Fast, Broad-Band Magnetic Resonance Spectroscopy with Diamond Widefield Relaxometry.

We present an alternative to conventional Electron Paramagnetic Resonance (EPR) spectroscopy equipment. Avoiding the use of bulky magnets and magnetron equipment, we use the photoluminescence of an ensemble of Nitrogen-Vacancy centers at the surface of a diamond. Monitoring their relaxation time (or T1), we detected their cross-relaxation with a compound of interest. In addition, the EPR spectra are encoded through a localized magnetic field gradient. While recording previous data took 12 min per data point with individual NV centers, we were able to reconstruct a full spectrum at once in 3 s, over a range from 3 to 11 G. In terms of sensitivity, only 0.5 µL of a 1 µM hexaaquacopper(II) ion solution was necessary.

Diamond-Based Nanoscale Quantum Relaxometry for Sensing Free Radical Production in Cells.

Diamond magnetometry makes use of fluorescent defects in diamonds to convert magnetic resonance signals into fluorescence. Because optical photons can be detected much more sensitively, this technique currently holds several sensitivity world records for room temperature magnetic measurements. It is orders of magnitude more sensitive than conventional magnetic resonance imaging (MRI) for detecting magnetic resonances. Here, the use of diamond magnetometry to detect free radical production in single living cells with nanometer resolution is experimentally demonstrated. This measuring system is first optimized and calibrated with chemicals at known concentrations. These measurements serve as benchmarks for future experiments. While conventional MRI typically has millimeter resolution, measurements are performed on individual cells to detect nitric oxide signaling at the nanoscale, within 10-20 nm from the internalized particles localized with a diffraction limited optical resolution. This level of detail is inaccessible to the state-of-the-art techniques. Nitric oxide is detected and the dynamics of its production and inhibition in the intra- and extracellular environment are followed.

Single-Particle Tracking and Trajectory Analysis of Fluorescent Nanodiamonds in Cell-Free Environment and Live Cells.

Diamond magnetometry can provide new insights on the production of free radicals inside live cells due to its high sensitivity and spatial resolution. However, the measurements often lack intracellular context for the recorded signal. In this paper, the possible use of single-particle tracking and trajectory analysis of fluorescent nanodiamonds (FNDs) to bridge that gap is explored. It starts with simulating a set of different possible scenarios of a particle's movement, reflecting different modes of motion, degrees of confinement, as well as shapes and sizes of that confinement. Then, the insights from the analysis of the simulated trajectories are applied to describe the movement of FNDs in glycerol solutions. It is shown that the measurements are in good agreement with the previously reported findings and that trajectory analysis yields meaningful results, when FNDs are tracked in a simple environment. Then the much more complex situation of FNDs moving inside a live cell is focused. The behavior of the particles after different incubation times is analyzed, and the possible intracellular localization of FNDs is deducted from their trajectories. Finally, this approach is combined with long-term magnetometry methods to obtain maps of the spin relaxation dynamics (or T1) in live cells, as FNDs move through the cytosol.

Quantum Sensing of Free Radicals in Primary Human Dendritic Cells.

Free radicals are crucial indicators for stress and appear in all kinds of pathogenic conditions, including cancer, cardiovascular diseases, and infection. However, they are difficult to detect due to their reactivity and low abundance. We use relaxometry for the detection of radicals with subcellular resolution. This method is based on a fluorescent defect in a diamond, which changes its optical properties on the basis of the magnetic surroundings. This technique allows nanoscale MRI with unprecedented sensitivity and spatial resolution. Recently, this technique was used inside living cells from a cell line. Cell lines differ in terms of endocytic capability and radical production from primary cells derived from patients. Here we provide the first measurements of phagocytic radical production by the NADPH oxidase (NOX2) in primary dendritic cells from healthy donors. The radical production of these cells differs greatly between donors. We investigated the cell response to stimulation or inhibition.

Nanodiamond Relaxometry-Based Detection of Free-Radical Species When Produced in Chemical Reactions in Biologically Relevant Conditions.

Diamond magnetometry is a quantum sensing method involving detection of magnetic resonances with nanoscale resolution. For instance, T1 relaxation measurements, inspired by equivalent concepts in magnetic resonance imaging (MRI), provide a signal that is equivalent to T1 in conventional MRI but in a nanoscale environment. We use nanodiamonds (between 40 and 120 nm) containing ensembles of specific defects called nitrogen vacancy (NV) centers. To perform a T1 relaxation measurement, we pump the NV center in the ground state (using a laser at 532 nm) and observe how long the NV center can remain in this state. Here, we use this method to provide real-time measurements of free radicals when they are generated in a chemical reaction. Specifically, we focus on the photolysis of H2O2 as well as the so-called Haber-Weiss reaction. Both of these processes are important reactions in biological environments. Unlike other fluorescent probes, diamonds are able to determine spin noise from different species in real time. We also investigate different diamond probes and their ability to sense gadolinium spin labels. Although this study was performed in a clean environment, we take into account the effects of salts and proteins that are present in a biological environment. We conduct our experiments with nanodiamonds, which are compatible with intracellular measurements. We perform measurements between 0 and 108 nM, and we are able to reach detection limits down to the nanomolar range and typically find T1 times of a few 100 µs. This is an important step toward label-free nano-MRI signal quantification in biological environments.

