using a dynamical decoupling pulse sequence to extend its coherence time. Specifically, a sequence called XY8-k consisting of 8 π pulses repeated k times is used. This sequence allows the NV to probe the local AC field from the nuclear spin bath. e) Frequency filter narrowing the NV sensitivity to NMR signals with central frequency υ = 1/2τ[17] by repeating the sequence k times. f) Spectrum of the NV spin-echo contrast obtained by varying the pulse spacing τ, providing a dip in the time domain at half the nuclear spins Larmor precession period (τL), which, by deconvolution in the frequency domain, corresponds to a peak at the Larmor precession frequency (νL).[24]
resolution and the ultimate limit of MRI at the single-protein level. We will now discuss the key concepts underlying NV magnetometry and the present sensitivity. References [13–15] proposed the optical readout of spin transitions associated with NV centers in diamond for magnetometry applications. This was achieved in the presence of small CW and AC magnetic fields using techniques such as ODMR, Rabi oscillation, spin-echo sequences, and universal dynamic decoupling protocols (see Figure 1).[16] The design of compact magnetic-field sensors calls for solid-state magnetometers like diamond-based devices. Due to their atomic size, they can achieve very close proximity with other spins, such as nuclear spins that can interact with NV electron spins, and change the photoluminescence modulation. Numerous materials allow optical spin readouts, however, NVs in diamond can operate under ambient conditions. The optical pumping of the NV center excited-level triplet state is achieved through a dark-state pathway, whose transition is spin-dependent. This allows the exploitation of a spin–orbit coupling effect, as the triplet excited E state spin levels ms = ±1 can decay to the triplet ground state A ms = ±1 via the transition to a dark, metastable singlet state, while the small 2015, 11, No. 34, 4229–4236
triplet E spin level ms = 0 state cannot. This implies that, after few cycles, the 0 spin will be polarized, meaning that most of the electrons will populate the ms = 0 ground state via optical excitation. For the ms = ±1 transition going through the darkstate crossing, the detectable fluorescence will be reduced. When the NV center couples to a static magnetic field, ms = ±1 levels will split and, since this transition can be triggered by applying a GHz microwave tuned to the ms = 0 → ±1 transitions, the fluorescence can be modulated with an amplitude that tracks the local magnetic field, giving a position accuracy on the order of the NV-center size (see Figure 1). Using single NV centers in ultrapure bulk diamond, this method has a reached sensitivity ≈ 40 nT/√Hz by DC magnetometry and ≈ 10 nT/√Hz in AC magnetometry.[11] The magnitude of the magnetic field and the fluctuations caused by a single electron spin exceeds these values at nanoscopic proximities, while a single nuclear spin magnetic field variation is about 10 nT when 10 nm distant from another spin, thus, small ensembles (104) of statistically polarized nuclear spins are within NV magnetometry sensitivity. AC magnetometry is based on increasing the coherence time of the NV electron spin. This is obtained by using different spin
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manipulation techniques based on microwave sequences such as Hahn-echo or spin-echo and a universal dynamic decoupling sequence. In the spin-echo method, first, a green laser pulse of about 8 µs polarizes the NV electronic spins in the ms = 0 state, |0>. The resonance to the NV transition microwave pulse π/2 prepares the spin in a superposition of ms = |0>, |1>, i.e., (|0>+ |1>) = √2. The π pulse flips the spins after the first free-precession time in such a way that the two spin states, after the second precession time, have accumulated the same average phase that cancels out (also known as refocusing the pulse). The final π/2 pulse projects the spin into a |0>, |1> state population difference. This allows measurement of the NV electron spin coherence time, decoupled from background spin noise. The universal dynamic decoupling sequence replaces the refocusing π pulse with a sequence of them, and further decouples the NV spin from the local nuclear spin in the diamond, greatly extending its coherence time. Two other strategies are used to further push the sensitivity. One strategy is based on the use of ensembles of NVs, where the magnetic sensitivity scales as √N, N being the number of NV spins. In this case a sensitivity of 100 pT/√Hz has been reached at room temperature in AC magnetometry. The other strategy uses the infrared emission from the singlet metastable state, together with an ensemble of NVs. This method achieved sensitivities of 10 pT/√Hz,[18] and there is an expectation to reach the fT/√Hz range.[19] Regardless of such high sensitivity achieved in ensemble magnetometry, this approach may not be applicable for detecting few nuclear spins due to the larger sample volume and the excessive distance of the NV defects from the target nuclei. Magnetometry demonstrations in biological science include the magnetic imaging of living cells (magnetotactic bacteria) under ambient conditions and with subcellular resolution.[20] Single NV AC magnetometry was recently applied to sense other spins such as electron and nuclear spins, providing methods to prove the possibility of NMR by NVs at the nanoscale and under ambient conditions. The first demonstration of the potential of NV magnetometry first proved the imaging of another NV target spin,[21] creating the basis for the successful sensing of nuclear spins. To achieve this first breakthrough, the NV magnetometer was based on a diamond nanopillar functioning as a scanning probe, nanofabricated from ultrapure material. The nanopillar contained the NV spin sensor which scanned target NV spins in bulk diamond in nanometric proximity. This permitted the imaging of the magnetic dipole of a single electron spin under ambient conditions using AC mode and a dynamical decoupling pulse sequence. The use of NVs to sense nuclear spin was initially established in sensing 13C nuclear spins embedded in the spin bath of the diamond.[22] However, the relevance of NV-NMR is demonstrated in sensing other nuclear spins external to the diamond. The first recent breakthrough showed that NV is capable of detecting proton-NMR signals in an organic sample external to the diamond.[23] Similarly, it was proven useful for the detection of NMR signals within volumes of external samples (5 nm3).[24] In one study,[23] NV centers were formed 20 nm from the diamond surface within a 12C enriched grown diamond to avoid noise from the 13C nuclear spin bath naturally present
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in synthetic diamonds. Due to the NV’s close proximity to the surface, the sensitivity was limited by a 600 µs singleNV coherence time, measured using a Hahn-echo sequence. The sensed protons were contained in a 60 nm-thick layer of polymethylmethacrylate (acrylic; PMMA) on top of the diamond, and NVs probed a volume of protons of 24 nm3. After optical polarization, NVs underwent a Hahn-echo microwave sequence, synchronous, with two identical radio-frequency pulses, to produce an inversion of the proton polarization during the NV spin-echo sequence. The NV spin-echo response was dependent on the radio-frequency NMR transition of the protons. In another study,[24] NV was 5 nm from the diamond surface while 1H nuclear spins were contained in solid and liquid samples on the diamond surface. The spin noise from this ensemble of nuclear spins generates an oscillating field. This magnetic field has an amplitude and phase statistically fluctuating and could be detected by using a dynamical decoupling AC magnetometer based on an XY8-k sequence, as shown in the illustration in Figure 1. After the first π/2 pulse, the spin state is (|0> + eIφ|1>)/√2, where φ is the spin phase that evolves from 0 at the beginning of the sequence to a final random value Δφ, which is read out optically after the final π/2 pulse, as a population difference from the NV spin states 0 and 1. By averaging over many sequence repetitions, the variance of the phase is reduced. Every refocusing π pulse makes the phase sensitive to specific frequency components, providing a spectral filter centered around ν = 1/(2τ), where τ is the π pulse temporal spacing. Finally, by changing the value of τ, it is possible to read out from the NV fluorescence its coherence time change contrast, with a dip at half the Larmor magnetic field precession period of the probed nuclear spins. This corresponds in the frequency domain to a peak at the Larmor frequency of the statistically polarized nuclear spins (Figure 1). The probed volume contained 104 nuclear spins, while only 102 nuclear spins were statistically polarized.
