Relaxometry with Nitrogen Vacancy (NV) Centers in Diamond

[Image: see text] Relaxometry is a technique which makes use of a specific crystal lattice defect in diamond, the so-called NV center. This defect consists of a nitrogen atom, which replaces a carbon atom in the diamond lattice, and an adjacent vacancy. NV centers allow converting magnetic noise into optical signals, which dramatically increases the sensitivity of the readout, allowing for nanoscale resolution. Analogously to T1 measurements in conventional magnetic resonance imaging (MRI), relaxometry allows the detection of different concentrations of paramagnetic species. However, since relaxometry allows very local measurements, the detected signals are from nanoscale voxels around the NV centers. As a result, it is possible to achieve subcellular resolutions and organelle specific measurements. A relaxometry experiment starts with polarizing the spins of NV centers in the diamond lattice, using a strong laser pulse. Afterward the laser is switched off and the NV centers are allowed to stochastically decay into the equilibrium mix of different magnetic states. The polarized configuration exhibits stronger fluorescence than the equilibrium state, allowing one to optically monitor this transition and determine its rate. This process happens faster at higher levels of magnetic noise. Alternatively, it is possible to conduct T1 relaxation measurements from the dark to the bright equilibrium by applying a microwave pulse which brings NV centers into the −1 state instead of the 0 state. One can record a spectrum of T1 at varying strengths of the applied magnetic field. This technique is called cross-relaxometry. Apart from detecting magnetic signals, responsive coatings can be applied which render T1 sensitive to other parameters as pH, temperature, or electric field. Depending on the application there are three different ways to conduct relaxometry experiments: relaxometry in moving or stationary nanodiamonds, scanning magnetometry, and relaxometry in a stationary bulk diamond with a stationary sample on top. In this Account, we present examples for various relaxometry modes as well as their advantages and limitations. Due to the simplicity and low cost of the approach, relaxometry has been implemented in many different instruments and for a wide range of applications. Herein we review the progress that has been achieved in physics, chemistry, and biology. Many articles in this field have a proof-of-principle character, and the full potential of the technology still waits to be unfolded. With this Account, we would like to stimulate discourse on the future of relaxometry

Elastic moduli of polyelectrolyte multilayer films regulate endothelium-blood interaction under dynamic conditions

A broad spectrum of biomaterials has been explored in order to design cardiovascular implants of sufficient hemocompatibility. Most of them were extensively tested for the ability to facilitate repopulation by patient cells. It was shown that stiffness, surface roughness, or hydrophilicity of polyelectrolyte films have an impact on adhesion, proliferation, and differentiation of cells. At the same time, it is still unknown how these properties influence cell functionality and as a consequence interactions with blood components under dynamic conditions. In this study, we aimed to determine the impact of chemical cross-linking of Chitosan (Chi) and Chrondroitin Sulphate (CS) on endothelium-blood cross-talk. We have found that the morphology of the endothelium monolayer was not altered by changes in coating properties. However, free radical generation by endothelial cells varied depending on the elastic properties of the coating. Simultaneously, we have observed a significant decrease in the level of adhering and circulating active platelets as well as aggregates when the endothelium monolayer was formed on stiffer films than on the other coating variants. Moreover, the same type of films has promoted significantly higher adhesion of blood morphotic elements when they were not functionalized by endothelium. The observed changes in hemocompatibility indicate the importance of a design of coatings that will promote cellularization in vivo in a relatively short time and which will regulate cell function.</p

Not all cells are created equal - endosomal escape in fluorescent nanodiamonds in different cells

Successful delivery of fluorescent nanodiamonds (FNDs) into the cytoplasm is essential to many biological applications. Other applications require FNDs to stay within the endosomes. The diversity of cellular uptake of FNDs and following endosomal escape are less explored. In this article, we quantify particle uptake at a single cell level. We report that FNDs enter into the cells gradually. The number of internalized FNDs per cell differs significantly for the cell lines we investigated at the same incubation time. In HeLa cells we do not see any significant endosomal escape. We also found a wide distribution of FND endosomal escape efficiency within the same cell type. However, compared with HeLa cells, FNDs in HUVECs can easily escape from the endosomes and less than 25% FNDs remained in the vesicles after 4 h incubation time. We believe this work can bring more attention to the diversity of the cells and provide potential guidelines for future studies