The Fate of Lipid-Coated and Uncoated Fluorescent Nanodiamonds during Cell Division in Yeast.

Fluorescent nanodiamonds are frequently used as biolabels. They have also recently been established for magnetic resonance and temperature sensing at the nanoscale level. To properly use them in cell biology, we first have to understand their intracellular fate. Here, we investigated, for the first time, what happens to diamond particles during and after cell division in yeast (Saccharomyces cerevisiae) cells. More concretely, our goal was to answer the question of whether nanodiamonds remain in the mother cells or end up in the daughter cells. Yeast cells are widely used as a model organism in aging and biotechnology research, and they are particularly interesting because their asymmetric cell division leads to morphologically different mother and daughter cells. Although yeast cells have a mechanism to prevent potentially harmful substances from entering the daughter cells, we found an increased number of diamond particles in daughter cells. Additionally, we found substantial excretion of particles, which has not been reported for mammalian cells. We also investigated what types of movement diamond particles undergo in the cells. Finally, we also compared bare nanodiamonds with lipid-coated diamonds, and there were no significant differences in respect to either movement or intracellular fate.

Toward Using Fluorescent Nanodiamonds To Study Chronological Aging in Saccharomyces cerevisiae.

One of the theories aiming to explain cellular aging is the free radical theory of aging, which describes the possible role of increased production and accumulation of free radicals. Fluorescent nanodiamonds (FNDs) are proposed to provide a tool to detect these radicals, as they function as magnetic sensors that change their optical properties depending on their magnetic surrounding. Therefore, they could enable the study of aging at a molecular level and unravel the exact role of free radicals in this process. In this study, important steps toward this goal are made. FNDs are introduced in chronologically aging yeast cells. Furthermore, the behavior of FNDs in these aging cells is studied to demonstrate the potency of using FNDs in the search for causes of cellular aging.

Nanodiamonds and Their Applications in Cells.

Diamonds owe their fame to a unique set of outstanding properties. They combine a high refractive index, hardness, great stability and inertness, and low electrical but high thermal conductivity. Diamond defects have recently attracted a lot of attention. Given this unique list of properties, it is not surprising that diamond nanoparticles are utilized for numerous applications. Due to their hardness, they are routinely used as abrasives. Their small and uniform size qualifies them as attractive carriers for drug delivery. The stable fluorescence of diamond defects allows their use as stable single photon sources or biolabels. The magnetic properties of the defects make them stable spin qubits in quantum information. This property also allows their use as a sensor for temperature, magnetic fields, electric fields, or strain. This Review focuses on applications in cells. Different diamond materials and the special requirements for the respective applications are discussed. Methods to chemically modify the surface of diamonds and the different hurdles one has to overcome when working with cells, such as entering the cells and biocompatibility, are described. Finally, the recent developments and applications in labeling, sensing, drug delivery, theranostics, antibiotics, and tissue engineering are critically discussed.

The interaction of fluorescent nanodiamond probes with cellular media.

Fluorescent nanodiamonds (FNDs) are promising tools to image cells, bioanalytes and physical quantities such as temperature, pressure, and electric or magnetic fields with nanometer resolution. To exploit their potential for intracellular applications, the FNDs have to be brought into contact with cell culture media. The interactions between the medium and the diamonds crucially influence sensitivity as well as the ability to enter cells. The authors demonstrate that certain proteins and salts spontaneously adhere to the FNDs and may cause aggregation. This is a first investigation on the fundamental questions on how (a) FNDs interact with the medium, and (b) which proteins and salts are being attracted. A differentiation between strongly binding and weakly binding proteins is made. Not all proteins participate in the formation of FND aggregates. Surprisingly, some main components in the medium seem to play no role in aggregation. Simple strategies to prevent aggregation are discussed. These include adding the proteins, which are naturally present in the cell culture to the diamonds first and then inserting them in the full medium. Graphical abstractSchematic of the interaction of nanodiamonds with cell culture medium. Certain proteins and salts adhere to the diamond surface and lead to aggregation or to formation of a protein corona.