3. Towards Imaging Single Biomolecules The magnetic noise produced by unpaired electrons at or near the diamond surface thus far is a limitation for the detection by an NV sensor of very few or single external nuclear spins. Although earlier studies proved that it is possible to greatly reduce the numbers of unpolarised diamond surface spins, they have not been eliminated completely. The ultimate sensitivity limit of single-molecule detection and higher-resolution imaging of nuclear and electron spins have been achieved using improved detection strategies in very recent demonstrations. One approach is based on relying on the strong coupling between the sensor and nuclear spins. Figure 2 illustrates the concepts of inductive NMR with a large number of the nuclear spins where a high B field polarizes them, nano-MRI with very low magnetic field, working with a dilute ensemble of nuclear spins (104) and using NVs as a sensor. In quantum MRI, very few or one nuclear spin magnetic field can be sensed. The quantum MRI approach results in a nuclear magnetic resonance signal which is independent from the number of the polarized spins. To
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Figure 2. a) Concept of high magnetic field inductive NMR, where a large ensemble of nuclear spins is probed (≥1012 spins). In this case, the sensor coupling (ΓS) with individual nuclear spins is much weaker than interactions between nuclei (ΓN), i.e., ΓN > ΓS. The nuclei are polarized by the high B field. The time-averaged measured signal, < S > , is proportional to the sample net polarized number of nuclear spins multiplied by the spin magnetic moment, µ, as < S > ∝ µ (N↓ – N↑). b) Nanoscale-NMR (detailed in Figure 1) refers to the detection of small ensembles (≈104 nuclear spins) with B ≈ 0, where the NV electron spin is the sensor and is still weakly coupled with the nuclear spins (ΓN > ΓS). The average signal < S > ∝ µ √N is still proportional to the sample magnetization where N spins are statistically polarized as statistical fluctuations may exceeds thermal polarization. c) Quantum-NMR corresponds to the detection of individual or few nuclei when the interaction of each nuclear spin with the sensor is stronger than the coupling to the surrounding bath (ΓS > ΓN). In this strong coupling regime, the detected signal is then proportional to < S > ∝ Nµ, where N is the number of nuclear spins, regardless their net polarization. Images reproduced with permission.[25] Copyright 2014, Macmillan Publishers, Ltd.
prove the quantum-MRI concept, four silicon nuclei (Si29) from silica deposited on diamond were strongly coupled with a 2 nm shallow NV electron spin sensor in bulk diamond.[25] Specifically fabricated glass-like coatings positioned on the diamond surface were probed by very close NV centers. The weakness of nuclear spin interactions with NV sensors has, up to now, placed a limit on single nuclear spin magnetic resonance imaging. A nano-MRI method using a single NV center has, however, been successfully applied to achieve the 2D imaging of 1H NMR from a polymer test with a spatial resolution of 12 nm.[26] This work removed a limit of previous NV-based nano-NMR probing a dilute sample of nuclear spins. The experimental apparatus developed in ref. [26] involved a scanning probe system based on a quartz tuning fork with a PMMA sample attached to it, and hanging over a diamond substrate with the NV center embedded by surface ion implantation. A XY8–96 sequence was used to measure the coherence of NV spins in the presence of the proton spin, and a clear dip was observed when the π pulse’s temporal separation matched the half of the proton precession period. By scanning the sample, NMR images were obtained measuring the proton resonance signal. Figure 3 presents the 2D NMR images from two distinct PMMA samples with two diverse NVs. The z height is proportional to the NMR signal small 2015, 11, No. 34, 4229–4236
s(x,y). Photon shot noise is predominantly responsible for the apparent roughness. This result makes it much closer to opportunities for visualizing the full 3D morphology of complex nanostructures including biomolecules, in vivo and nondestructively. Further development of the technique promises to bring resolution and signal-to-noise ratio enhancements, to the point where 3D imaging of proteins can be achieved. Multiple nuclear spins such as 1H, 19F, and 31P have been sensed with NMR and imaged using nano-MRI in confocal and wide-field microscopy imaging modalities.[17] The wide-field modality makes use of ensemble NV magnetometry concepts, combined with repeated sequence XY8-k, previously discussed. Figure 4 presents the nano-MRI imaging of multi-species sample with sub-micrometer structures in wide field modality. It has also been proven that NV is capable of measuring the magnetic resonance spectrum of a single molecule labeled using single-electron spin from nitroxide, a common spin label used in conventional electron spin paramagnetic resonance imaging.[27] The authors selected as the experimental sample MAD2 (mitotic arrest deficient-2), an essential spindle checkpoint protein. A single nitroxide spin label can easily modify this protein. The protein embedded in a poly-L-lysine layer is well restrained on the diamond surface. Its magnetic dipole interaction with a single NV center
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Figure 3. 2D images based on 1H NMR signals contained in two PMMA samples, probed using two different NV centers. The z height is proportional to the NMR signal s(x,y). Images reproduced with permission.[26] Copyright 2015, Macmillan Publishers, Ltd.
