Imaging Extreme Ultraviolet Radiation Using Nanodiamonds with Nitrogen-Vacancy Centers.

Extreme ultraviolet (EUV) radiation with wavelengths of 10-121 nm has drawn considerable attention recently for its use in photolithography to fabricate nanoelectronic chips. This study demonstrates, for the first time, fluorescent nanodiamonds (FNDs) with nitrogen-vacancy (NV) centers as scintillators to image and characterize EUV radiations. The FNDs employed are ∼100 nm in size; they form a uniform and stable thin film on an indium-tin-oxide-coated slide by electrospray deposition. The film is nonhygroscopic and photostable and can emit bright red fluorescence from NV0 centers when excited by EUV light. An FND-based imaging device has been developed and applied for beam diagnostics of 50 nm and 13.5 nm synchrotron radiations, achieving a spatial resolution of 30 µm using a film of ∼1 µm thickness. The noise equivalent power density is 29 µW/(cm2 Hz1/2) for the 13.5 nm radiation. The method is generally applicable to imaging EUV radiation from different sources.

Fluorescent nanodiamond-based spin-enhanced lateral flow immunoassay for detection of SARS-CoV-2 nucleocapsid protein and spike protein from different variants.

SARS-CoV-2 viruses, responsible for the COVID-19 pandemic, continues to evolve into new mutations, which poses a significant threat to public health. Current testing methods have some limitations, such as long turnaround times, high costs, and professional laboratory requirements. In this report, the novel Spin-Enhanced Lateral Flow Immunoassay (SELFIA) platform and fluorescent nanodiamond (FND) reporter were utilized for the rapid detection of SARS-CoV-2 nucleocapsid and spike antigens from different variants, including wild-type (Wuhan-1), Alpha (B.1.1.7), Delta (B.1.617.2), and Omicron (B.1.1.529). The SARS-CoV-2 antibodies were conjugated with FND via nonspecific binding, enabling the detection of SARS-CoV-2 antigens via both direct and competitive SELFIA format. Direct SELFIA was performed by directly adding the SARS-CoV-2 antibodies-conjugated FND on the antigens-immobilized nitrocellulose (NC) membrane. Conversely, the SARS-CoV-2 antigen-containing sample was first incubated with the antibodies-conjugated FND, and then dropped on the antigen-immobilized NC membrane to carry out the competitive SELFIA. The results suggested that S44F anti-S IgG antibody can be efficiently used for the detection of wild-type, Alpha, Delta, and Omicron variants spike antigens. Findings were comparable in direct SELFIA, competitive SELFIA, and ELISA. A detection limit of 1.94, 0.77, 1.14, 1.91, and 1.68 ng/mL can be achieved for SARS-CoV-2 N protein, wild-type, Alpha, Delta, and Omicron S proteins, respectively, via competitive SELFIA assay. These results suggest that a direct SELFIA assay can be used for antibody/antigen pair screening in diagnosis development, while the competitive SELFIA assay can serve as an accurate quantitative diagnostic tool. The simplicity and rapidity of the SELFIA platform were demonstrated, which can be leveraged in the detection of other infectious diseases in the near future.

Thermometric lateral flow immunoassay with colored latex beads as reporters for COVID-19 testing.

Temperature sensing is a promising method of enhancing the detection sensitivity of lateral flow immunoassay (LFIA) for point-of-care testing. A temperature increase of more than 100 °C can be readily achieved by photoexcitation of reporters like gold nanoparticles (GNPs) or colored latex beads (CLBs) on LFIA strips with a laser power below 100 mW. Despite its promise, processes involved in the photothermal detection have not yet been well-characterized. Here, we provide a fundamental understanding of this thermometric assay using non-fluorescent CLBs as the reporters deposited on nitrocellulose membrane. From a measurement for the dependence of temperature rises on the number density of membrane-bound CLBs, we found a 1.3-fold (and 3.2-fold) enhancement of the light absorption by red (and black) latex beads at 520 nm. The enhancement was attributed to the multiple scattering of light in this highly porous medium, a mechanism that could make a significant impact on the sensitivity improvement of LFIA. The limit of detection was measured to be 1 × 105 particles/mm2. In line with previous studies using GNPs as the reporters, the CLB-based thermometric assay provides a 10× higher sensitivity than color visualization. We demonstrated a practical use of this thermometric immunoassay with rapid antigen tests for COVID-19.

