Fluorescent particles are routinely used to probe biological processes. The quantum properties of single spins within fluorescent particles have been explored in the field of nanoscale magnetometry, but not yet in biological environments. Here, we demonstrate optically detected magnetic resonance of individual fluorescent nanodiamond nitrogen-vacancy centres inside living human HeLa cells, and measure their location, orientation, spin levels and spin coherence times with nanoscale precision. Quantum coherence was measured through Rabi and spin-echo sequences over long (>10 h) periods, and orientation was tracked with effective 1° angular precision over acquisition times of 89 ms. The quantum spin levels served as fingerprints, allowing individual centres with identical fluorescence to be identified and tracked simultaneously. Furthermore, monitoring decoherence rates in response to changes in the local environment may provide new information about intracellular processes. The experiments reported here demonstrate the viability of controlled single spin probes for nanomagnetometry in biological systems, opening up a host of new possibilities for quantum-based imaging in the life sciences.

The detection of small numbers of magnetic spins is a significant challenge in the life, physical and chemical sciences, especially when room temperature operation is required. Here we show that a proximal nitrogen-vacancy spin ensemble serves as a high precision sensing and imaging array. Monitoring its longitudinal relaxation enables sensing of freely diffusing, unperturbed magnetic ions and molecules in a microfluidic device without applying external magnetic fields. Multiplexed charge-coupled device acquisition and an optimized detection scheme permits direct spin noise imaging of magnetically labelled cellular structures under ambient conditions. Within 20 s we achieve spatial resolutions below 500 nm and experimental sensitivities down to 1,000 statistically polarized spins, of which only 32 ions contribute to a net magnetization. The results mark a major step towards versatile subcellular magnetic imaging and real-time spin sensing under physiological conditions providing a minimally invasive tool to monitor ion channels or haemoglobin trafficking inside live cells.

Here we report the increase of the coherence time T 2 of a single electron spin at room temperature by using dynamical decoupling. We show that the Carr-Purcell-Meiboom-Gill (CPMG) pulse sequence can prolong the T 2 of a single Nitrogen-Vacancy center in diamond up to 2.44 ms compared to the Hahn echo measurement where T 2 = 390 µs. Moreover, by performing spin locking experiments we demonstrate that with CPMG the maximum possible T 2 is reached. On the other hand, we do not observe strong increase of the coherence time in nanodiamonds, possibly due to the short spin lattice relaxation time T 1 = 100 µs (compared to T 1 = 5.93 ms in bulk). An application for detecting low magnetic field is demonstrated, where we show that the sensitivity using the CPMG method is improved by about a factor of two compared to the Hahn echo method.

Electron spin resonance (ESR) describes a suite of techniques for characterizing electronic systems with applications in physics, chemistry, and biology. However, the requirement for large electron spin ensembles in conventional ESR techniques limits their spatial resolution. Here we present a method for measuring ESR spectra of nanoscale electronic environments by measuring the longitudinal relaxation time of a single-spin probe as it is systematically tuned into resonance with the target electronic system. As a proof of concept, we extracted the spectral distribution for the P1 electronic spin bath in diamond by using an ensemble of nitrogen-vacancy centres, and demonstrated excellent agreement with theoretical expectations. As the response of each nitrogen-vacancy spin in this experiment is dominated by a single P1 spin at a mean distance of 2.7 nm, the application of this technique to the single nitrogen-vacancy case will enable nanoscale ESR spectroscopy of atomic and molecular spin systems.