permits detection of this electron spin label. By simultaneously driving the NV probe and the spin label of the sample, the magnetic dipole interaction between the NV spin and the spin label was measured. Single NV centers were generated approximately 5 nm beneath the surface of the diamond. This demonstration is very important, as it first proves the possibility to extend the resolution of conventional spin label approaches to single-molecule spin tracking. Using the same optical detection and microwave manipulation of spins based on XY8-N microwave frequency sequence (similar to ref. [17] and [26], but combined with a scanning probe cantilever (as in Ref. [26] the distinctive measurement of a signal contrast from 1H and 19F nuclear spins was achieved using an NV in a bulk diamond, positioned 5 nm below the surface.[28] This work achieved a resolution of 10 nm in ambient conditions. By using a scanning probe labelled with 19F nuclear spins, an NMR resonant peak at the Larmor frequency of the fluorine nuclear spins was observed when the probe was engaged with the NV spin. The NMR peak signal switched to a 1H Larmor frequency resonance when the probe was retracted from the diamond surface. 1H nuclei were contained in a proton-rich organic absorbent 9 nm beneath the diamond surface. In this case, the sampling
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nuclear spins was a few nm3 out of the diavolume for mond surface. The method also allowed distinguishing subsurface chemical species, namely 19F and 1H, using the same fluorinated tip indented into the diamond surface. This method proves the possibility to achieve MRI contrasts for different nuclei within 10 nm, and it can be extended to arbitrary samples, opening new avenues in materials science and in biological sample analysis. A distinctively novel detection method that could improve tenfold currently demonstrated spatial resolutions has been proposed.[29] The method utilizes the coupling of the NV center to a nitrogen nuclear spin, which constitutes a very sharp dynamical frequency filter for NV to effectively sense fewer coupled nuclear spins and allows a longer acquisition time. In addition, the main thrust of this method is to make use, as in conventional MRI, of an effective magnetic field gradient, originating from the NV magnetic dipolar field spatial variation on the diamond surface. In this case it would be possible to decouple the NMR frequency of different nuclei within a complex biomolecule, such as the chemokine receptor CXCR4. A NV center positioned 1–2 nm from the surface is needed, and its magnetic field gradient created on the diamond surface would allow imaging of single nuclear spins in a molecule anchored on this surface. This method could achieve 2D-NMR with sub-nanometer resolution. Another method proved sub-nanometre resolution in nano-MRI applied to dark electronic spins,[30] with 0.8 nm and 1.5 nm lateral and vertical resolutions, respectively. The method provides the opportunity of 3D imaging. The demonstration combines the scanning magnetic field gradient used in magnetic force microscopy with a single NV magnetometer positioned 10 nm from the diamond surface (see Figure 5). The dark electron spins on the surface interact with a scanning magnetic atomic force microscope tip. The dark electron spins are driven by a radio-frequency signal (resonant dark spin in blue in Figure 5). The magnetic tip position relative to the diamond surface produces a local magnetic-field gradient. The tip magnetic field provides a narrower sampling of the spin spatial volume, as for different tip positions different dark spins precession are in resonance with the driving radiofrequency field. NV detects only dark spin magnetic signals in resonance with the driving radiofrequency field, while nonresonant spins (black spins in Figure 5) are not detected. Dark spin signal detection via a NV optical readout is achieved using a confocal optical microscope and NV spin is manipulated using a microwave excitation spin-echo sequence, whose π pulse is synchronous with the π pulse radiofrequency driving field. For each magnetic tip position, different dark spins are driven by the radio-frequency signal and accumulate a net NV phase. The radio-frequency sequence is known in conventional electron paramagnetic resonance methods as double electron–electron resonance.
4. Discussion and Conclusions Nano-MRI methods using NV centers in diamond are based on the concept of sensing spins using an in-principle atomicsize sensor exerting magnetic dipole–dipole interactions
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Figure 4. a) White-light transmission image of a fabricated structure by atomic layer deposition of SiO2 on the surface of a diamond containing a shallow (10 nm) high-density NV layer. b) Optical MRI achieved using a shallow ensemble of NVs in wide-field microscopy mode. The NMR signal is from the 19F nuclear spin density of a fluorinated sample on the diamond bare surface (A). The color bar indicates the NMR contrast, with blue representing a deep 19F NMR contrast dip and hence a high fluorine concentration on the bare diamond surface. Red represents no 19F NMR signal detected by the NV ensemble under the SiO2 structure (B), as the SiO2 layer displaces the fluorinated sample ≈90 nm away from the diamond surface and NV sensors. c,d) The same images from a second SiO2 structure/fluorinated sample on diamond surface. Images reproduced with permission.[17] Copyright 2015, Macmillan Publishers, Ltd.