Magnetically Modulated Fluorescence of Nitrogen-Vacancy Centers in Nanodiamonds for Ultrasensitive Biomedical Analysis.

The negatively charged nitrogen-vacancy center in fluorescent nanodiamonds (FNDs) is a point defect with unique magneto-optical properties. It emits far-red fluorescence at ∼700 nm, and its intensity can be magnetically modulated with a depth of more than 10% at a field strength of 30 mT. We have closely examined this property and illustrated its practical use in biomedicine by applying a periodic, time-varying magnetic field to FNDs deposited on a surface or dispersed in a solution with a lock-in detection method. We achieved selective and sensitive detection of 100 nm FNDs on a nitrocellulose membrane at a particle density of 0.04 ng/mm2 (or ∼2 × 104 particles/mm2) and in an aqueous solution with a particle concentration of 1 ng/mL (or ∼1 fM) in 10 s as the detection limits. The utility and versatility of the technique were demonstrated with an application to background-free detection of FNDs as reporters for FND-based lateral flow immunoassays as well as selective quantification of FNDs in tissue digests for in vivo studies.

Mapping Dynamical Magnetic Responses of Ultrathin Micron-Size Superconducting Films Using Nitrogen-Vacancy Centers in Diamond.

Two-dimensional superconductors have attracted growing interest because of their scientific novelty, structural tunability, and useful properties. Studies of their magnetic responses, however, are often hampered by difficulties to grow large-size samples of high quality and uniformity. We report here an imaging method that employed NV- centers in diamond as a sensor capable of mapping out the microwave magnetic field distribution on an ultrathin superconducting film of micron size. Measurements on a 33 nm thick film and a 125 nm thick bulklike film of Bi2Sr2CaCu2O8+δ revealed that the alternating current (ac) Meissner effect (or repulsion of ac magnetic field) set in at 78 and 91 K, respectively; the latter was the superconducting transition temperature of both films. The unusual ac magnetic response of the thin film presumably was due to thermally excited vortex-antivortex diffusive motion in the film. Spatial resolution of our ac magnetometer was limited by optical diffraction and the noise level was at 14 µT/Hz1/2. The technique could be extended with better detection sensitivity to extract local ac conductivity/susceptibility of ultrathin or monolayer superconducting samples as well as ac magnetic responses of other two-dimensional exotic thin films of limited lateral size.

Quantification and Imaging of Antigens on Cell Surface with Lipid-Encapsulated Fluorescent Nanodiamonds.

Quantifying the density and locating the position of antigens on cell surface has been a challenge in molecular biology research. The challenge lies in the need for a chemically and photophysically stable fluorophore to achieve the required sensitivity and accuracy. Here, we present a method suitable for the purpose by using lipid-encapsulated fluorescent nanodiamonds (FNDs) of 35 nm in diameter as biolabels. The encapsulation of FNDs in biotinylated phospholipids not only facilitates good dispersion of the particles in biological buffers, but also endows them with high specific targeting ability. We demonstrated a viable application of the technique for biotin-mediated immunostaining of antigens on fixed human cells, identifying their positions by two-color confocal fluorescence imaging, and determining their densities by magnetically modulated fluorescence detection. A binding capacity of 6 ± 1 × 104 antigens/cell was measured specifically for CD44 on HeLa cell surface. The result agreed well with the assay of R-phycoerythrin-conjugated antibodies by flow cytometry, supporting the reliability of this new nanoparticle-based method.

