David R. Glenn

Optical magnetic imaging of living cells

Magnetic imaging is a powerful tool for probing biological and physical systems. However, existing techniques either have poor spatial resolution compared to optical microscopy and are hence not generally applicable to imaging of sub-cellular structure (for example, magnetic resonance imaging), or entail operating conditions that preclude application to living biological samples while providing submicrometre resolution (for example, scanning superconducting quantum interference device microscopy, electron holography and magnetic resonance force microscopy). Here we demonstrate magnetic imaging of living cells (magnetotactic bacteria) under ambient laboratory conditions and with sub-cellular spatial resolution (400 nanometres), using an optically detected magnetic field imaging array consisting of a nanometre-scale layer of nitrogen–vacancy colour centres implanted at the surface of a diamond chip. With the bacteria placed on the diamond surface, we optically probe the nitrogen–vacancy quantum spin states and rapidly reconstruct images of the vector components of the magnetic field created by chains of magnetic nanoparticles (magnetosomes) produced in the bacteria. We also spatially correlate these magnetic field maps with optical images acquired in the same apparatus. Wide-field microscopy allows parallel optical and magnetic imaging of multiple cells in a population with submicrometre resolution and a field of view in excess of 100 micrometres. Scanning electron microscope images of the bacteria confirm that the correlated optical and magnetic images can be used to locate and characterize the magnetosomes in each bacterium. Our results provide a new capability for imaging bio-magnetic structures in living cells under ambient conditions with high spatial resolution, and will enable the mapping of a wide range of magnetic signals within cells and cellular networks.

Magnetic field imaging with nitrogen-vacancy ensembles

We demonstrate a method of imaging spatially varying magnetic fields using a thin layer of nitrogen-vacancy (NV) centers at the surface of a diamond chip. Fluorescence emitted by the two-dimensional NV ensemble is detected by a CCD array, from which a vector magnetic field pattern is reconstructed. As a demonstration, ac current is passed through wires placed on the diamond chip surface, and the resulting ac magnetic field patterns are imaged using an echo-based technique with sub-micron resolution over a 140µm◊140µm field of view, giving single-pixel sensitivity 100nT/ p Hz. We discuss ongoing efforts to further improve the sensitivity, as well as potential bioimaging applications such as real-time imaging of activity in functional, cultured networks of neurons.

Optical magnetic detection of single-neuron action potentials using quantum defects in diamond

Significance We demonstrate noninvasive detection of action potentials with single-neuron sensitivity, including in whole organisms. Our sensor is composed of quantum defects within a diamond chip, which detect time-varying magnetic fields generated by action potentials. The sensor is biocompatible and can be brought into close proximity to the organism without adverse effect, allowing for long-term observation and superior resolution of neuron magnetic fields. Optical magnetic detection with quantum defects also provides information about action potential propagation that is not easily available with existing methods. The quantum diamond technique requires no labeling or genetic modification, allows submillisecond time resolution, does not bleach, and senses through opaque tissue. With further development, we expect micrometer-scale magnetic imaging of a variety of neuronal phenomena. Magnetic fields from neuronal action potentials (APs) pass largely unperturbed through biological tissue, allowing magnetic measurements of AP dynamics to be performed extracellularly or even outside intact organisms. To date, however, magnetic techniques for sensing neuronal activity have either operated at the macroscale with coarse spatial and/or temporal resolution—e.g., magnetic resonance imaging methods and magnetoencephalography—or been restricted to biophysics studies of excised neurons probed with cryogenic or bulky detectors that do not provide single-neuron spatial resolution and are not scalable to functional networks or intact organisms. Here, we show that AP magnetic sensing can be realized with both single-neuron sensitivity and intact organism applicability using optically probed nitrogen-vacancy (NV) quantum defects in diamond, operated under ambient conditions and with the NV diamond sensor in close proximity (∼10 µm) to the biological sample. We demonstrate this method for excised single neurons from marine worm and squid, and then exterior to intact, optically opaque marine worms for extended periods and with no observed adverse effect on the animal. NV diamond magnetometry is noninvasive and label-free and does not cause photodamage. The method provides precise measurement of AP waveforms from individual neurons, as well as magnetic field correlates of the AP conduction velocity, and directly determines the AP propagation direction through the inherent sensitivity of NVs to the associated AP magnetic field vector.

Single-cell magnetic imaging using a quantum diamond microscope.