Figure 5. Conceptual scheme to detect dark spins on a diamond surface using a scanning magnetic tip placed 100 nm from the diamond and a single NV sensor. A NV center in diamond located in a confocal laser spot with nearby dark spins is used as a sensor via the optical read-out of its electron spins, manipulated by applying a microwave (MW) signal. A radio-frequency (RF) signal drives the resonance of few electron dark spins, selected by the gradient magnetic field exerted by the scanning magnetic tip. These dark spins in resonance are detected by applying a double electron–electron resonance sequence for each tip position, while a MW spin-echo sequence is executed on the NV, allowing independent coherent control of the NV and dark spins. Images reproduced with permission.[30] Copyright 2015, Macmillan Publishers Ltd. small 2015, 11, No. 34, 4229–4236
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with very close nuclear or electron spins. This approach does not require a high magnetic field as in conventional inductive MRI (only small magnetic fields are applied, depending on the method), and therefore allows spatial nanometric resolution and a high signal-to-noise ratio over a larger frequency band. Different techniques to image few nuclei spins in polymer samples and to detect NMR spectra have been demonstrated based on optical confocal and wide-field microscopy, adding more complex microwave pulses sequence to narrow the frequency sensitivity of NV electron spin coherence time variation. Improvement of the initial methodology was achieved by adding scanning probes functionalized with nuclear spins and a scanning magnetic tip, permitting 2D imaging of protons with nanometric resolution and 3D imaging of dark electronic spins with sub-nanometric resolution. The methods developed using NV have evidenced the potential to achieve unprecedented resolution at room temperature in detecting few nuclear spins, electron dark spins, and electronic-spin labeled single biomolecules, pushing the boundary of previous realizations based on magnetic resonance force microscopy. However, the weakness of nuclear spins coupling at the nanoscale has placed a limit on 3D imaging of single nuclear spins. Further development of the technique is required to bring resolution and signal-to-noise ratio enhancements to the point where 3D imaging of nuclear spins within proteins can be achieved. Such breakthroughs over the state of the art may certainly open new avenues for medicine by revolutionizing nano-MRI imaging techniques. Presently, several different approaches have evidenced a fast growing field with relevant advances towards fully establishing a consolidated technique to respond to the quest of a molecular microscope that can provide 2D or 3D imaging, as it is well-established in conventional MRI scanning systems at the micrometer level. Several groups have provided remarkable progress with major stepping stones to deliver the expected future key breakthroughs. As NV centres are highly sensitive, they may sense the magnetic noise produced by other spin sources.[31] Therefore, the key to achieve the here-described major results relied on the opportunity to reduce the distance between the NV centre and the target
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nuclei by generating NV centres a few nanometres beneath the diamond surface.[17,26,29] Yet the spectral resolution of the resonance frequency of the nuclei is a long way from the resolution of conventional specrometers and the synthesis of diamond with near surface NV spins of long coherence time is also very difficult.[31] Nanofabrication of single crystal diamond probes and cantilivers containg a single NV with a long coherence time could allow further improvement. Finally, as the nuclear spin coupling degrades from nuclei more than few nanometres from the NV centre, 3D imaging of large molecules may be troublesome.[31] In this case, the use of small ensembles of NVs could allow an increase in the volume sampled, maintaining high spatial resolution albeit a shorter coherence time of the NV ensemble.
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Received: March 18, 2015 Revised: April 27, 2015 Published online: June 25, 2015
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Towards Single Biomolecule Imaging via Optical Nanoscale Magnetic Resonance Imaging Alberto Boretti,* Lorenzo Rosa, and Stefania Castelletto
Nuclear
magnetic resonance (NMR) spectroscopy is a physical marvel in which electromagnetic radiation is charged and discharged by nuclei in a magnetic field. In conventional NMR, the specific nuclei resonance frequency depends on the strength of the magnetic field and the magnetic properties of the isotope of the atoms. NMR is routinely utilized in clinical tests by converting nuclear spectroscopy in magnetic resonance imaging (MRI) and providing 3D, noninvasive biological imaging. While this technique has revolutionized biomedical science, measuring the magnetic resonance spectrum of single biomolecules is still an intangible aspiration, due to MRI resolution being limited to tens of micrometers. MRI and NMR have, however, recently greatly advanced, with many breakthroughs in nano-NMR and nano-MRI spurred by using spin sensors based on an atomic impurities in diamond. These techniques rely on magnetic dipole–dipole interactions rather than inductive detection. Here, novel nano-MRI methods based on nitrogen vacancy centers in diamond are highlighted, that provide a solution to the imaging of single biomolecules with nanoscale resolution in-vivo and in ambient conditions.
Prof. A. Boretti Department of Mechanical and Aerospace Engineering (MAE) Benjamin M. Statler College of Engineering and Mineral Resources West Virginia University (WVU), PO Box 6106 325 Engineering Sciences Building, Morgantown, WV 26506, USA E-mail: [email protected] and [email protected] Dr. L. Rosa, Prof. S. Castelletto Swinburne University of Technology Centre for Micro-Photonics (H74), PO Box 218 Hawthorn, VIC 3122, Australia Dr. L. Rosa University of Parma Department of Information Engineering Viale G.P. Usberti 181/A, 43124 Parma, Italy Prof. S. Castelletto School of Aerospace Mechanical and Manufacturing Engineering RMIT University, PO Box 71 Bundoora, VIC 3083, Australia DOI: 10.1002/smll.201500764 small 2014, 11, No. 34, 4229–4236
1. Introduction The study of spin polarization by nuclear magnetic resonance (NMR) has been, for a long time, an integral part of scientific fields ranging from medicine to materials science to semiconductor electronics. It was subsequently applied to sensing magnetic fields in now-conventional NMR with the opportunity to obtain a 3D nuclear spin image using magnetic resonance imaging (MRI) inductive detection techniques, based on a high magnetic-field gradient. These clinical methods rely on substantial high-field superconducting magnets, utilized to build the polarization of a number of nuclear spins of the order of 1015. This allows a reduction of the noise from naturally dephasing spins in the large probed volume. The sensitivity of this inductive detection depends on the first time derivative of the magnetic flux, scaling with the detected frequency, which increases with the strength of the applied magnetic field. This leads to extensive magnets with high cost, limited transportability of NMR/MRI instrumentation, and a resolution at best of tens of micrometers in present research