Light Emission from Plasmonic Nanostructures Enhanced with Fluorescent Nanodiamonds.

In the surface-enhanced fluorescence (SEF) process, it is well known that the plasmonic nanostructure can enhance the light emission of fluorescent emitters. With the help of atomic force microscopy, a hybrid system consisting of a fluorescent nanodiamond and a gold nanoparticle was assembled step-by-step for in situ optical measurements. We demonstrate that fluorescent emitters can also enhance the light emission from gold nanoparticles which is judged through the intrinsic anti-Stokes emission owing to the nanostructures. The light emission intensity, spectral shape, and lifetime of the hybrid system were dependent on the coupling configuration. The interaction between gold nanoparticles and fluorescent emitter was modelled based on the concept of a quantised optical cavity by considering the nanodiamond and the nanoparticle as a two-level energy system and a nanoresonator, respectively. The theoretical calculations reveal that the dielectric antenna effect can enhance the local field felt by the nanoparticle, which contributes more to the light emission enhancement of the nanoparticles rather than the plasmonic coupling effect. The findings reveal that the SEF is a mutually enhancing process. This suggests the hybrid system should be considered as an entity to analyse and optimise surface-enhanced spectroscopy.

Fluorescent nanodiamonds enable quantitative tracking of human mesenchymal stem cells in miniature pigs.

Cell therapy is a promising strategy for the treatment of human diseases. While the first use of cells for therapeutic purposes can be traced to the 19th century, there has been a lack of general and reliable methods to study the biodistribution and associated pharmacokinetics of transplanted cells in various animal models for preclinical evaluation. Here, we present a new platform using albumin-conjugated fluorescent nanodiamonds (FNDs) as biocompatible and photostable labels for quantitative tracking of human placenta choriodecidual membrane-derived mesenchymal stem cells (pcMSCs) in miniature pigs by magnetic modulation. With this background-free detection technique and time-gated fluorescence imaging, we have been able to precisely determine the numbers as well as positions of the transplanted FND-labeled pcMSCs in organs and tissues of the miniature pigs after intravenous administration. The method is applicable to single-cell imaging and quantitative tracking of human stem/progenitor cells in rodents and other animal models as well.

Fluorescent Nanodiamond: A Versatile Tool for Long-Term Cell Tracking, Super-Resolution Imaging, and Nanoscale Temperature Sensing.

Fluorescent nanodiamond (FND) has recently played a central role in fueling new discoveries in interdisciplinary fields spanning biology, chemistry, physics, and materials sciences. The nanoparticle is unique in that it contains a high density ensemble of negatively charged nitrogen-vacancy (NV(-)) centers as built-in fluorophores. The center possesses a number of outstanding optical and magnetic properties. First, NV(-) has an absorption maximum at ∼550 nm, and when exposed to green-orange light, it emits bright fluorescence at ∼700 nm with a lifetime of longer than 10 ns. These spectroscopic properties are little affected by surface modification but are distinctly different from those of cell autofluorescence and thus enable background-free imaging of FNDs in tissue sections. Such characteristics together with its excellent biocompatibility render FND ideal for long-term cell tracking applications, particularly in stem cell research. Next, as an artificial atom in the solid state, the NV(-) center is perfectly photostable, without photobleaching and blinking. Therefore, the NV-containing FND is suitable as a contrast agent for super-resolution imaging by stimulated emission depletion (STED). An improvement of the spatial resolution by 20-fold is readily achievable by using a high-power STED laser to deplete the NV(-) fluorescence. Such improvement is crucial in revealing the detailed structures of biological complexes and assemblies, including cellular organelles and subcellular compartments. Further enhancement of the resolution for live cell imaging is possible by manipulating the charge states of the NV centers. As the "brightest" member of the nanocarbon family, FND holds great promise and potential for bioimaging with unprecedented resolution and precision. Lastly, the NV(-) center in diamond is an atom-like quantum system with a total electron spin of 1. The ground states of the spins show a crystal field splitting of 2.87 GHz, separating the ms = 0 and ±1 sublevels. Interestingly, the transitions between the spin sublevels can be optically detected and manipulated by microwave radiation, a technique known as optically detected magnetic resonance (ODMR). In addition, the electron spins have an exceptionally long coherence time, making FND useful for ultrasensitive detection of temperature at the nanoscale. Pump-probe-type nanothermometry with a temporal resolution of better than 10 µs has been achieved with a three-point sampling method. Gold/diamond nanohybrids have also been developed for highly localized hyperthermia applications. This Account provides a summary of the recent advances in FND-enabled technologies with a special focus on long-term cell tracking, super-resolution imaging, and nanoscale temperature sensing. These emerging and multifaceted technologies are in synchronicity with modern imaging modalities.