Quantum Sensing for Detection of Zinc-Triggered Free Radicals in Endothelial Cells

Oxidative stress originating from the overproduction of free radicals poses a major threat to cell fate, therefore it is of great importance to address the formation of free radicals in cells subjected to various pathological stimuli. Here we investigate the free radical response of endothelial cells to biodegradable zinc. In addition to the standard free radical assays, relaxometry was used for determining the production of free radicals in cells exposed to non-physiological concentrations of zinc ions. The cellular morphology, intracellular zinc accumulation, as well as the levels of reactive oxygen/nitrogen species, are determined using standard fluorescent methods. For endothelial cells subjected to 50% zinc extracts, deviations from the normal cell shape and cell agglomeration tendency are observed. The culture medium containing the highest amount of zinc ions caused nuclei fragmentation, blebbing, and cell shrinkage, indicating cell death. A potential explanation for the observed phenomena is an overproduction of free radicals. In the case of 1% and 10% zinc extracts, the formation of free radicals is clearly confirmed by relaxometry, while the results obtained by using fluorescent techniques are unambiguous. It is revealed that high concentrations of zinc ions released from biodegradable samples induce a deleterious effect on endothelial cells.</p

Fluorescent Nanodiamonds for Detecting Free-Radical Generation in Real Time during Shear Stress in Human Umbilical Vein Endothelial Cells

Free-radical generation is suspected to play a key role in cardiovascular diseases. Another crucial factor is shear stress. Human umbilical vein endothelial cells (HUVECS), which form the lining of blood vessels, require a physiological shear stress to activate many vasoactive factors. These are needed for maintaining vascular cell functions such as nonthrombogenicity, regulation of blood flow, and vascular tone. Additionally, blood clots form at regions of high shear stress within a blood vessel. Here, we use a new method called diamond magnetometry which allows us to measure the dynamics of free-radical generation in real time under shear stress. This quantum sensing technique allows free-radical detection with nanoscale resolution at the single-cell level. We investigate radical formation in HUVECs in a microfluidic environment under different flow conditions typically found in veins and arteries. Here, we looked into free-radical formation before, during, and after flow. We found that the free-radical production varied depending on the flow conditions. To confirm the magnetometry results and to differentiate between radicals, we performed conventional fluorescent reactive oxygen species (ROS) assays specific for superoxide, nitric oxide, and overall ROS

Measuring free radicals with relaxometry:Pioneering steps for measurements in human semen

A possible biological mechanism for unexplained male infertility is due to the effect of oxidative stress (OS), defined by the imbalance of reactive oxygen species (ROS) production, and the capacity of the antioxidant defence system to counteract it. In physiological concentrations, ROS and especially free radicals play an essential role in sperm maturation and fertilization, while an overabundance could lead to OS-induced damage to spermatozoa. To date, there are no direct detection techniques available that can measure the total amount of free radicals real time and identify where and when free radicals are generated. This study applies a quantum sensing technique using fluorescent nanodiamonds (FNDs), called T1 relaxometry, which is uniquely sensitive and specific for free radicals allowing measurements of the current radical load for nanoscale detection in living cells and body fluids. This proof-of-principle study investigates if we can use this technique to detect the free radical generation in human whole and separated, using density gradient centrifugation, semen. This method could be potentially used as new diagnostic measure for unexplained infertility or to track the effect of therapeutic interventions such as lifestyle changes. We adapted the existing relaxometry technique to measure free radicals in semen. The measured relaxation time (T1 time) was correlated to sperm concentration and progressive motility. Additionally, we explored the influence of the oxidative trigger hydrogen peroxide and the antioxidant glutathione on the free radical concentration measured. No significant correlations were found, which indicates that measurements in more proximity of the sperm cell are required to use relaxometry as a potential diagnostic tool for unexplained male infertility

Biomechanical and morphological stability of acellular scaffolds for tissue-engineered heart valves depends on different storage conditions