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using magnetic resonance microscopy. This resolution is insufficient for imaging at the molecular scale.[1] A key engineering challenge in imaging and sensing in biomedical applications is tracking processes that occur across different scales from micrometers to nanometers. The understanding of processes at the molecular level important to assess biological functions requires MRI with nanometric sensitivity. Thorough modifications to the structure and electron configuration of individual biomolecules such as proteins or DNA could be tracked by using nano-MRI. Another application of nano-MRI is in imaging the dynamics of molecular engineering. To move away from high magnetic fields, either hyperpolarization techniques and/or more sensitive detection methods are required. Novel approaches based on optical probes acting as atomic size spin sensors that can function at the nanoscale could provide a revolutionary paradigm for magnetic resonance microscopy at the molecular level. The idea behind atomic size optical spin probes was proposed many decades ago by Kastler.[2] It consists of using the energy-level transitions corresponding to an optical emission of the ground state of paramagnetic atoms. The detecting atoms in a specific spin level are excited and then polarized by an optical field.[3,4] Optical spin detection methods were established with the discovery of optically detected magnetic resonance (ODMR),[3] attractive for the ability to precisely detect local small magnetic fields due to electrons or small ensembles of nuclear spins. These methods relied on the tracking of spin echoes and the free precession of electrons from initially phosphorescent molecules, excited optically in their higherenergy triplet state in a zero magnetic field. The typical decay type of their spins was determined by observing the modulation of their phosphorescence intensity during the application of a specific microwave field sequence such as Hanh-Echo. Once these systems are subject to other spins, a change in their dephasing time occurs. This allows the quantification of the presence of small magnetic fields or the concentration of nuclear spins with, in principle, a very high resolution. The problem that initially hampered these methods/systems from entering more applied NMR/MRI protocols was the need for a cryogenic operation temperature and the intrinsic short dephasing time of the chosen sensors, in the range of microseconds. Long dephasing times in the range of seconds for sensing electron/nuclear spins are in fact needed to increase the resolution of these techniques. A nitrogen vacancy (NV) is an extrinsic defect available in diamond. It consists of a nitrogen atom substituting a carbon in the lattice close to a vacant carbon site.[5,6] The NV has been extensively studied and provides a solution to some of the original problems related to MRI at the nanoscale. The major concept underlying the use of NVs in diamond in nano-MRI relies on sensing only a small number of sampled spins. These spins are detected and imaged by using their intrinsic statistical polarization or spin–spin coupling between the sampled spins and the atomic size NV sensor. This sensor is positioned in very close proximity to the target spins, without resorting to the application of high magnetic fields. NV is a photostable fluorescent atomic-size spin probe and can be used as an NMR sensor even in nanodiamonds (60 µs were also observed via universal dynamical decoupling protocols in nano diamonds.[12] This result is, however, not sufficient for application in nanoMRI with the required resolution due to a lack of highquality, single-digit-sized nanodiamonds. In this concept article, we will highlight the key findings of the recent developments of nanoscale NMR and MRI based on NV sensors, where the high sensitivity of NV magnetometry methods have been applied to detect and image nuclear and electron spins. We will follow the processes that led to several relevant breakthroughs in this field, focusing on the different methodologies adopted.
2. Optical Nanoscale Nuclear Magnetic Resonance Engineering NV spins for high-sensitivity magnetometry was the first important milestone to achieve present nano-MRI
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Figure 1. a) NV center atomistic illustration and NV electronic levels showing the ground and excited states with spins 0; ±1 (NV total spin S = 1) with the NV optical transition (637 nm) and the microwave transition (2.78 GHz in zero magnetic field). The transition into the singlet state is dark (no photoluminescence) and is spin selective, and is therefore responsible for the photoluminescent modulation of the defect if excited using a microwave in the presence of a small magnetic field B. b) Sequence to establish the optical spin polarization and the read-out (using a 532 nm laser pulse), and the microwave pulse time sequence to realize AC magnetometry, using a Hanh-echo or spin-echo sequence made of π/2–π–π/2 microwave pulses. c) Microwave sequence to realize universal dynamical decoupling to prolong the coherence time of a NV spin. This sequence consists of replacing single refocusing π pulses in the spin-echo sequence with a train of nπ–π pulses. d) Illustration of a confocal modality to sense a small ensemble of nuclear spins at a nanometric distance from a NV center sensor operating in AC magnetic field modality, using a dynamical decoupling pulse sequence to extend its coherence time. Specifically, a sequence called XY8-k consisting of 8 π pulses repeated k times is used. This sequence allows the NV to probe the local AC field from the nuclear spin bath. e) Frequency filter narrowing the NV sensitivity to NMR signals with central frequency υ = 1/2τ[17] by repeating the sequence k times. f) Spectrum of the NV spin-echo contrast obtained by varying the pulse spacing τ, providing a dip in the time domain at half the nuclear spins Larmor precession period (τL), which, by deconvolution in the frequency domain, corresponds to a peak at the Larmor precession frequency (νL).[24]
resolution and the ultimate limit of MRI at the single-protein level. We will now discuss the key concepts underlying NV magnetometry and the present sensitivity. References [13–15] proposed the optical readout of spin transitions associated with NV centers in diamond for magnetometry applications. This was achieved in the presence of small CW and AC magnetic fields using techniques such as ODMR, Rabi oscillation, spin-echo sequences, and universal dynamic decoupling protocols (see Figure 1).[16] The design of compact magnetic-field sensors calls for solid-state magnetometers like diamond-based devices. Due to their atomic size, they can achieve very close proximity with other spins, such as nuclear spins that can interact with NV electron spins, and change the photoluminescence modulation. Numerous materials allow optical spin readouts, however, NVs in diamond can operate under ambient conditions. The optical pumping of the NV center excited-level triplet state is achieved through a dark-state pathway, whose transition is spin-dependent. This allows the exploitation of a spin–orbit coupling effect, as the triplet excited E state spin levels ms = ±1 can decay to the triplet ground state A ms = ±1 via the transition to a dark, metastable singlet state, while the small 2015, 11, No. 34, 4229–4236