Magnetic field fluctuations arising from fundamental spins are ubiquitous in nanoscale biology, and are a rich source of information about the processes that generate them. However, the ability to detect the few spins involved without averaging over large ensembles has remained elusive. Here, we demonstrate the detection of gadolinium spin labels in an artificial cell membrane under ambient conditions using a single-spin nanodiamond sensor. Changes in the spin relaxation time of the sensor located in the lipid bilayer were optically detected and found to be sensitive to nearindividual (4 ± 2) proximal gadolinium atomic labels. The detection of such small numbers of spins in a model biological setting, with projected detection times of 1 s [corresponding to a sensitivity of ∼5 Gd spins per Hz 1/2 ], opens a pathway for in situ nanoscale detection of dynamical processes in biology.nitrogen-vacancy center | biophysics | nanomagnetometry T he development of sensitive and highly localized probes has driven advances in our understanding of the basic processes of life at increasingly smaller scales (1). In the last decade there has been a strong drive to expand the range of probes that can be used for studying biological systems (2-6), with emphasis on the detection of atoms and molecules in nanometer-sized volumes to gain access to information that may be hidden in ensemble averaging. However, at present there are no nanoprobes suitable for directly sensing the weak magnetic fields arising from small numbers of fundamental spins in nanoscale biology, occurring naturally (e.g., free radicals) or introduced (e.g., spin labels). These can be a rich source of information about processes at the atomic and molecular level. Magnetic resonance techniques such as electron spin resonance (ESR) have played an important role in the development of our understanding of membranes, proteins, and free radicals (7); however, ESR sensitivity and resolution are fundamentally limited to mesoscopic ensembles of at least 10 7 spins with a sensitivity of ∼2 × 10 9 spins per Hz 1/2 (8). In a typical ESR application, small electron spin label moieties are attached to the system of interest and their environment is investigated through spin measurements on the labels. Because of the large ensemble required, nanoscopic detail at the few-spin level can be lost in the averaging process. Recently, magnetic resonance force microscopy techniques have demonstrated single-spin detection (9-11), but these require cryogenic temperatures and vacuum. Here, we demonstrate a nanoparticle probe--a nitrogen-vacancy spin in a nanodiamond--which is situated in the target structure itself and acts as a nanoscopic magnetic field detector under ambient conditions with noncontact optical readout. We use this probe to detect near-individual spin labels in an artificial cell membrane at a projected sensitivity of ∼5 Gd spins per Hz 1/2 , effectively bridging the gap between traditional ESR ensemble-based techniques and the ultimate goal of few-spin nanoscale detection...

A quantitative understanding of the dynamics of biological neural networks is fundamental to gaining insight into information processing in the brain. While techniques exist to measure spatial or temporal properties of these networks, it remains a significant challenge to resolve the neural dynamics with subcellular spatial resolution. In this work we consider a fundamentally new form of wide-field imaging for neuronal networks based on the nanoscale magnetic field sensing properties of optically active spins in a diamond substrate. We analyse the sensitivity of the system to the magnetic field generated by an axon transmembrane potential and confirm these predictions experimentally using electronically-generated neuron signals. By numerical simulation of the time dependent transmembrane potential of a morphologically reconstructed hippocampal CA1 pyramidal neuron, we show that the imaging system is capable of imaging planar neuron activity non-invasively at millisecond temporal resolution and micron spatial resolution over wide-fields.

Optical biomarkers have been used extensively for intracellular imaging with high spatial and temporal resolution. Extending the modality of these probes is a key driver in cell biology. In recent years, the nitrogen-vacancy (NV) center in nanodiamond has emerged as a promising candidate for bioimaging and biosensing with low cytotoxicity and stable photoluminescence. Here we study the electrophysiological effects of this quantum probe in primary cortical neurons. Multielectrode array recordings across five replicate studies showed no statistically significant difference in 25 network parameters when nanodiamonds are added at varying concentrations over various time periods, 12-36 h. The physiological validation motivates the second part of the study, which demonstrates how the quantum properties of these biomarkers can be used to report intracellular information beyond their location and movement. Using the optically detected magnetic resonance from the nitrogen-vacancy defects within the nanodiamonds we demonstrate enhanced signal-to-noise imaging and temperature mapping from thousands of nanodiamond probes simultaneously. This work establishes nanodiamonds as viable multifunctional intraneuronal sensors with nanoscale resolution, which may ultimately be used to detect magnetic and electrical activity at the membrane level in excitable cellular systems.

We present a study of the spin properties of dense layers of near-surface nitrogen-vacancy (NV) centres in diamond created by nitrogen ion implantation. The optically detected magnetic resonance contrast and linewidth, spin coherence time, and spin relaxation time, are measured as a function of implantation energy, dose, annealing temperature and surface treatment. To track the presence of damage and surface-related spin defects, we perform in situ electron spin resonance spectroscopy through both double electron-electron resonance and cross-relaxation spectroscopy on the NV centres. We find that, for the energy (4−30 keV) and dose (5×10 11 −10 13 ions/cm 2 ) ranges considered, the NV spin properties are mainly governed by the dose via residual implantation-induced paramagnetic defects, but that the resulting magnetic sensitivity is essentially independent of both dose and energy. We then show that the magnetic sensitivity is significantly improved by high-temperature annealing at ≥ 1100 • C. Moreover, the spin properties are not significantly affected by oxygen annealing, apart from the spin relaxation time, which is dramatically decreased. Finally, the average NV depth is determined by nuclear magnetic resonance measurements, giving ≈ 10-17 nm at 4-6 keV implantation energy. This study sheds light on the optimal conditions to create dense layers of near-surface NV centres for high-sensitivity sensing and imaging applications.