Currently available bioprosthetic heart valves have been successfully used clinically; however, they have several limitations. Alternatively, tissue-engineering techniques can be used. However, there are limited data concerning the impact of storage conditions of scaffolds on their biomechanics and morphology. The aim of this study was to determine the effect of different storage conditions on the biomechanics and morphology of pulmonary valve dedicated for the acellular scaffold preparation to achieve optimal conditions to obtain stable heart valve prostheses. Scaffold can then be used for the construction of tissue-engineered heart valve, for this reason evaluation of these parameters can determine the success of the clinical application this type of bioprosthesis. Pulmonary heart valves were collected from adult porcines. Materials were divided into five groups depending on the storage conditions. Biomechanical tests were performed, both the static tensile test, and examination of viscoelastic properties. Extracellular matrix morphology was evaluated using transmission electron microscopy and immunohistochemistry. Tissue stored at 4 °C exhibited a higher modulus of elasticity than the control (native) and fresh acellular, which indicated the stiffening of the tissue and changes of the viscoelastic properties. Such changes were not observed in the radial direction. Percent strain was not significantly different in the study groups. The storage conditions affected the acellularization efficiency and tissue morphology. To the best of our knowledge, this study is the first that attributes the mechanical properties of pulmonary valve tissue to the biomechanical changes in the collagen network due to different storage conditions. Storage conditions of scaffolds for tissue-engineered heart valves may have a significant impact on the haemodynamic and clinical effects of the used bioprostheses

Quantum Sensing for Detection of Zinc-Triggered Free Radicals in Endothelial Cells

Oxidative stress originating from the overproduction of free radicals poses a major threat to cell fate, therefore it is of great importance to address the formation of free radicals in cells subjected to various pathological stimuli. Here we investigate the free radical response of endothelial cells to biodegradable zinc. In addition to the standard free radical assays, relaxometry was used for determining the production of free radicals in cells exposed to non-physiological concentrations of zinc ions. The cellular morphology, intracellular zinc accumulation, as well as the levels of reactive oxygen/nitrogen species, are determined using standard fluorescent methods. For endothelial cells subjected to 50% zinc extracts, deviations from the normal cell shape and cell agglomeration tendency are observed. The culture medium containing the highest amount of zinc ions caused nuclei fragmentation, blebbing, and cell shrinkage, indicating cell death. A potential explanation for the observed phenomena is an overproduction of free radicals. In the case of 1% and 10% zinc extracts, the formation of free radicals is clearly confirmed by relaxometry, while the results obtained by using fluorescent techniques are unambiguous. It is revealed that high concentrations of zinc ions released from biodegradable samples induce a deleterious effect on endothelial cells.</p

Relaxometry for detecting free radical generation during Bacteria's response to antibiotics

Free radical generation plays a key role in killing bacteria by antibiotics. However, radicals are short-lived and reactive, and thus difficult to detect for the state of the art. Here we use a technique which allows optical nanoscale magnetic resonance imaging (MRI) to detect radical generation on the scale of single bacteria. We demonstrate that the radical generation in Staphylococcus aureus increases in the presence of UV irradiation as well as vancomycin and is dependent on the antibiotic's dose. With a method based on ensembles of nitrogen vacancy (NV) centers in diamond, we were able to follow the radical formation near individual bacteria over the whole duration of the experiment to reveal the dynamics of radical generation. Using this new approach, we observed free radical concentrations within nanoscale voxels around the diamond particles and determined its exact timing depending on the antibiotic dose. Since changes in the response to antibiotics emerge in only a few bacteria of the entire population, such a single-cell approach can prove highly valuable for research into drug resistance

Evaluation of Bacterial Adhesion to the ZrO2 Atomic Layer Deposited on the Surface of Cobalt-Chromium Dental Alloy Produced by DMLS Method

The main purpose of the research was to analyze the influence of surface modification of the cobalt-based alloy used in dental prosthetics by applying zirconium oxide (ZrO2) layers using the ALD (Atomic Layer Deposition) method. The samples were made using the DMLS (Direct Metal Laser Sintering) technique, and their surfaces were prepared in accordance with the principles of removable partial dentures (RPDs). A 50 nm-thick zirconium oxide coating was applied to the prepared substrates. This paper deals with the issues of prosthetic stomatopathy, which is a complex of pathological changes occurring in approx. 40% of the Polish population using removable dentures. Often, these changes, occurring on the mucosa, are related to improper performance, allergic reactions or the multiplication of bacteria on the surface of partial dentures. An innovative method of surface modification was proposed, together with the analysis of its influence on the physicochemical properties of the alloy and the adhesion of bacteria to the surface