triplet E spin level ms = 0 state cannot. This implies that, after few cycles, the 0 spin will be polarized, meaning that most of the electrons will populate the ms = 0 ground state via optical excitation. For the ms = ±1 transition going through the darkstate crossing, the detectable fluorescence will be reduced. When the NV center couples to a static magnetic field, ms = ±1 levels will split and, since this transition can be triggered by applying a GHz microwave tuned to the ms = 0 → ±1 transitions, the fluorescence can be modulated with an amplitude that tracks the local magnetic field, giving a position accuracy on the order of the NV-center size (see Figure 1). Using single NV centers in ultrapure bulk diamond, this method has a reached sensitivity ≈ 40 nT/√Hz by DC magnetometry and ≈ 10 nT/√Hz in AC magnetometry.[11] The magnitude of the magnetic field and the fluctuations caused by a single electron spin exceeds these values at nanoscopic proximities, while a single nuclear spin magnetic field variation is about 10 nT when 10 nm distant from another spin, thus, small ensembles (104) of statistically polarized nuclear spins are within NV magnetometry sensitivity. AC magnetometry is based on increasing the coherence time of the NV electron spin. This is obtained by using different spin
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manipulation techniques based on microwave sequences such as Hahn-echo or spin-echo and a universal dynamic decoupling sequence. In the spin-echo method, first, a green laser pulse of about 8 µs polarizes the NV electronic spins in the ms = 0 state, |0>. The resonance to the NV transition microwave pulse π/2 prepares the spin in a superposition of ms = |0>, |1>, i.e., (|0>+ |1>) = √2. The π pulse flips the spins after the first free-precession time in such a way that the two spin states, after the second precession time, have accumulated the same average phase that cancels out (also known as refocusing the pulse). The final π/2 pulse projects the spin into a |0>, |1> state population difference. This allows measurement of the NV electron spin coherence time, decoupled from background spin noise. The universal dynamic decoupling sequence replaces the refocusing π pulse with a sequence of them, and further decouples the NV spin from the local nuclear spin in the diamond, greatly extending its coherence time. Two other strategies are used to further push the sensitivity. One strategy is based on the use of ensembles of NVs, where the magnetic sensitivity scales as √N, N being the number of NV spins. In this case a sensitivity of 100 pT/√Hz has been reached at room temperature in AC magnetometry. The other strategy uses the infrared emission from the singlet metastable state, together with an ensemble of NVs. This method achieved sensitivities of 10 pT/√Hz,[18] and there is an expectation to reach the fT/√Hz range.[19] Regardless of such high sensitivity achieved in ensemble magnetometry, this approach may not be applicable for detecting few nuclear spins due to the larger sample volume and the excessive distance of the NV defects from the target nuclei. Magnetometry demonstrations in biological science include the magnetic imaging of living cells (magnetotactic bacteria) under ambient conditions and with subcellular resolution.[20] Single NV AC magnetometry was recently applied to sense other spins such as electron and nuclear spins, providing methods to prove the possibility of NMR by NVs at the nanoscale and under ambient conditions. The first demonstration of the potential of NV magnetometry first proved the imaging of another NV target spin,[21] creating the basis for the successful sensing of nuclear spins. To achieve this first breakthrough, the NV magnetometer was based on a diamond nanopillar functioning as a scanning probe, nanofabricated from ultrapure material. The nanopillar contained the NV spin sensor which scanned target NV spins in bulk diamond in nanometric proximity. This permitted the imaging of the magnetic dipole of a single electron spin under ambient conditions using AC mode and a dynamical decoupling pulse sequence. The use of NVs to sense nuclear spin was initially established in sensing 13C nuclear spins embedded in the spin bath of the diamond.[22] However, the relevance of NV-NMR is demonstrated in sensing other nuclear spins external to the diamond. The first recent breakthrough showed that NV is capable of detecting proton-NMR signals in an organic sample external to the diamond.[23] Similarly, it was proven useful for the detection of NMR signals within volumes of external samples (5 nm3).[24] In one study,[23] NV centers were formed 20 nm from the diamond surface within a 12C enriched grown diamond to avoid noise from the 13C nuclear spin bath naturally present
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in synthetic diamonds. Due to the NV’s close proximity to the surface, the sensitivity was limited by a 600 µs singleNV coherence time, measured using a Hahn-echo sequence. The sensed protons were contained in a 60 nm-thick layer of polymethylmethacrylate (acrylic; PMMA) on top of the diamond, and NVs probed a volume of protons of 24 nm3. After optical polarization, NVs underwent a Hahn-echo microwave sequence, synchronous, with two identical radio-frequency pulses, to produce an inversion of the proton polarization during the NV spin-echo sequence. The NV spin-echo response was dependent on the radio-frequency NMR transition of the protons. In another study,[24] NV was 5 nm from the diamond surface while 1H nuclear spins were contained in solid and liquid samples on the diamond surface. The spin noise from this ensemble of nuclear spins generates an oscillating field. This magnetic field has an amplitude and phase statistically fluctuating and could be detected by using a dynamical decoupling AC magnetometer based on an XY8-k sequence, as shown in the illustration in Figure 1. After the first π/2 pulse, the spin state is (|0> + eIφ|1>)/√2, where φ is the spin phase that evolves from 0 at the beginning of the sequence to a final random value Δφ, which is read out optically after the final π/2 pulse, as a population difference from the NV spin states 0 and 1. By averaging over many sequence repetitions, the variance of the phase is reduced. Every refocusing π pulse makes the phase sensitive to specific frequency components, providing a spectral filter centered around ν = 1/(2τ), where τ is the π pulse temporal spacing. Finally, by changing the value of τ, it is possible to read out from the NV fluorescence its coherence time change contrast, with a dip at half the Larmor magnetic field precession period of the probed nuclear spins. This corresponds in the frequency domain to a peak at the Larmor frequency of the statistically polarized nuclear spins (Figure 1). The probed volume contained 104 nuclear spins, while only 102 nuclear spins were statistically polarized.