We apply a quantum diamond microscope for detection and imaging of immunomagnetically labeled cells. This instrument uses nitrogen-vacancy (NV) centers in diamond for correlated magnetic and fluorescence imaging. Our device provides single-cell resolution and a field of view (∼1 mm2) two orders of magnitude larger than that of previous NV imaging technologies, enabling practical applications. To illustrate, we quantified cancer biomarkers expressed by rare tumor cells in a large population of healthy cells.

Solar nebula magnetic fields recorded in the Semarkona meteorite

Magnetic fields are proposed to have played a critical role in some of the most enigmatic processes of planetary formation by mediating the rapid accretion of disk material onto the central star and the formation of the first solids. However, there have been no experimental constraints on the intensity of these fields. Here we show that dusty olivine-bearing chondrules from the Semarkona meteorite were magnetized in a nebular field of 54 ± 21 microteslas. This intensity supports chondrule formation by nebular shocks or planetesimal collisions rather than by electric currents, the x-wind, or other mechanisms near the Sun. This implies that background magnetic fields in the terrestrial planet-forming region were likely 5 to 54 microteslas, which is sufficient to account for measured rates of mass and angular momentum transport in protoplanetary disks. Magnetic field strength in the early solar system is recorded in chondrules within a meteorite born of the asteroid Vesta. Magnetic moments in planetary history To know the magnetic history of the solar nebula in the age of planet formation, researchers turn to the most primitive meteorites. Samples such as the Semarkona chondrite are composed partly of chondrules, which reflect the strength of the ambient magnetic field when this material was last molten. Fu et al. used a SQUID microscope to measure the remnant magnetization in a section of Semarkona. The findings reveal secrets about what goes on inside protoplanetary disks. Science, this issue p. 1089

Correlative light and electron microscopy using cathodoluminescence from nanoparticles with distinguishable colours

Correlative light and electron microscopy promises to combine molecular specificity with nanoscale imaging resolution. However, there are substantial technical challenges including reliable co-registration of optical and electron images, and rapid optical signal degradation under electron beam irradiation. Here, we introduce a new approach to solve these problems: imaging of stable optical cathodoluminescence emitted in a scanning electron microscope by nanoparticles with controllable surface chemistry. We demonstrate well-correlated cathodoluminescence and secondary electron images using three species of semiconductor nanoparticles that contain defects providing stable, spectrally-distinguishable cathodoluminescence. We also demonstrate reliable surface functionalization of the particles. The results pave the way for the use of such nanoparticles for targeted labeling of surfaces to provide nanoscale mapping of molecular composition, indicated by cathodoluminescence colour, simultaneously acquired with structural electron images in a single instrument.

Silicon-Vacancy Color Centers in Nanodiamonds: Cathodoluminescence Imaging Markers in the Near Infrared

We recently demonstrated [ 18 ] multi-color cathodoluminescence (CL) of nanodiamonds as a powerful tool for nanoscale imaging of biological structures. CL is the emission of light by matter as the result of electron bombardment. CL imaging of bulk matter is typically carried out in an electron microscope outfi tted with an optical detector, and is widely used in materials characterization. [ 19 ] However, application of CL to imaging biological structures has been hindered by low photon count rates and rapid signal degradation due to the destruction of biomolecules and organic fl uorophores under electron beam irradiation. [ 20 ] These problems may be overcome with correlated CL and secondary electron (SE) imaging of samples tagged with surface-functionalizable nanoparticles containing defects that are robust under electron beam illumination and emit stable, spectrally distinct CL: e.g., A-band defects and NV centers in nanodiamonds, as well as Ce:LuAG nanophosphors. [ 18 ] In this approach, the CLemitting particles function as color-distinguishable nanoscale markers of targeted epitopes, while the correlated SE image provides high-resolution information about the cellular structure. The combination of nanoscale molecular localization and structural imaging acquired simultaneously and in the same instrument constitutes a uniquely powerful new imaging modality. For applications with large intrinsic CL background, e.g., correlated CL and SE imaging of unfi xed/ living cells in an environmental chamber in a scanning electron microscope (SEM), [ 21 ] it is desirable to use spectrally narrow CL markers with emission peaks at wavelengths distinct from the CL background (such as from proteins, nucleic acid, and fl uorophore-conjugated antibodies, which usually emit CL at short optical wavelengths). [ 22 ] However, many of the nanoparticle species investigated to date have relatively broad (∼100 nm) CL emission spectra at room temperature. [ 20 , 23,24 ] Here, we show that silicon-vacancy (Si-V) color centers [ 25 ] in nanodiamonds provide a promising solution to this challenge. Specifi cally, we demonstrate experimentally that nanodiamonds fabricated to incorporate Si-V color centers provide bright, spectrally narrow (∼5 nm) CL emission in the near-infrared (∼740 nm), which lies within the near-infrared transmission window of biological tissue and is DOI: 10.1002/smll.201303582 Nanodiamonds