Wide-field imaging and flow cytometric analysis of cancer cells in blood by fluorescent nanodiamond labeling and time gating.

Nanodiamonds containing high density ensembles of negatively charged nitrogen-vacancy (NV(-)) centers are promising fluorescent biomarkers due to their excellent photostability and biocompatibility. The NV(-) centers in the particles have a fluorescence lifetime of up to 20 ns, which distinctly differs from those (<10 ns) of cell and tissue autofluorescence, making it possible to achieve background-free detection in vivo by time gating. Here, we demonstrate the feasibility of using fluorescent nanodiamonds (FNDs) as optical labels for wide-field time-gated fluorescence imaging and flow cytometric analysis of cancer cells with a nanosecond intensified charge-coupled device (ICCD) as the detector. The combined technique has allowed us to acquire fluorescence images of FND-labeled HeLa cells in whole blood covered with a chicken breast of ~0.1-mm thickness at the single cell level, and to detect individual FND-labeled HeLa cells in blood flowing through a microfluidic device at a frame rate of 23 Hz, as well as to locate and trace FND-labeled lung cancer cells in the blood vessels of a mouse ear. It opens a new window for real-time imaging and tracking of transplanted cells (such as stem cells) in vivo.

Polarization modulation spectroscopy of single fluorescent nanodiamonds with multiple nitrogen vacancy centers.

A technique based on polarization modulation spectroscopy (PMS) has been developed to determine quantitatively the number of fluorophores in nanoparticles at the single-molecule level. The technique involves rotation of the polarization of the excitation laser on a millisecond time scale, leading to fluorescence intensity modulation. By taking account of the heterogeneous orientation among the dipoles of the fluorophores and simulating the modulation depth distribution with Monte Carlo calculations, we show that it is possible to deduce the ensemble average and number distribution of the fluorophores. We apply the technique to fluorescent nanodiamonds (FNDs) containing multiple nitrogen vacancy (NV) centers. Comparing the experimental and simulated modulation depth distributions of 11 nm FNDs, we deduce an average number of = 3, which is in good agreement with independent photon correlation measurements. The method is general, rapid, and applicable to other nanoparticles, polymers, and molecular complexes containing multiple and randomly orientated fluorophores as well.

Two-photon fluorescence correlation spectroscopy of lipid-encapsulated fluorescent nanodiamonds in living cells.

Dynamics of fluorescent diamond nanoparticles in HeLa cells has been studied with two-photon fluorescence correlation spectroscopy (FCS). Fluorescent nanodiamond (FND) is an excellent fluorescent probe for bioimaging application, but they are often trapped in endosomes after cellular uptake. The entrapment prohibits FCS from being performed in a time frame of 60 s. Herein, we show that the encapsulation of FNDs within a lipid layer enhances the diffusion of the particles in the cytoplasm by more than one order of magnitude, and particles as small as 40 nm can be probed individually with high image contrast by two-photon excited luminescence. The development of the technique together with single particle tracking through one-photon excitation allows probing of both short-term and long-term dynamics of single FNDs in living cells.