3. Towards Imaging Single Biomolecules The magnetic noise produced by unpaired electrons at or near the diamond surface thus far is a limitation for the detection by an NV sensor of very few or single external nuclear spins. Although earlier studies proved that it is possible to greatly reduce the numbers of unpolarised diamond surface spins, they have not been eliminated completely. The ultimate sensitivity limit of single-molecule detection and higher-resolution imaging of nuclear and electron spins have been achieved using improved detection strategies in very recent demonstrations. One approach is based on relying on the strong coupling between the sensor and nuclear spins. Figure 2 illustrates the concepts of inductive NMR with a large number of the nuclear spins where a high B field polarizes them, nano-MRI with very low magnetic field, working with a dilute ensemble of nuclear spins (104) and using NVs as a sensor. In quantum MRI, very few or one nuclear spin magnetic field can be sensed. The quantum MRI approach results in a nuclear magnetic resonance signal which is independent from the number of the polarized spins. To
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Figure 2. a) Concept of high magnetic field inductive NMR, where a large ensemble of nuclear spins is probed (≥1012 spins). In this case, the sensor coupling (ΓS) with individual nuclear spins is much weaker than interactions between nuclei (ΓN), i.e., ΓN > ΓS. The nuclei are polarized by the high B field. The time-averaged measured signal, < S > , is proportional to the sample net polarized number of nuclear spins multiplied by the spin magnetic moment, µ, as < S > ∝ µ (N↓ – N↑). b) Nanoscale-NMR (detailed in Figure 1) refers to the detection of small ensembles (≈104 nuclear spins) with B ≈ 0, where the NV electron spin is the sensor and is still weakly coupled with the nuclear spins (ΓN > ΓS). The average signal < S > ∝ µ √N is still proportional to the sample magnetization where N spins are statistically polarized as statistical fluctuations may exceeds thermal polarization. c) Quantum-NMR corresponds to the detection of individual or few nuclei when the interaction of each nuclear spin with the sensor is stronger than the coupling to the surrounding bath (ΓS > ΓN). In this strong coupling regime, the detected signal is then proportional to < S > ∝ Nµ, where N is the number of nuclear spins, regardless their net polarization. Images reproduced with permission.[25] Copyright 2014, Macmillan Publishers, Ltd.
prove the quantum-MRI concept, four silicon nuclei (Si29) from silica deposited on diamond were strongly coupled with a 2 nm shallow NV electron spin sensor in bulk diamond.[25] Specifically fabricated glass-like coatings positioned on the diamond surface were probed by very close NV centers. The weakness of nuclear spin interactions with NV sensors has, up to now, placed a limit on single nuclear spin magnetic resonance imaging. A nano-MRI method using a single NV center has, however, been successfully applied to achieve the 2D imaging of 1H NMR from a polymer test with a spatial resolution of 12 nm.[26] This work removed a limit of previous NV-based nano-NMR probing a dilute sample of nuclear spins. The experimental apparatus developed in ref. [26] involved a scanning probe system based on a quartz tuning fork with a PMMA sample attached to it, and hanging over a diamond substrate with the NV center embedded by surface ion implantation. A XY8–96 sequence was used to measure the coherence of NV spins in the presence of the proton spin, and a clear dip was observed when the π pulse’s temporal separation matched the half of the proton precession period. By scanning the sample, NMR images were obtained measuring the proton resonance signal. Figure 3 presents the 2D NMR images from two distinct PMMA samples with two diverse NVs. The z height is proportional to the NMR signal small 2015, 11, No. 34, 4229–4236
s(x,y). Photon shot noise is predominantly responsible for the apparent roughness. This result makes it much closer to opportunities for visualizing the full 3D morphology of complex nanostructures including biomolecules, in vivo and nondestructively. Further development of the technique promises to bring resolution and signal-to-noise ratio enhancements, to the point where 3D imaging of proteins can be achieved. Multiple nuclear spins such as 1H, 19F, and 31P have been sensed with NMR and imaged using nano-MRI in confocal and wide-field microscopy imaging modalities.[17] The wide-field modality makes use of ensemble NV magnetometry concepts, combined with repeated sequence XY8-k, previously discussed. Figure 4 presents the nano-MRI imaging of multi-species sample with sub-micrometer structures in wide field modality. It has also been proven that NV is capable of measuring the magnetic resonance spectrum of a single molecule labeled using single-electron spin from nitroxide, a common spin label used in conventional electron spin paramagnetic resonance imaging.[27] The authors selected as the experimental sample MAD2 (mitotic arrest deficient-2), an essential spindle checkpoint protein. A single nitroxide spin label can easily modify this protein. The protein embedded in a poly-L-lysine layer is well restrained on the diamond surface. Its magnetic dipole interaction with a single NV center
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Figure 3. 2D images based on 1H NMR signals contained in two PMMA samples, probed using two different NV centers. The z height is proportional to the NMR signal s(x,y). Images reproduced with permission.[26] Copyright 2015, Macmillan Publishers, Ltd.
permits detection of this electron spin label. By simultaneously driving the NV probe and the spin label of the sample, the magnetic dipole interaction between the NV spin and the spin label was measured. Single NV centers were generated approximately 5 nm beneath the surface of the diamond. This demonstration is very important, as it first proves the possibility to extend the resolution of conventional spin label approaches to single-molecule spin tracking. Using the same optical detection and microwave manipulation of spins based on XY8-N microwave frequency sequence (similar to ref. [17] and [26], but combined with a scanning probe cantilever (as in Ref. [26] the distinctive measurement of a signal contrast from 1H and 19F nuclear spins was achieved using an NV in a bulk diamond, positioned 5 nm below the surface.[28] This work achieved a resolution of 10 nm in ambient conditions. By using a scanning probe labelled with 19F nuclear spins, an NMR resonant peak at the Larmor frequency of the fluorine nuclear spins was observed when the probe was engaged with the NV spin. The NMR peak signal switched to a 1H Larmor frequency resonance when the probe was retracted from the diamond surface. 1H nuclei were contained in a proton-rich organic absorbent 9 nm beneath the diamond surface. In this case, the sampling
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nuclear spins was a few nm3 out of the diavolume for mond surface. The method also allowed distinguishing subsurface chemical species, namely 19F and 1H, using the same fluorinated tip indented into the diamond surface. This method proves the possibility to achieve MRI contrasts for different nuclei within 10 nm, and it can be extended to arbitrary samples, opening new avenues in materials science and in biological sample analysis. A distinctively novel detection method that could improve tenfold currently demonstrated spatial resolutions has been proposed.[29] The method utilizes the coupling of the NV center to a nitrogen nuclear spin, which constitutes a very sharp dynamical frequency filter for NV to effectively sense fewer coupled nuclear spins and allows a longer acquisition time. In addition, the main thrust of this method is to make use, as in conventional MRI, of an effective magnetic field gradient, originating from the NV magnetic dipolar field spatial variation on the diamond surface. In this case it would be possible to decouple the NMR frequency of different nuclei within a complex biomolecule, such as the chemokine receptor CXCR4. A NV center positioned 1–2 nm from the surface is needed, and its magnetic field gradient created on the diamond surface would allow imaging of single nuclear spins in a molecule anchored on this surface. This method could achieve 2D-NMR with sub-nanometer resolution. Another method proved sub-nanometre resolution in nano-MRI applied to dark electronic spins,[30] with 0.8 nm and 1.5 nm lateral and vertical resolutions, respectively. The method provides the opportunity of 3D imaging. The demonstration combines the scanning magnetic field gradient used in magnetic force microscopy with a single NV magnetometer positioned 10 nm from the diamond surface (see Figure 5). The dark electron spins on the surface interact with a scanning magnetic atomic force microscope tip. The dark electron spins are driven by a radio-frequency signal (resonant dark spin in blue in Figure 5). The magnetic tip position relative to the diamond surface produces a local magnetic-field gradient. The tip magnetic field provides a narrower sampling of the spin spatial volume, as for different tip positions different dark spins precession are in resonance with the driving radiofrequency field. NV detects only dark spin magnetic signals in resonance with the driving radiofrequency field, while nonresonant spins (black spins in Figure 5) are not detected. Dark spin signal detection via a NV optical readout is achieved using a confocal optical microscope and NV spin is manipulated using a microwave excitation spin-echo sequence, whose π pulse is synchronous with the π pulse radiofrequency driving field. For each magnetic tip position, different dark spins are driven by the radio-frequency signal and accumulate a net NV phase. The radio-frequency sequence is known in conventional electron paramagnetic resonance methods as double electron–electron resonance.