A Genetic Strategy for Probing the Functional Diversity of Magnetosome Formation

Model genetic systems are invaluable, but limit us to understanding only a few organisms in detail, missing the variations in biological processes that are performed by related organisms. One such diverse process is the formation of magnetosome organelles by magnetotactic bacteria. Studies of model magnetotactic α-proteobacteria have demonstrated that magnetosomes are cubo-octahedral magnetite crystals that are synthesized within pre-existing membrane compartments derived from the inner membrane and orchestrated by a specific set of genes encoded within a genomic island. However, this model cannot explain all magnetosome formation, which is phenotypically and genetically diverse. For example, Desulfovibrio magneticus RS-1, a δ-proteobacterium for which we lack genetic tools, produces tooth-shaped magnetite crystals that may or may not be encased by a membrane with a magnetosome gene island that diverges significantly from those of the α-proteobacteria. To probe the functional diversity of magnetosome formation, we used modern sequencing technology to identify hits in RS-1 mutated with UV or chemical mutagens. We isolated and characterized mutant alleles of 10 magnetosome genes in RS-1, 7 of which are not found in the α-proteobacterial models. These findings have implications for our understanding of magnetosome formation in general and demonstrate the feasibility of applying a modern genetic approach to an organism for which classic genetic tools are not available.

High-resolution magnetic resonance spectroscopy using a solid-state spin sensor

Quantum systems that consist of solid-state electronic spins can be sensitive detectors of nuclear magnetic resonance (NMR) signals, particularly from very small samples. For example, nitrogen–vacancy centres in diamond have been used to record NMR signals from nanometre-scale samples, with sensitivity sufficient to detect the magnetic field produced by a single protein. However, the best reported spectral resolution for NMR of molecules using nitrogen–vacancy centres is about 100 hertz. This is insufficient to resolve the key spectral identifiers of molecular structure that are critical to NMR applications in chemistry, structural biology and materials research, such as scalar couplings (which require a resolution of less than ten hertz) and small chemical shifts (which require a resolution of around one part per million of the nuclear Larmor frequency). Conventional, inductively detected NMR can provide the necessary high spectral resolution, but its limited sensitivity typically requires millimetre-scale samples, precluding applications that involve smaller samples, such as picolitre-volume chemical analysis or correlated optical and NMR microscopy. Here we demonstrate a measurement technique that uses a solid-state spin sensor (a magnetometer) consisting of an ensemble of nitrogen–vacancy centres in combination with a narrowband synchronized readout protocol to obtain NMR spectral resolution of about one hertz. We use this technique to observe NMR scalar couplings in a micrometre-scale sample volume of approximately ten picolitres. We also use the ensemble of nitrogen–vacancy centres to apply NMR to thermally polarized nuclear spins and resolve chemical-shift spectra from small molecules. Our technique enables analytical NMR spectroscopy at the scale of single cells.

Nanodiamond-enhanced MRI via in situ hyperpolarization

Nanodiamonds are of interest as nontoxic substrates for targeted drug delivery and as highly biostable fluorescent markers for cellular tracking. Beyond optical techniques, however, options for noninvasive imaging of nanodiamonds in vivo are severely limited. Here, we demonstrate that the Overhauser effect, a proton–electron polarization transfer technique, can enable high-contrast magnetic resonance imaging (MRI) of nanodiamonds in water at room temperature and ultra-low magnetic field. The technique transfers spin polarization from paramagnetic impurities at nanodiamond surfaces to 1H spins in the surrounding water solution, creating MRI contrast on-demand. We examine the conditions required for maximum enhancement as well as the ultimate sensitivity of the technique. The ability to perform continuous in situ hyperpolarization via the Overhauser mechanism, in combination with the excellent in vivo stability of nanodiamond, raises the possibility of performing noninvasive in vivo tracking of nanodiamond over indefinitely long periods of time.