4. Discussion and Conclusions Nano-MRI methods using NV centers in diamond are based on the concept of sensing spins using an in-principle atomicsize sensor exerting magnetic dipole–dipole interactions
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Figure 4. a) White-light transmission image of a fabricated structure by atomic layer deposition of SiO2 on the surface of a diamond containing a shallow (10 nm) high-density NV layer. b) Optical MRI achieved using a shallow ensemble of NVs in wide-field microscopy mode. The NMR signal is from the 19F nuclear spin density of a fluorinated sample on the diamond bare surface (A). The color bar indicates the NMR contrast, with blue representing a deep 19F NMR contrast dip and hence a high fluorine concentration on the bare diamond surface. Red represents no 19F NMR signal detected by the NV ensemble under the SiO2 structure (B), as the SiO2 layer displaces the fluorinated sample ≈90 nm away from the diamond surface and NV sensors. c,d) The same images from a second SiO2 structure/fluorinated sample on diamond surface. Images reproduced with permission.[17] Copyright 2015, Macmillan Publishers, Ltd.
Figure 5. Conceptual scheme to detect dark spins on a diamond surface using a scanning magnetic tip placed 100 nm from the diamond and a single NV sensor. A NV center in diamond located in a confocal laser spot with nearby dark spins is used as a sensor via the optical read-out of its electron spins, manipulated by applying a microwave (MW) signal. A radio-frequency (RF) signal drives the resonance of few electron dark spins, selected by the gradient magnetic field exerted by the scanning magnetic tip. These dark spins in resonance are detected by applying a double electron–electron resonance sequence for each tip position, while a MW spin-echo sequence is executed on the NV, allowing independent coherent control of the NV and dark spins. Images reproduced with permission.[30] Copyright 2015, Macmillan Publishers Ltd. small 2015, 11, No. 34, 4229–4236
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with very close nuclear or electron spins. This approach does not require a high magnetic field as in conventional inductive MRI (only small magnetic fields are applied, depending on the method), and therefore allows spatial nanometric resolution and a high signal-to-noise ratio over a larger frequency band. Different techniques to image few nuclei spins in polymer samples and to detect NMR spectra have been demonstrated based on optical confocal and wide-field microscopy, adding more complex microwave pulses sequence to narrow the frequency sensitivity of NV electron spin coherence time variation. Improvement of the initial methodology was achieved by adding scanning probes functionalized with nuclear spins and a scanning magnetic tip, permitting 2D imaging of protons with nanometric resolution and 3D imaging of dark electronic spins with sub-nanometric resolution. The methods developed using NV have evidenced the potential to achieve unprecedented resolution at room temperature in detecting few nuclear spins, electron dark spins, and electronic-spin labeled single biomolecules, pushing the boundary of previous realizations based on magnetic resonance force microscopy. However, the weakness of nuclear spins coupling at the nanoscale has placed a limit on 3D imaging of single nuclear spins. Further development of the technique is required to bring resolution and signal-to-noise ratio enhancements to the point where 3D imaging of nuclear spins within proteins can be achieved. Such breakthroughs over the state of the art may certainly open new avenues for medicine by revolutionizing nano-MRI imaging techniques. Presently, several different approaches have evidenced a fast growing field with relevant advances towards fully establishing a consolidated technique to respond to the quest of a molecular microscope that can provide 2D or 3D imaging, as it is well-established in conventional MRI scanning systems at the micrometer level. Several groups have provided remarkable progress with major stepping stones to deliver the expected future key breakthroughs. As NV centres are highly sensitive, they may sense the magnetic noise produced by other spin sources.[31] Therefore, the key to achieve the here-described major results relied on the opportunity to reduce the distance between the NV centre and the target
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nuclei by generating NV centres a few nanometres beneath the diamond surface.[17,26,29] Yet the spectral resolution of the resonance frequency of the nuclei is a long way from the resolution of conventional specrometers and the synthesis of diamond with near surface NV spins of long coherence time is also very difficult.[31] Nanofabrication of single crystal diamond probes and cantilivers containg a single NV with a long coherence time could allow further improvement. Finally, as the nuclear spin coupling degrades from nuclei more than few nanometres from the NV centre, 3D imaging of large molecules may be troublesome.[31] In this case, the use of small ensembles of NVs could allow an increase in the volume sampled, maintaining high spatial resolution albeit a shorter coherence time of the NV ensemble.
[15]
[16] [17]
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© 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Received: March 18, 2015 Revised: April 27, 2015 Published online: June 25, 2015
small 2015, 11, No. 34, 4229–4236
